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The Effects of Added Fiber to the Diets of Chronic Kidney Disease Patients on Quality of Life, Clinical Markers And, Gas...

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Title:
The Effects of Added Fiber to the Diets of Chronic Kidney Disease Patients on Quality of Life, Clinical Markers And, Gastrointestinal and Kidney Function
Physical Description:
1 online resource (150 p.)
Language:
english
Creator:
Salmean, Younis A
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Dahl, Wendy Joanne
Committee Members:
Henken, Robin J
Sitren, Harry S
Mai, Volker

Subjects

Subjects / Keywords:
bowel -- fermentable -- fiber -- fos -- function -- inulin -- kidney -- peahull
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre:
Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Chronic kidney disease (CKD) patients may consume lower than recommended amounts of dietary fiber due to typical dietary habits, dietary restrictions, and uremic symptoms. Progressive decline in kidney function causes an accumulation of uremic molecules that contribute to further progression of the disease and reduced quality of life. The objective was to conduct clinical trials to determine the effects of added fiber in the diet of CKD patients on uremic molecules, kidney function, uremia, and quality of life.  Two single blind, intervention clinical trials with patients with moderate to severe decline in kidney function (eGFR of = 50 mL/min/1.73 m2) were conducted. The first study was 6 weeks in duration; 2 weeks of control and 4 weeks intervention with 23 g/d of mixed fiber sources. The second study was 12 weeks in duration; 2 weeks control, followed by 4 weeks intervention with 10 g/d of pea hull fiber, followed by 6 weeks of an additional 13.5 g/d of fiber from inulin. Study 1: Provision of 23 g/d of added fiber lowered serum creatinine from 2.44±0.30 mg/dL (mean±SE) at baseline to 2.21±0.26 mg/dL after 4 weeks of intervention (p(MDRD) from 29.6±3.5 mL/min/1.73m2 at baseline to 32.5±3.6 mL/min/1.73m2 after 4 weeks of intervention (p±2 at baseline to 35±3 (pStudy 2: Foods fortified with 10 g/d of pea hull fiber did not improve uremic profile or eGFR(creatinine-cystatin C). Inulin supplement at 3 weeks improved eGFR (47.6±5.6 mL/min/1.73 m2) compared to control period (42.4±5 mL/min/1.73 m2), but not baseline (43.9±4.8 mL/min/1.73 m2). However, this improvement diminished after 6 weeks of inulin supplementation when supplement compliance dropped from 90% to 77%. Plasma p-cresol decreased by 20% from baseline at study end. Supplementing the diet of CKD patients with insoluble and fermentable fibers may reduce uremic molecules and thus improve renal function and improve some aspects of quality of life.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Younis A Salmean.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Dahl, Wendy Joanne.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045366:00001

MISSING IMAGE

Material Information

Title:
The Effects of Added Fiber to the Diets of Chronic Kidney Disease Patients on Quality of Life, Clinical Markers And, Gastrointestinal and Kidney Function
Physical Description:
1 online resource (150 p.)
Language:
english
Creator:
Salmean, Younis A
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Dahl, Wendy Joanne
Committee Members:
Henken, Robin J
Sitren, Harry S
Mai, Volker

Subjects

Subjects / Keywords:
bowel -- fermentable -- fiber -- fos -- function -- inulin -- kidney -- peahull
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre:
Food Science and Human Nutrition thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Chronic kidney disease (CKD) patients may consume lower than recommended amounts of dietary fiber due to typical dietary habits, dietary restrictions, and uremic symptoms. Progressive decline in kidney function causes an accumulation of uremic molecules that contribute to further progression of the disease and reduced quality of life. The objective was to conduct clinical trials to determine the effects of added fiber in the diet of CKD patients on uremic molecules, kidney function, uremia, and quality of life.  Two single blind, intervention clinical trials with patients with moderate to severe decline in kidney function (eGFR of = 50 mL/min/1.73 m2) were conducted. The first study was 6 weeks in duration; 2 weeks of control and 4 weeks intervention with 23 g/d of mixed fiber sources. The second study was 12 weeks in duration; 2 weeks control, followed by 4 weeks intervention with 10 g/d of pea hull fiber, followed by 6 weeks of an additional 13.5 g/d of fiber from inulin. Study 1: Provision of 23 g/d of added fiber lowered serum creatinine from 2.44±0.30 mg/dL (mean±SE) at baseline to 2.21±0.26 mg/dL after 4 weeks of intervention (p(MDRD) from 29.6±3.5 mL/min/1.73m2 at baseline to 32.5±3.6 mL/min/1.73m2 after 4 weeks of intervention (p±2 at baseline to 35±3 (pStudy 2: Foods fortified with 10 g/d of pea hull fiber did not improve uremic profile or eGFR(creatinine-cystatin C). Inulin supplement at 3 weeks improved eGFR (47.6±5.6 mL/min/1.73 m2) compared to control period (42.4±5 mL/min/1.73 m2), but not baseline (43.9±4.8 mL/min/1.73 m2). However, this improvement diminished after 6 weeks of inulin supplementation when supplement compliance dropped from 90% to 77%. Plasma p-cresol decreased by 20% from baseline at study end. Supplementing the diet of CKD patients with insoluble and fermentable fibers may reduce uremic molecules and thus improve renal function and improve some aspects of quality of life.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Younis A Salmean.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: Dahl, Wendy Joanne.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045366:00001


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1 THE EFFECTS OF ADDED FIBER TO THE DIETS OF CHRONIC KIDNEY DISEASE PATIENTS ON QUALITY OF LIFE, CLINICAL MARKERS AND, GASTROINTESTINAL AND KIDNEY FUNCTION By YOUNIS ALI SALMEAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Younis Ali Salmean

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3 To my wife Maryam, my son Zaid, my da ughter, Jenna, and my m other and f amily

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4 ACKNOWLEDGMENTS I would like to specially thank my wife for her tremendous sacrifices and continued support during this journey. I would like to acknowledge Dr. Fatma Huffman and Dr. Jiwan Sidhu who believed in me and nominated me above all to earn the scholarship from Kuwait University that allowed me to go on and earn this degree. F inally I would like to thank my advisor Dr. Wendy Dahl for her mentoring and guidance during this difficult journey.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 1 INTRODUCTION ................................ ................................ ................................ .... 15 2 REVIEW OF THE LITERATURE ................................ ................................ ............ 19 Fiber Definition ................................ ................................ ................................ ........ 28 Pea Hull Fiber ................................ ................................ ................................ ......... 29 Inulin and FOS Fiber ................................ ................................ ............................... 30 Resistan t Corn Dextrin Fiber ................................ ................................ ................... 31 Fiber Recommendations and Current Intakes in General Population ..................... 31 Dietary Reference Intakes (DRIs) for Fiber and Current Intakes in CKD Population ................................ ................................ ................................ ............ 31 Chronic Kidney Disease Prevalence Overview ................................ ....................... 32 Stages of Chronic Kidney Disease ................................ ................................ ......... 33 The Kidneys ................................ ................................ ................................ ............ 34 Filtration ................................ ................................ ................................ .................. 37 Serum Creatinine ................................ ................................ ................................ .... 38 Serum Cystatin C ................................ ................................ ................................ .... 40 Uremic Load, Progressive Hyperfiltration, Uremic Toxins and Disease Progression ................................ ................................ ................................ ......... 41 C Reactive Protein ................................ ................................ ................................ .. 42 Fermentation ................................ ................................ ................................ ........... 43 ................................ ................................ ....................... 45 Description ................................ ................................ ................................ ....... 45 Urea ................................ ................................ ................................ ................. 45 p Cresol ................................ ................................ ................................ ............ 46 ................................ ................................ .................... 47 Health Related Quality of Life and Symptoms ................................ ........................ 48 Gastrointestinal Health and Well being ................................ ................................ ... 4 9 Appetite and Sleepiness ................................ ................................ ......................... 51 3 MATERIALS AND METHODS ................................ ................................ ................ 53 Study 1: Overview ................................ ................................ ................................ ... 53 Study Design ................................ ................................ ................................ .......... 53 Screening, Recru itments and Obtaining Consent ................................ ................... 54 Study Foods ................................ ................................ ................................ ............ 55

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6 Study Food Packaging ................................ ................................ ............................ 56 Clinic Visits ................................ ................................ ................................ ............. 56 Qualitative Questionnaires ................................ ................................ ...................... 56 Gastrointestinal Symptoms and Daily Diary ................................ ..................... 56 Quality of Life, Appetite and Risk of Weight Loss ................................ ............. 57 Anthropometric Measurements ................................ ................................ ............... 57 Food Intake Assessment ................................ ................................ ........................ 57 Nutrie nt Assessment ................................ ................................ ............................... 58 Blood Samples Collection ................................ ................................ ....................... 58 Assays ................................ ................................ ................................ .................... 58 Blood Urea Nitrogen ................................ ................................ ............................... 58 Serum Creatinine ................................ ................................ ................................ .... 59 Serum Trigly cerides (TG) ................................ ................................ ........................ 59 Total Serum Cholesterol ................................ ................................ ......................... 60 High Density Lipoprotein (HDL) ................................ ................................ .............. 60 Low Density Lipoprotein (LDL) ................................ ................................ ................ 60 Glucose ................................ ................................ ................................ ................... 61 eGFR Calculati on ................................ ................................ ................................ ... 61 General Statistical Approach ................................ ................................ .................. 61 Study 2: Overview ................................ ................................ ................................ ... 61 Study Design ................................ ................................ ................................ .......... 62 Screening and Re cruitments and Obtaining Consent ................................ ............. 62 Study Food ................................ ................................ ................................ ............. 63 Study Food Packaging ................................ ................................ ............................ 64 Demographic and Baseline Data Collection ................................ ............................ 64 Food Intake and Nutrient Assessment ................................ ................................ .... 65 Qualitative Questionnaires ................................ ................................ ...................... 65 Clinic Visits ................................ ................................ ................................ ............. 65 Quest Diagnostics ................................ ................................ ................................ ... 66 Assays ................................ ................................ ................................ .................... 66 BUN and Serum Creatini ne ................................ ................................ .................... 66 Ammonia ................................ ................................ ................................ ................. 66 CRP ................................ ................................ ................................ ........................ 66 Cystatin C ................................ ................................ ................................ ............... 67 GC MS Analysis of Total p Cresol in Plasma ................................ ......................... 67 eGFR Calculations ................................ ................................ ................................ .. 67 General Statistical Approach ................................ ................................ .................. 68 4 RESULTS STUDY 1 ................................ ................................ ............................... 73 Demographics Data ................................ ................................ ................................ 73 Weight ................................ ................................ ................................ ..................... 73 Nutrient and Fiber Intake ................................ ................................ ........................ 73 Compliance Data ................................ ................................ ................................ .... 73 Bowel Movement Frequency ................................ ................................ .................. 74 Lipid and Blood Glucose ................................ ................................ ......................... 74 Tota l Cholesterol, Dietary Fat and Fiber Correlations ................................ ............. 75

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7 BUN, Serum Creatinine and eGFR ................................ ................................ ......... 75 Qualitative Results ................................ ................................ ................................ .. 75 SNAQ ................................ ................................ ................................ ............... 75 Epworth Sleepiness Scale (ESS) ................................ ................................ ..... 75 GSRS ................................ ................................ ................................ ............... 76 Health Related Quality of Life ................................ ................................ ........... 76 5 RESULTS STUDY 2 ................................ ................................ ............................... 80 Demographic Characteristics ................................ ................................ .................. 80 Nutrient and Fiber Intake ................................ ................................ ........................ 80 Compliance ................................ ................................ ................................ ............. 80 Reported Sym p toms ................................ ................................ ............................... 80 Bowel Movement Frequency ................................ ................................ .................. 81 BUN, Ammonia, Serum Creatinine, Cystatin C, CRP and eGFR ............................ 81 p Cresol ................................ ................................ ................................ .................. 81 KDQOL ................................ ................................ ................................ .......... 82 6 DISCUSSION AND CONCLUSIONS ................................ ................................ ...... 87 Quality of Life and Symptoms ................................ ................................ ................. 87 Bowel Movement Frequency and Fiber Intake Impact and Recommendations ...... 89 Uremic Symptoms ................................ ................................ ................................ .. 91 Impact of Higher Fiber Intakes ................................ ................................ ................ 93 Cardiovascular Lipid Panel Markers ................................ ................................ ....... 94 Uremic Molecules ................................ ................................ ................................ ... 95 Plasma p Cresol ................................ ................................ ................................ ..... 97 Framework ................................ ................................ ................................ .............. 98 Conclusions ................................ ................................ ................................ .......... 101 APPENDIX A KIDNEY DISEASE QUALITY OF LIFE ................................ ................................ 103 B GASTROINTESTINAL RATING SCALE ................................ ............................... 112 C SIMPLIFIED NUTRITIONAL APPETITE QUESTIONNAIRE ................................ 114 D EPWORTH SLEEPINESS SCALE ................................ ................................ ....... 115 E TELEPHONE SCRIPT ................................ ................................ .......................... 116 F DAILY DIARY ................................ ................................ ................................ ....... 118 G FOOD RECORD ................................ ................................ ................................ ... 120 H FOOD JOURNAL WORKBOOK ................................ ................................ ........... 121

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8 I TELEPHONE SCRIPT ................................ ................................ .......................... 126 J PEA HU LL FIBER MUFFINS FORMULATIONS NUTRITION FACTS .................. 128 K FRUITAFIT HD PRODUCT SHEET ................................ ................................ ... 130 L BASELINE QUESTIONNAIRE ................................ ................................ .............. 131 M FOOD RECORD ................................ ................................ ................................ ... 133 N BOWEL FREQUE NCY QUESTIONNAIRE ................................ ........................... 135 LIST OF REFERENCES ................................ ................................ ............................. 136 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150

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9 LIST OF TABLES Table page 3 1 Nutrient composition of foods provided in the control and fiber intervention periods ................................ ................................ ................................ ................ 69 3 2 Nutrient composition of foods and supplements provided ................................ .. 70 4 1 Energy, fiber, and macronutrients intakes ................................ .......................... 77 4 2 Participants weight, clinical markers, quality of life and symptoms scores ......... 78 5 1 characteristics ................................ ................................ ................ 83 5 2 Macronutrients, energy and fiber intakes compared between each period ......... 83 5 3 ................................ ....... 84 5 4 Individual responses for uremic symptoms listed in the Symptom/Problem List subscale of the KDQOL ................................ ................................ ....... 85

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10 LIST OF FIGURES Figure page 3 1 Renal Study 1: screening and recruitment ................................ .......................... 70 3 2 Renal Study 1: study design ................................ ................................ ............... 71 3 3 Renal Study 2: study design ................................ ................................ ............... 71 3 4 Renal Study 2: screening and recruitment ................................ .......................... 72 4 1 Average bowel movement per day. ................................ ................................ .... 79 5 1 Average bowel movement frequencies. ................................ .............................. 86 5 2 Total p cresol comparisons before and after intervention. ................................ .. 86

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11 LIST OF ABBREVIATION S AI Adequate Intake AOAC Association of Official Analytical Chemists BUN Blood Urea Nitrogen CHD Coronary Heart Disease CKD Chronic Kidney Disease CKD EPI Chronic Kidney Disease Epidemiology Collaboration CRP C reactive protein CSFII Continuing Survey Food Intakes by Individuals CVD Cardiovascular Disease DRI Dietary Reference Intakes eGFR Estimated Glomerular Filtration Rate ESS Epworth Sleepine ss Scale FOS Fructo oligosaccharides GC MS Gas Chromatography Mass Spectrometry GFR Glomerular Filtration Rate GSRS Gastrointestinal Symptom Rating Scale HD Hemodialysis HDL High Density Lipoprotein IOM Institute of Medicine KD Q O I Kidney Disease Quality Outcome Initiative KDQOL Kidney Disease Quality of Life LDL Low Density Lipoprotein MDRD Modification of Diet in Renal Disease

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12 NHANES III Third National Health and Examination Survey NAD Nicotinamide Adenine Dinucleotide p Cresol Para cresol, 4 methylphenol SNAQ Simplified Nutritional Appetiite Questionnaire SCFA Short Chain Fatty Acids Tr TIMP 1 Tissue Inhibitor of Metalloproteinase 1 TG Triglycerides

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE EFFECTS OF ADDED FIBER TO THE DIETS OF CHRONIC KIDNEY DISEASE PATIENTS ON QUALITY OF LIFE, CLINICAL MAR KERS AND, GASTROINTESTINAL AND KIDNEY FUNCTION By Younis Ali Salmean August 2013 Chair: Wendy Dahl Major: Food Science and Human Nutrition Chronic kidney d isease (CKD) patients may consume lower than recommended amounts of dietary fiber due to typical d ietary habits, dietary restrictions, and uremic symptoms. Progressive decline in kidney function causes an accumulation of uremic molecules that contribute to fur ther progression of the disease and reduced quality of life. The objective was to conduct clin ical trials to determine the effects of added fiber in the diet of CKD patients on ure mic molecules, kidney function uremia, and quality of life. Two single blind, intervention clinical trials with patients with moderate to severe decline in kidney funct 2 ) were conducted. The firs t study was 6 weeks in duration; 2 weeks of control and 4 weeks intervention with 23 g/d of mixed fiber sources. The second study was 12 weeks in duration; 2 weeks control, followed by 4 weeks intervention with 10 g/d of pea hull fiber, followed by 6 weeks of an additional 13.5 g/d of fiber from inulin. Study 1 : Provision of 23 g/d of added fiber lowered serum creatinine from 2.44 0.30 mg/dL (mean SE) at baseline to 2.21 0.26 mg/dL after 4 week s of

PAGE 14

14 intervention (p<0.05). Blood urea nitrogen (BUN) decreased from 41.9 5.9 during control to 33.5 4.9 mg/dL after 4 weeks of fiber intervention (p<0.05). The decline in serum creatinine corresponded with an increase in eGFR (MDRD) from 29.6 3.5 mL/min/1. 73m 2 at baseline to 32.5 3.6 mL/min/1.73m 2 after 4 weeks of intervention ( p< 0.05). Functional Physical Health Related Quality of Lif e increased from 30 2 at baseline to 35 3 (p<0.05) post intervention Study 2 : Foods fortified with 10 g/d of pea hull fiber did not improve uremic profile or eGFR ( creatinine cystatin C ) Inulin supplement at 3 weeks improved eGFR (47.6 5 .6 mL/min/1.73 m 2 ) compared to control period (42 .4 5 mL/min/1.73 m 2 ), but not baseline ( 4 3.9 4. 8 mL/min/1.73 m 2 ). However, this improvement diminished after 6 weeks of inulin supplementation when supplement compliance dropped from 90% to 77%. P lasma p cresol decreased by 2 0 % from baseline at study end Supplementing the diet of CKD patients with insoluble and fermentable fibers may reduce uremic molecules and thus improve renal function and improve some aspects of quality of life.

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15 CHAPTER 1 INTRODUCTION Dietary fiber intake s in the US fall short of the reco mmendations. Moreover, chronic kidney d isease (CKD) patients may be more likely to consume lesser amounts of dietary fiber in the diet due to typical dietary habits, dietary restrictions, and symptoms. Reduced kidney function leads to accumulation of nitrogenous waste and uremic molecules that can alter normal physiological state and thus introduce uremic symptoms in this population. Uremic molecules originate from exogenous sources and/or are generated endogenously through various metabolic and bacterial processes. Uremic syndrome or uremia is a broad term to describe symptoms associated with the disease but cannot be explained by derangeme nts in extracellular volume, inorganic ion concentrations or known renal synthetic products (1) It commonly develops with the later stages of CKD and is believed to be due to accumulation of uremic molecules above physiological tolerance. U remic molecules such as urea and creatinine are excreted primarily via the urinary route. However kidney disease causes partial or near complete loss of the ability to excrete these uremic molecules Thus the loss of filtration capacity increases the uremic burden caused by the accumulation of these molecules in the blood. In a study where kidney patients were supplemented with mixed fiber (insoluble and fermentable) nitrogen excretion was shifted away from the urinary route toward the fecal route (2) The increase in fecal nitrogen and decrease in urinary nitrogen was coupled by a significant decline in blood urea nitrogen (BUN), suggesting an approach to reduce uremic molecules through fiber intervention and reducing the filtration burden on the remaining nephrons By lowering uremic molecules, potential improvements in

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16 symptoms and thus, quality of life can be speculated. A similar approach to lower another uremic molecule by using fib er was carried out in both healthy and hemodialysis (HD) patients. In a study of healthy volunteers, decreased, urinary p cresol excretion was observed by ingestion of a 50/50 v/v mixture of inulin and fructooligosaccharides (FOS) (3) A 4 week trial in H D patients using a total of 20 g/d of the inulin and FOS confirmed that p cresol generation and p cresol sulfate concentrations were lowered in these patients (4) Dietary therapy that may provide means to reduce urea, phenols and potentially other molecules may be effective to reduce overall uremic load which may impact GFR by reducing the need for the remaining nephrons to hyperfiltrate a process in which nephrons adapt higher filtration to compensate for the loss of nephrons mass Several uremic molecules are toxic and can induce oxidative stress and inflammatory response in various parts of the nephron Uremic toxic molecules propagates disease progression by inducing fibrosis related g enes and oxidative stress causing tubulointerstitial fibrosis and glomerular sclerosis (4 13) Dietary intervention that can reduce the generation, uptake and improve excretion of these molecules may provide additional protective benefits to preserve the remaining nephrons and slow their loss. In addition, lowering of uremic molecules may contribute to improved health and wellbeing by reducing uremic symptoms that are typically cau sed by increased uremic molecules concentration in the blood due to declining clearance capacity. Supplementing the diet of CKD patients with mixed sources of fiber is potentially a safe and practical way to increase fiber content in the diet while providi ng the various benefits attributed to the various types of fibers An increase of insoluble fiber may

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17 contribute to improvement in bulking and bowel frequency, while increasing intake of soluble fermentable fibers may enhance utilization of carbohydrates as a source of energy while depressing protein putrefaction in the large gut. Enhancing the role of the large intestine to capture and eliminate uremic molecules may pro vide a viable approach to reducing uremic load, which may impact kidney health and func tion. Ultimately such impacts on uremic molecules genera tion and elimination may result in improved uremic symptoms and quality of life. Increasing fiber intake is associated with many health benefits and is shown to have many protective effects. There ar e a limited number of studies investigating the effect of fiber on uremic molecules and eGFR. These studies focused on late stage CKD and dialysis patients. Omission of stage 3 patients may have hindered the potential effect that fiber may play in altering uremic load, serum creatinine, and health related quality of life. It is likely that some benefits maybe more measurable at more advanced stage of the disease, but it also likely that other factors are less affected with such decline in kidney function, m aking any benefits from dietary therapy more subtle. Additionally, studies with fiber as nutritional therapy to impact uremic molecules in late stage CKD and HD patien ts have not investigated health related quality of life and uremic symptoms. The potenti al of a nutritional intervention to restore fiber in the diet of renal patients to maintain healthy gut function and help decrease uremic load has great merits to the area of applied nutrition and dietetics. A study to determine the effect of early diet ary intervention in the course of the disease and the impact on various parameters including symptoms and quality of life is essential to the area of medical nutrition t herapy.

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18 The major aim of this research was to determine the impact of increased fiber intake on uremic molecules, kidney function, quality of life and symptoms in CKD patients who have experienced over 50 % loss of their kidney function. While previous studies focused on maximizing fiber intervention (40 50 g/d), the aim was to use practical and achievable fiber intake level s. In addition, the focus was to explore the potential effects in patients with moderate to severe decline in kidney func tion compared with previous tria ls. The first study was to investigate the effects of 23 g/d of fiber from pea hull, resistant corn dextrin and inulin on BUN, serum creatinine, and eGFR; uremic symptoms ; and quality of life. The second study was carried out to further assess the impact of pea hull fiber alone and in combin ation with supplemental inulin /FOS on quality of life, bowel movement frequency, and uremic molecules.

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19 CHAPTER 2 REVIEW OF THE LITERATURE Chronic diseases are worldwide problems and are leading causes of death in developed countries. In the United States, chronic diseases account for 70% of all deaths (14) There are several contributing factors to chronic diseases includin g smoking, sedentary life style and diet. Early interventions are critical to reduce risk, severity and mortality of chronic diseases. For example, interventions that focus on smoking cessation result in reduced risk of cardiovascular disease (CVD) and certain cancers, while increasing physical activity may help reduce the risk of many diseases such CVD and diabetes (15) Nutritional interventions may also help with reducing risk of various diseases and improve management. It is established that lowering trans fat intake and cholesterol may help reduce the risk of CVD while fiber has been proposed to be protective against chronic diseases such as C VD (16) Lack of fiber in the diet has been implicated in many of the chronic diseases seen today in modern societies lending the assumption that fiber, although not currently defi ned as a nutrient, is of importance comparable to nutrients (17) Chronic diseases contribute significantly to the financial burdens experienced both by individuals and economies as a whole. In the past, nutrition research was mainly focused on prevention of nutritional de ficiencies and malnourishment. In the early 1900s, researchers began to understand the link between nutrient s deficiencies and illnesses. In the first half of the 20th century, the focus was to eradicate nutritional deficiencies in the United States. Iodine fortification started in the 1922, vitamin D fortification in 1932, iron, niacin, riboflavin, and thiamin were added to foods in 1941 and the RDA was developed in (18)

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20 Later, and once most deficiencies became less prevalent and the health status of the population improved; the focus shifted toward promotion of optimal health. Once the link between nutrients, intakes, and chronic disease risk was elucidat ed, dietary recomme ndations were set to address these links from nutritional stand point Die ts that emphasize better health and reduced risk of chronic diseases are promoted for specific populations. Nutritional and governmental agencies aim to reduce fin ancial burdens associated with such diseases and improve the quality of life for people by publishing and promoting these dietary recommendations. One particular disease of high cost is CKD with high direct burden costs, and indirect burden costs related t o its co morbidities. Diabetes and hypertension are the leading causes of CKD (19, 20) Chronic kidney disease patients are at very high risk for developing CVD (21) The stage and severity of kidney disease are determined by kidney function and/or damage of the kidneys. Patients with lower kidney function have a higher stage disease and health care costs associated with each stage increases significantly ranging anywhere from as low as $16,000 per year for total costs in stage 2 to as high as $44,500 per year in stage 4 (22) One of the most cost ly co morbidities of diabetes and hypertension, in terms of health care expenditures and patient quality of life, is that of CKD. Health care costs are significantly higher in CKD patients that have other co morbidities compared with those only diagnosed w ith CKD (22) The decline in kidney function is typically slow and take years before approaches kidney failure. Any potential therapy to reduce or slow the decline in kidney functi on, manage co mor bidities, alleviate symptoms will contribute significantly to reduced total health care costs and improve d quality of life.

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21 It is estimated that 26 million adults are affected by a decline in kidney function in the United States (23) Th e Kidney Disease Quality Outcome Initiative (KD Q O I) guidelines define CKD as either kidney damage or a decreased kidney glomerular filtration rate (GFR) of less than 60 mL/min/1.73 m 2 for three or more months irrespective of etiology (24) maintain electrolyte balance. When kidney function declines, molecules that are usually filtered efficiently accumulate in the blood and tissues. Molecules that accumulate and to as uremic retention solutes, uremic toxins, or uremic molecules (25) It is suggested that the progressive accumulation of these uremic molecules or toxins causes a decline in health and increase uremic symptoms severity (25) Uremia is a broad term to descri be symptoms experienced by renal patients that concentrations, or lack of known renal synthetic products (1) It commonly develops with CKD in the later stages of the disease. Accumulation of organic waste products or uremic molecules have been identified as contributors of uremia A n estimated 90 retention uremic molecules identified so far that may explain uremia and contribute to the uremic load (25) Uremic molecules can originate from endogenous sources such as amino acids and protein catabolism, can be ingested, or generated from proteins bacterial fermentation (26) Uremic molecules have various properties that contribute to their diverse physiological effect in the body including water solubility, dimensions, charge distribution, concentration, molecular mass, and prote in binding (26) Declining renal

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22 function and thus clearance capacity can result in elevated concentrations of such molecules in bloo d and tissues, some of which can contribute to uremic symptoms and derangement in health and wellbeing. Uremic toxic molecules may accelerate glomeruli and tubular structural damage and stimulate disease progression by stimulating renal expression of fibro sis related genes such as tr (8) induction of renal oxidative stress and free radical s production (13, 27) (5) and inhibition of cellular proliferation (28) Creatinine is a byproduct of the breakdown of creatine phosphate and is not viewed as a harmful molecule, but its elevation indicates an increase in muscle breakdown or decline in renal func tion. Ammonia is a very toxic molecule and blood levels need to st ay very low. Ammonia forms by removal of an amino group from amino acids by deamination, via glutamic acid deamination by glutamate dehydrogenase, or from ammonia generation by bacteria in the large intestine. As it forms, the body keeps ammonia levels fro m elevation by converting it to urea, which is relatively non toxic and biologically inactive. Urea is the primary end product of nitrogen metabolism that is produced via the urea cycle and is the largest pool of uremic nitrogenous molecules in the blood. Urea is normally excreted to maintain a narrow range but can become very elevated with declining kidney function. A bout 70% of the urea produced in healthy adults is eliminated in the urine while 30% is hydrolyzed and a portion of that is retained in the body (29) This occurs because urea in circulation can diffuse through the colonic wall d ue to its small molecular mass of 60 Da. It is suggested that hydrolyzed urea in the large intestine ranges from 20 25% to as high as 40% of total urea production (30) In patients with reduced kidney function, urea accumulates in the blood contributing to the rise in pool of uremic molecules.

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23 Uremic molecules and toxins become elevated in the blood with inadequate renal clearance and are associated with uremia (31 33) These uremic molecules can affect biochemical, biological and physiological functions which may explain the broad uremic symptoms profile. p Cresol for example is a microbiota product that diminishes oxygen uptake by brain cells, disrupts endothelial progenitor cell function is related to growth retardation in the weanling pig, and causes alteration of cell membrane permeability (34, 35) Symptoms of uremia i nclude fatigue, intense itching, anorexia, cognitive dysfunction and constipation (1) As blood and excrete urine decline, nitrogenous compounds become elevated. It is possible that by inducing bacterial demand for nitrogen in the large intestine by providing the preferred substrate to support bacterial proliferation, and subsequently depress the return of nitrogen to circulation that the uremic load can be mitigated. In addition, interfering with bacteri al generation of uremic molecules such as p cresol would also provide a viable approach to reduce the uremic load. Eventually, by targeting certain uremic molecules and reducing their retention and/or generation, the uremic load can be reduced assuming no increase in other uremic molecules. Such reduction may lead to improved renal hemodynamics and excretion due to reduced concentration of inflammatory molecules and/or by reductions in volume of molecules to be filt ered through the renal tubules. It is ther efore very valuable to have a nutritional inter vention that target s the colon and enhance s its role in reducing uremic mo lecules generation and uptake. Reduction of such molecules may provide valuable treatment for uremia and the burden associated with suc h symptoms. Fiber may provide such potential therapy due to its fermenta tion

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24 Fermentable fibers stimulate bacterial proliferation by increasing carbohydrate substrate availability, thus leading to increase nitrogen demand for bacterial protein synth esis Saccharolytic fermentation, as opposed to proteolytic, results in an increase in the production of short chain fatty acids (SCFA), which is thought to provide benefits to the host. Butyrate for example, is shown to be a potent trophic factor that stimulat es cell wall hypertrophy and maintenance (36, 37) Induction of fermen tation utilizing fermentable carbohydrate sources in animal models show significant hypertrophy of the cecal cell wall, which increases blood flow into the cecum (38 40) Increasing blood flow into the cecum, along with increased nitrogen demand, will lead to an enhanced entry and re tention of nitrogenous molecules such as urea and ammonia in the colon (39 41) Animal studies show that addition of ferment able fiber to the diet of animals lead s to nitrogen shift from urinary to fecal excretion (41 46) T he addition of inulin to the rat diet causes cecum enlargement with declining urinary nitrogen and increased urea flow into the cec um with significant increase in acid production (40, 41, 47) Increase d acidity creates a unfavorable environment for a number of pathogenic bacteria l species (48) which typically contribute to the uremic load by generating uremic molecules such as amines and phenols. Although scarce, reports from human stu dies involving renal patients are also suggestive of a more interesting role of the colon in removing, capturing and /or reducing uremic molecules appearance in the blood (2, 7, 49 51) In a study where CKD pa tients were supplemented with soluble fibers (30 g/d arabinogalactan or 7 g/d ispaghula) for 6 8 weeks, BUN decreased by 11% 19% (50) Fifty gram per day of gum a rabic was shown to increase fecal nitrogen excretion by 41% while decreasing BUN levels in 16

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25 CKD patients (51) Younes et al. demonstrated tha t five weeks of feeding 40 g/d of mixed fiber sources (4.5 g inulin, 10.5 g resistant starch, and 2 5 g of fiber from wholemeal bread ) resulted in a 51% increase in fecal N excretion and 12% reduction in urinary nitrogen excretion in 9 uremic CKD patients a long with a significant reduction in BUN levels ( 23%) (2) There was a 1.7 fold increase in stool content of nitrogen with bacterial fraction of feces accounting for (59%) of this increase suggesting with the observed decrease in BUN that the colonic bacteria are largely res ponsible for the decrease in serum urea Fermentable substrates can alter the luminal pH, and selectively enhance the growth of bacterial species which can alter uremic molecules generation. Several uremic molecules are primarily generated by gut bacteri a and contribute to the uremic load. p Cresol is generated by tyrosine fermenting bacteria and can be elevated in uremic patients (52) Similar to BUN, lowering of p cresol, can be achieved via fiber supplementation and may contribute to lowering the uremic load. In a 4 week study, inulin and FOS supplementation resulted in lower concentrations of p c resol and its conjugated form p cresol sulfate in HD patients (7) However, lowering uremic and toxic molecules in CKD may be dependent on several factors in cluding fiber dose and type stage of kidney disease colonic bacterial make up, fiber intake, and transit time. Parillo et al investigated th e metabolic effect of feeding high and low fiber diets to CKD patients (49) One group received a high fiber diet (65 g/d) (legumes, vegetables and fruit) with moderate protein intake (69 g/d), and the other group received a low fiber (22 g) diet with l ow er protein content (50 g/d or 9%). The high fiber diet induced a significant improvement in blood glucose control and a significant decrease in serum

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26 cholesterol. While some beneficial metabolic outcomes were observed, no changes in serum creatinin e, BUN or eGFR were observed. There was however a significant increase in fecal nitrogen losses with no changes in urinary nitrogen excretion. The study by Younes et al showed a significant impact of fiber on BUN but not serum creatinine with an increase in fecal nitrogen loss and a parallel decline in urinary nitrogen loss (2) unlike the observation of Parillo et al. These results suggest a potential impact of the use of fiber as a therapeutic nut ritional component to reduce various uremic molecules and impact various markers depending on the fiber source and dosage. This potential positive ef fect may impact kidney function, uremic symptoms and subsequently health related quality of life. Because fiber intake is likely to be low in this population, restoration of adequate fiber intake may also provide additional health benefits such as improved lipid profile, improved glycemic control, and improved bowel habits. Dietary fibers such as wheat and o at brans, coarse and wheat flours have been used for centuries as a laxative and bulking agents. In the past few decades, the role fiber plays in health maintenance and disease prevention have been expanded to include risk reduction of CVD, stroke, hyperte nsion, diabetes, and certain gastrointestinal disorders, along with improvements in lipid profile (53) Moreover the effect of fiber on gut health was expanded to include symbiosis by establishing the importance of certain fibers as prebiotic substances. A p rebiotic is n on digestible food ingredient that beneficially affect the host by selectively stimulating the growth and/or activity of one of more limited number of bacteria in the colon and thus improve host (54) M ore recently a prebiotic has been

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27 gastrointestinal microbiota that confers benef its upon host well being and h ealth (55) Examp les of prebiotic fibers include: r esistant s tarch (56 58) i nulin, lactulose, l actosucrose xylo oligosaccharides, soy bean oligosaccharides, g lacto oligosaccharides among others (59) Several experimental studies and some human trials have shown potential benefits of prebiotics in inflammatory bowel disease, mineral bioavailability and constipation. In addit ion, human studies have shown that prebiotic fibers reduce some serum uremic molecules and fecal nitrogenous byproducts (2, 7, 40, 41, 60) It is therefore important to determine the potential beneficial role that fiber may play in the diet of CKD patients, particularly those with uremia, beyond that usually as cribed to healthy individuals. This is particularly important when considering that over half of CKD patients are diagnosed as stage 3 CKD patients and thus any beneficial therapy can have significant impact Fiber intakes of CKD populations have not been documented. Current estimation of fiber intake in the general population is about half of the adequate intake (AI) recommenda tion of 31 38 g/d for men and 21 25 g/d for women (16) Although there are predict that individuals with advanced CKD may consume less fiber than the general public due to diet restrictions and uremic symptoms such as anorex ia (61) Restricted sodium, restricted phosphorus and high calcium intake are required for the management of late stages of CKD, sinc e with the loss of kidney function, electrolyte, mineral and fluid balance are disrupted. High phosphorus foods may be

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28 restricted. These include primarily dairy products, beans, bran cereals and whole grains. Limitations on potassium intake for some patien ts necessitate the exclusion of beans and milk, as well as nuts and many fruits and vegetables. The restriction of beans, whole grains and many fruits and vegetables may leave a diet seriously depleted of dietary fiber, and may lead to constipation and ina dequate fermentable substrate for colonic and general health. Although it is recommended that CKD patients consume diets high in fiber, no kidney disease diet recommendations provide guidelines as to how to achieve this recommendation. There is evidence s uggesting reduced risk of CVD, improved management of diabetes and constipation with higher fiber intakes, but no specific benefits have been established between increased fiber intake and the health of CKD patients. Emerging evidence suggest that fiber ma y provide benefits for CKD patients beyond that of reducing CVD risk, specifically reducing uremic molecules which are implicated in uremia. However, there is very limited research on the benefits fiber may play in the treatment of uremia in kidney disease population and the effect on quality of life. Fiber Definition In the United States, fiber is typically categorized as soluble and insoluble. The categorization is derived based on the Association of Official Analytical Chemists (AOAC) method, typically t he AOAC 985.29 (16) The Institute of Medicine (IOM) suggests that the solubility based definitions are limiting and should be replaced with dietary fiber, functional fiber, and tot al f iber when defining types of fiber (16) The IOM defines dietary f digestible carbohydrates and lignin that are intrinsic functional f digestible carbohydrates that have beneficial physiol ogical eff

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29 fiber and functional f iber (16) Dietary fibers are of plant origin and are composed of non digestible polysaccharides and lignin while their matrix is largely intact. These fibers maintain plant cells three dimensional interrelationships that are typically responsible for some of the p hysicochemical properties attributed to dietary fiber (16) Added fibers may be functional fibers. As defined by the IOM, they are functional fibers if they are non digestible carbo hydrates that are isolated and extracted and are shown to have beneficial physiological effects in human (16) They can be made available by modification of plant sources, or can be synthetically made by enzymatic reactions such as oligosaccharides and resistant starches (16) Whether dietary or functional, fiber must be a carbohydrate that is non digestible b y the human intestinal enzymes. The different properties of fibers such as solubility, viscosity, bulking and fermentability among others determine the way fiber behaves in the small and large intestines, and the physiological role it may play during its p assage throughout the digestive tract. Fiber according to the IOM classification must be 1) part of an edible plant cell; 2) carbohydrates (both oligosaccharides and polysaccharides); 3) resistance to hydrolysis by human digestive enzymes; 4) resistance to abs orption in the small intestine (62) While dieta ry fiber is the intact part of the plant, functional fibers are added to products after being isolated, extracted, or synthesized. In order to bare the definition functional, however, they must be non digestible carbohydrates that have beneficial physiolo gical effects in human (16) Pea Hull Fiber Plant seeds are an importan t agricultural product that are consumed as a whole, or used as food ingredients. Generally, the outer layer of a seed is composed of insoluble polysaccharides, pectin, and lignin while the inner polysaccharides

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30 components varies in their solubility and are mainly composed of non starch polysaccharides. Peas belong to the Leguminosae family. The main tissues in a pea seed are the seed coat (hull) and the cotyledons (inner plant storage tissue). The two tissues are distinctively different. The outer layer is relatively higher in cellulose hemicellulose and lignin. The cellulosic content of the hull fibers is comparable to fibers isolated from cereal bran (63) Pea hull fiber is light in color, has a smooth mouth feel and yields products neutral in flavor and odor with moderate water holding capacity characteristics. It is composed of 89% dietary fiber, 82% of which is insoluble with the primary insoluble fiber being hemicellulose and only 7% soluble fiber (63, 64) Inulin and FOS Fiber Fruct ans are predominantly linear chains of fructose units 1) linkage. Fructans can be found in a variety of plants such as garlic, asparagus, wheat, rye, onions, chicory roots, and Jerusalem artichoke with later ones having the highest content Inulin is a generic term that covers all 1) linear fructans with mostly one terminal glucose unit, and with a degree of polymerization (DP) ranging from 2 60 (average of 10 12) (16, 59, 65) Inulin is typically produced commercially by extraction from chicory roots ( Cicgirium intybus) using hot water extraction process; the extracted native inulin is then purified into a white powder. Partial enzymatic hydrolysis of chicory inulin using endo inulinase yields oligofructoses (66) In addition, FOS fructans are produced from sucrose using enzymatic transfer of fructosyl groups. Oligofructose have a DP ranging from 2 8 while FOS have DP ranging from 2 4 (16) Unlike inulin, oligofructose and FOS may or may n ot have a terminal glucose unit.

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31 Resistant Corn D e xtrin Fiber Dextrins are hydrolysates obtained from the breakdown of starch by hydrolysis. The treatment of these low molecular weight s imple carbohydrates with heat followed by enzymatic treatment results in resistant malto dextrins. On average, resistant maltodextrins have a molecular weight of about 2 kDa consisting of glucose polymers 6) glucosidic bonds and 1 2 and 1 3 linkages (16) Specifically, treatment of c ornstarch by heat/acid and amylase treatment produce resistant corn dextrin. Fiber Recommendations and Current Intakes in General Population In the United States, fiber recommendations are established as Adequate Intakes (AI). The recommendations deri ved as AI because protec tive evidence against c oronary heart d isease (CHD) is found across continuous ranges of intakes and therefore a n Estimated Average Requirement (EAR) was not possible to establish (16) The current AI recommendations for adults younger than 50 years of age are 25 g/d for women and 38 g/d for men, while for those 50 and older will need to consume 21 g/d and 3 1 g/d for women and men respectively, or 14 g per 1000 kcal (16) Current fiber intakes in the United States fall s hort of these recommendations. From the 1994 1996, 1998 Continuing Survey Food Intakes by Individuals (CSFII), median fi ber intakes ranged from 16.5 to 17.9 g/d and 12.1 to 13.8 g/d for men and women respectively suggesting Americans consume about half the recommendations (67) Dietary Reference Intakes (DRIs) for Fiber and Current Intakes in CKD Population Current dietary fiber recommendation s for kidney disease population are not different from that for healthy individuals. There are no specific recommendation s for consuming high fiber intakes in this population. The recommendations are similar to

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32 those for the general population in which consuming high fiber is protective against heart disease. Fiber intake however falls dramatically short of the amount recommended for good health The m ajority of Americans consume an average fiber intake that is half of the recommendations (68) Fiber i ntake in populations with CKD has not been established but is expected to be lower than the current intakes in the healthy population. This is because CKD patients, especially those with advanced stages of the disease, may need to restrict foo ds that are typically high in fiber such as whole grains, legumes and certain fruits and vegetables due to their content of phosphorous and /or potassium. These minerals are usually restricted in later stages of CKD Uremic symptoms also may lead to loss of appetite and anorexia which results in lower food intakes and subsequently lower fiber intakes. Chronic Kidney Disease Prevalence Overview The Kidney Diseas e Quality Outcome Initiative defines CKD as either kidney damage or a decreased kidney GFR of less than 60 mL/min/1.73 m 2 for 3 or more months irrespective of etiology (24) Fourteen percent of U.S. women ages 20 and older have CKD compared to 11% of males (69) Fourteen percent of w hite have CKD compared to 12% African Americans and 8% Mexican Americans (69) Whites make up 72.6% o f CKD population wh ile African Americans represent 10.5% (70) The leading risk factors for kidney diseas e are hypertension (19, 20) and diabetes (71) About 7% of the CKD population has diabetes and 27% are hypertensive (70) In 2006, 7 out of 10 people who had developed renal failure had diabetes or hypertension (69) D iabetes is the leading cause of kidney failure (69) It is estimated that the prevalence of CKD in the United States as of 2008 was 13.1% (72) Prevalence of CKD by stages according to

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33 the 2004 Third National Health and Examination Survey (NHANES III), using e GFR as a bench marker, is as follows: stage 1 (1.8%), stage 2 (3.2%), stage 3 (7.7%), stage 4 (0.35%) (70) and 570,000 patients on transplant functioning kidney or dialysis as of 2 011 (72) Health care costs associated with each stage increases significantly and can range anywhere from as low as $16,00 0 for total costs in stage 2 to as high as $44,500 per year in stage 4 (22) One of the most costly co morbidities of diabetes and hypertension, in terms of health care expenditure s and patient quality of life, is that of CKD. Health care costs are significantly higher in CKD patients that have other co morbidities compared with those only diagnosed with CKD (2 2) Stages of Chronic Kidney Disease The Kidney Disease Quality Outcome Initiative guidelines define CKD as persisting structural or functional abnormalities for over three months as determined by kidney damage or GFR of less than 60 mL/min/1.73m 2 Dam age can be structural as determined by imaging tests or functional as determined by abnormal blood, or urine tests (e.g. proteinuria). The calculated GFR is typically based on the Modification of Diet in Renal Disease (MDRD) study equation, which uses seru m creatinine, age, gender and race (African American o r other). Based on the above KD Q OI criteria, CKD is 2 with evidence of kidney damage; stage 2 (Mild): GFR of 60 to 89 mL/min/1.73m 2 with evidence of kidney damage; stage 3 (Moderate): GFR of 30 to 59 mL/min/1.73m 2 ; stage 4 (Severe): GFR of 15 to 29 mL/min/1.73m 2 ; and stage 5 (Kidney failure): GFR <15 mL/min/1.73m 2 Without any structural or functional abnormalities, CKD is only diagnosed as such when GFR is <60 mL/min/1.73m 2 The threshold of GFR is established because it is less than half the adult level of GFR, and lower levels are associated with increasing complications of

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34 CKD and adverse outcomes such as cardiovascular disease morbidity and mortal ity. In addition this level can be detected with current estimating equations for GFR based on serum creatinine, and is substantially above level associated with kidney failure for prevention therapy to take place in a timely manner (24) The Kidneys Kidneys are impo rtant organs that provide homeostatic roles in addition to being the primary site of filtration in the body. They are the essential components of the urinary system and homeostatic r egulation of electrolytes, acid base balance, and regulation of blood pres sure. The primary function of the kidney is the removal of cellular metabolites toxins and waste products. Removal of uremic molecule s is via urine production in the kidneys, which is produced by the process of filtration, reabsorption, and tubular secret ion. The basic functioning urine producing unit of the kidney is the nephron. There are about two million nephrons in both kidneys. The renal artery branches to form several other smaller arteries that eventually give rise to the glomerular capillaries whe re the renal consists of renal corpuscle and a renal tubule. The renal corpuscle consists of l tubule is divided into several segments, the proximal tubule (convoluted and proximal straight tubule), the loop of Henle (proximal straight tubule, thin limb, and thick ascending limb), and the distal convoluted tubule. The connecting tubule will conver ge the distal convoluted tubule with the next segment at the cortical collecting duct where several [T] he entire renal tubule and collecting duct system consists of a single layer of epithelial cells surrounding fluid in the tubule or duct lumen (73)

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35 Due to pressure gradient s that exist s between blood in the capillaries and fluid in from the glomerulus collected stream from the tubule lumen where the composition and volume are altered by tubular activity (73) Tubular reabs orption (active or passive) allows substances to be transported out of tubular filtrate (out of urine), where they are returned to the peri t ubular capillaries surrounding the tubules. These reabsorbed substances include: ions, water, glucose, amino acids, and uremic molecules such as urea, creatinine and uric acid. Tubular secretions (active or passive) allow the disposal of unfiltered substances, passively reabsorbed substances such as urea and uric acid, and excessive potassium ions, in addition to mainte nance of blood pH. Unlike glomerular filtration, which is non selective, tubular transport is selective. In the glomerulus, all molecules are filtered via the same and only mechanism, while in the tubules various mechanisms are involved in transportatio n of the molecules and substances. The transport process can involve absorption, secretion or both, and it can be passive or active depending on the partic ular substance and conditions. In the proximal tubule, organic anions (mainly protonated carboxylic an d sulfonic acids) are actively secreted in the proximal tubule and become saturated at high plasma concentrations, which leads to organic anions secretion competition (73) Organic cations (mainly amine and ammonium compounds) are also secreted via active transport mechanism. The broad substrate specificity of these anions and cations

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36 transporters allow for secretion of a variety of chemically diverse compounds. Org anic acids can also be passively transported by accepting/releasing hydrogen ions. Ammonia is a lipid soluble base and its excretion to form ammonium is favored to increase acid losses, as unbound hydrog en ions are not well excreted This occurs when blood pH decrease s, which signal s the tubules to increase secretion of hydrogen ions along with increasing the retention of bicarbonate and potassium ions to allow the urine to drain more acids. If blood pH rises, more chloride anions are reabsorbed and bi carbonate is eliminated in the urine while ammonium is reabsorbed to recycle the hydrogen ions to further furnish the bicarbonate formation Post proximal convoluted tubule, urea travels through the thick ascending limb, distal convoluted tubule, connecti ng tubule, cortical collecting duct, and outer medullary collecting duct with very limited absorption action due to impermeability of these segments to urea (73) Because a large volume of water through these segments has been reabsorbed, urea concentration rises in the inner medulla segment of the nephron. Concentrated urea is then transported via urea transporter into the interstitial fluid of the inner medulla (73) Urea can also re enter the loop of Henle to be recycled into the inner medulla or can be added to the inner medulla by diffusion from urine (73) Secretion and absorption mechanisms can vary in various parts of the nephron and collecting duct segments. F iltered b lood travels out the renal vein while urine collects i n the collecting tubule. Approximately, 1200 mL of blood flows through the kidneys e ach minute generating 180 L of filtrate a day and a pproximately 2 L of urine.

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37 Filtration Glomerular filtration rate is defined as the volume of plasma that can be complete ly cleared of a particular substance by the kidneys in a unit of time (74) The kidneys produce approximately 180 L of ultrafiltrate per day or 125 mL/min. The adult normal GFR range is 120 130 mL/min/1.73m 2 It is similar in men and women after adjusting for body surface are a. The functional state of the kidneys is routinely evaluated by estimating GFR. It is done indirectly by measuring the flow rate of filtered fluid from glomerular capillaries into ellent index of functioning renal mass or filtering capacity (75) Measuring GFR can only be done physiologically inert, freely filtered at the glomerulus, and neither, secreted, reabsorbed, synthesized, nor metabolized by the kidney, the amount of that substance filtered at the glomerulus is equal to the amount excreted in the urine (21) The f ructose polysaccharide, inulin, has been considered the ideal substance to estimate GFR by injecting the substance into the blood and collecting it in the urine using timed urine collections. Inulin meets all the above requirements as a substance and thus its clearance rate equals GFR. However, the inherent problem of intravenous infusion and timed collections make this method very impractical, costly and cumbersome, especially in a clinical setting and for screening purposes Because inulin use to estimate GFR is impractical, serum creatinine is widely used to estimate GFR. Both urinary creatinine clearance and plasma cre atinine can be used to estimate GFR. Once measured, estimation equations such as the MDRD or the Chronic Kidney Disease Epidemiol ogy Collaboration (CKD EPI) equation are used to calculate GFR.

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38 Briefly, the MDRD equation is a 4 variable equation (serum creatinine, age, gender and race) developed in 1999 using a large data set from over 1 600 CKD patients with GFR ranging from 5 90 mL /min/1.73 m 2 It was re expressed in 2005 using standardized serum creatinine assay. In 2009, the CKD EPI equation was developed to estimate GFR using serum creatinine, age, sex and race (76) It is more accurate than MDRD in predicting GFR in patients with function greater than 60 mL/min/1.73 m 2 but is not different when estimating lower functions (77) Althoug h not widely used, cystatin C also can be used to estimate GFR using CKD EPI cystatin C based equation (78) Equations with a single marker however underestimate the measured GFR and lack precision. Two recent meta analysis studies suggest that using both serum creatinine and cystatin C with age, sex, and race would be bet ter than equations that use only one of these serum markers (79, 80) and that the combined equation is more precise to estimate higher GFRs (76) Recently, Inker et al confirmed that the combined creatinine and cystatin C equation performs better than equations with a single marker (78) Serum C reatinine Creatinine is small organ ic molecule (113 Da) that is not bound to plasma proteins. Creatinine is the end product generated in muscle from conversion of creatine and creatine phosphate. About 98% of creatine pool is in muscle and of that amount, about 1.6% 1.7% is converted to c reatinine per day (81) Creatinine breakdown rate is fairly stable and depends on mu sc le mass. Serum creatinine is used as an index of kidney function. Under conditions of normal renal function, 90% 95% of creatinine is filtered by glomerular filtration with 5% 10% tubular secretions and reabsorption with the latter being less of a factor (82) Serum creatinine is the acceptable and preferable method

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39 used in clinical settings to index kidney function to date with the use of estimating equations. However, several factors can affect serum creatinine independen t of actual 1.53 mg/dL (83) depending on gender, age and race. Dietary changes su ch as an increased protein intake can induce a transi en t rise in serum creatinine levels and urinary excretion as tubular secretions are induced (81) Similarly, urea generation is induced with such dietary changes. Thus, a decrease in serum creatinine that is not related to improve d kidney function has clinical implications for the interpr etation of serum creatinine that will lead to false positive results. In healthy individuals, almost all creatinine elimination happens via the urinary route. However, with severe renal function impairment such as in CKD stage 5, limited evidence suggests that elevated creatinine pool may induce higher losses from external rout e s (84, 85) Degradation of creatinine by bacterial creatininase in the colon is thought to be responsible for the removal of creatinine (86, 87) Burnett and Jones suggested that creatinine, similar to urea and uric acid can enter the gut from the blood when elevated in the blood where it can be recycled through the gut or utilized by gut flora (84) Rats fed creatinine had higher colonic creatininase activities compared to controls (87) and earlier w ork with rats showed that creatinine fed to rats was recovered in the feces (88) A more recent study suggested that retained creatinine induces creatininase activity throughout the bowel and causes creatinine degradation by gut flora (86) In the study creatinine degradation was determined by meas uring creatininase activity in stool isolates in advanced renal patients (stage 5) compared to normal. In patients with serum creatinine above 6 mg/dL, higher stool creatinine

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40 degradation was seen compared to controls and to patients with creatinine levels lower than 6 mg/dL who did not use antibiotics for a period of three months or more (86) Very e arl y work by Goldman however did not show creatinine in the feces (89) While the evidence is inconc lusive at best, there is no evidence that in CKD patients with moderately elevated creatinine levels creatininase activities will be induced. Furthermore, studies supplementing CKD patients with fiber have not shown any significant reduction in serum crea tinine suggesting that it is unlikely that significant quantities of serum creatinine can enter the colon and become substrate for microbiota. Serum C ystatin C Cystatin C is a non glycosylated protein 120 residue polypeptide chain (90) that belongs to the cystatin super family of cysteine protease inhibitors and damage to cysteine proteases is regulated by these proteins (91) N ucleated cells produce cystatin C at a constant rate where it is concentrated in the fluid rich environments of the body such as seminal fluid, cerebrospinal fluid and synovial fluid (91) Elimination of cystatin C is through the glomerular filtration wi th no extra renal elimination. The n ormal ref e rence range is 0.5 1.0 mg/L (92) Filt e red cystatin C is comp letely reabsorb ed by the tubule cells and rapidly degraded (93) thus it does not re enter circulation nor does it appears in the urine. Cystatin C has a low molecular mass of 13 kDa with positive charge at physiological pH leading to free filtration by the glomerulus (91) Its correlation with GFR is not influe nced by gender, muscle mass, age, or protein intake (93, 94) Therefore, the use of cystatin C as an additional indexing molecule is becoming more re levant.

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41 Uremic Load, Progressive Hyperfiltration Uremic Toxins and Disease Progression Several factors can contribute to the progression of CKD although the exact reasons are not known. Because the rate of glomerular filtra tion is dependent on the number of nephrons and the single nephron GFR, when nephrons decline in number, the load on the remaining nephrons will be higher. Filtration is measured as the flow of filtrate from the glomerular capillaries (glomerulus) out into th space per minute. In CKD, the number of residual nephrons decline progressively leading to reduced volume of filtrate formed per minute. As such, the adaptive response of nephrons to hyperfiltrate to compensate for the loss of nephrons a nd maintain optimal filtration capacity progressively increases Hyperfiltration is characterized by reduced afferent arteriolar resistance, which increases glomerular capillary plasma flow rate This allow greater fraction of the systemic blood pressure t o be transmitted into the glomerular capillary network therapy raising the glomerular capillary hydraulic pressure without affecting perfusion pressure While this can lead to temporarily improved filtration capacity chronically, it c ause s permanent glome rular damage (95, 9 6) This is because the distention of the glomeruli capillary lumen leads to the stretching of the m esangial cells associated with the glomerular capillaries that stimulates the release of fibrosis related genes such as that accelerates glomerul ar scle rosis (95, 97, 98) In addition, a declining number of nephrons mean s less tubular surface area available for tubular secretions and absorption as well as increase competition for the available transporters. Plasma becomes satura ted with uremic toxins that are otherwise filtered easily leading to build up of these toxins that can induce chronic inflammation and damage the tubular cells.

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42 Changes in dietary therapy are likely to affect hyperfiltration by means of altering uremic mol ecules in the blood. It has been shown that d ietary protein restriction limit s the adaptive increase in single nephron GFR and glomeru lar capillary hydraulic pressure that lead s to amelioration of glomerular damage and loss of function (96, 99) Animal studies show that the addition of uremic toxic molecules leads to accelerated disease progression and decreased filtration capacity while removal of such toxins is nephron protective. Anima l treated with these uremic toxic molecules score significantly higher on the glomerular sclerosis index and exhibit a more advanced interstitial fibrosis with increased glomerular dysfunction (lower GFR) compared to controls (11, 12) Uremic toxins can accumulate in tubular cells and induce oxidative stress (13, 27) and inhibit cellular proliferation (28) It is thought that chronic exposure to oxidative uremic molecules induces the production of vasoactive and inflammatory substanc es t hat increase expression of 1 (TIMP 1), and pro proximal tubule cells (8, 11, 95) leading to accelerated interstitial fibrosis, glomerular sc intervention to reduce uremic toxins maybe effective to delay the progression of renal (11) C R eactive P rotein C reactive protein (CRP) is produced by the liver (100) and typically measured to assess general inflammation with no specificity to the location of infection or inflammation. Values of circulating CRP have been found to co rrelate closely with other markers of inflammation (100) Normal values are <0.80 mg/dL (101) CRP is a predictor of type 2 diabetes and coronary events in the general population (100) Inflammation plays a significant role in the progression of CKD (102) Systemically,

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43 elevated CRP levels are associated with hyperfiltration, glomerulosclerosis, and cortex scarring (102, 103) The inflammatory process induces the release of inflammatory mediators that can accumulate in tissues without clearance Such process es lead to maladaptation and sustained inflammation (104) Fermentation The human large intestine is approximately 150 cm long and is slightly acidic with a pH range of 6.0 7.0. The resident microbial community is complex, diverse, and biochemically very active with a bacterial population count of 10 11 10 12 per g content wit hin the colon (26) .The princip al role of the colon is water and solute retention and waste disposal. Other roles include immunity, and energy and nitrogen salvaging from carbohydrates and proteins. On average, 0.3 4.1g of nitrogen, primarily protein s (50%) and peptides (20 30%), enters the colon daily (105) The bacterial mass and diversity in the colon are dep endent on several factors, one of which is substrate availability. Typical fermentation substrates are sloughed cells and mucous, unabsorbed sugars/starch and fiber. Protein fermentation occurs in the large intestine and increases when carbohydrate substra te is lacking. Fermentation is largely dependent on substrate availability, substrate physical properties, and colonic conditions such as pH and transit time. Carbohydrate fermentation by saccharolytic activity results in short chain fatty acids ( SCFAs ) CO 2 and H 2 production while protein and amino acid ferm entation by proteolytic activity yields CO 2 CH 4 and H 2 phenols, amines and branched SCFAs, but comparatively fewer SCFAs (106, 107) Protein bacterial fermentation in the colon can result in the generation of tumorigenic and uremic molecules (26, 108) I t was first proposed by Niw a et al that uremic metabolites originating from bacterial protein fermentation in the colon have key

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44 effect in the progression of CKD (9, 10) It is shown that a high protein diet can increase uremic molecules and is associated with accelerated glomerulosclerosis while low protein diet ameliorate hyperfiltration (109) Along the length of the colon, the ratio of available fermentable substrate to nitrogen progressively decline which impacts bacterial composition and metabolism (110) This decline of fermentable substrate, allows the fermentation of proteins to be more significant. Saccharolytic and proteoly tic activity predominate in the right colon and left colon, respectively (26) Reduced transit time can cause a reduction in the carbohydrate substrate reaching distal parts of the colon and as a result proteolytic activity leading to increase generation of toxins ( 26) Increasing intake of fermentable fiber substrate can lead to interference with microbial metabolites generation by selectively increasing saccharolytic and reducin g proteolytic bacteria activities and metabolites (26) Bacterial generated uremic toxins such as amines, phenols, and indoles are increasingly recognized as contributors of uremia (26) These microbial metabolites believed to be increased in the blood under two conditions, reduced renal clearance and increased colonic generation and absorption (26) Fermentable fibers alter uremic toxins positively by depressing the colonic protein fermentation as a result of improved carbohydrate availability (108) Factors that are thought to promote uremic toxins generation and absorption include an increased ratio of dietary protein to carbohydrate due to insufficient intake of fiber and /or reduced intestinal protein assimilation, as well as prolonged colonic transit time (26) While prebiotic fibers can provide the p referred substrate to promote the proliferation of saccharolytic bacteria, i nsoluble fiber s can promote regularity, and increase bowel

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45 movement frequency thus allowing rapid removal of nitrogenous molecules while potentially expand ing the availability of fermentable substrates distally. Uremic Molecule Description Loss of kidney function leads to accumulation of uremic molecules above uremic mo lecule that can be singled out as a cause of uremia because retained solutes have variable characteristics such as (25) which result in various symptoms among patients. The re are 90 retention molecules that are identified as uremic as they become elevated above normal ranges (25) Elevated concentration and potential accumulation of uremic molecules and toxins in the interstitial fluids, inner medulla, collecting duct or any part of the renal co rtex may lead to ischemia, inflammation or interstitial fibrosis due to potential toxic effect of some of these uremic toxic molecules on the glomerulus and tubules cells (5, 6, 8, 11, 13, 27, 28) Urea Urea is an organic compound with molecular mass of 60 Da Urea makes up the largest proportion of nitrogenous waste in urine of human s Blood urea nitrogen is a measure of the amount of nitrogen in the blood in the form of urea used as a measurement of kidney function. Normal levels f or adults are between 7 25 mg/dL Elevat ion of BUN greater than 60 mg/dL can be interpreted as moderate to severe kidney disease. However, BUN is not sensitive enough because it c an be elevated independent of kidney disease but is mostly used as an indicator of uremic molecules levels in the blood.

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46 p Cresol p Cresol is volatile phenol with a low molecular mass of 108 .1 D a It is partially lipophilic and strongly binds to plasma p rotein under normal condition s (111) p Cresol is generated from partial breakdown of tyrosine and to a lesser extent phenylalanine mainly by aerobic enterobacteria and the anaerobic c lostridium perfringerns bacterial species (26, 52, 112) They are eliminated primarily in the urine as conjugates (113) with the main circulating form p cresol sulfate (114, 115) p Cresol is metabolized through inorganic sulfate via conjugation (sulfation) pathway and to a lesser extent to glucuronic acid (glucuronization) (113) (116, 117) However the removal of the unconjugated p cresol is via the kidneys (111) p Cresol is associated with uremia (33) a nd along with its sulfate and glucuronide conjugates, is associated with CVD in HD patients, and is implicated in uremic immunodeficiency and endothelial dysfunction (118) Serum p cresol levels are increasingly suggested as a cardiovascular risk marker (119) and cardiovascular death is the leading cause among renal patients. While these correlations point toward a relationship, it is not clear how modifiable is p cresol as a risk factor for cardiovascular disease in CKD patients. In a prospective observational study in 499 CKD patients, p cresol was a predictor of cardiovascular events independent of GFR with higher baseline concentrations of free p cresol directly associated with cardiovascular events (120) p Cresol and its main conjugated derivative, p cresol sulfate, have been shown to contribute to endothelial dysfunction in HD patients (119) have pro inflammatory effect on unstimulated leucocyt es and activates leucocyte free radical production (121) and caused a significant in crease in cellular inflammation in cultured mouse proximal renal tubular cells (5) Median total p cresol for controls was 1.58 mg/L compared to

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47 20.10 mg/L for HD patients (114) In another study, serum concentratio n averaged 1.14 mg/L for healthy individuals (122) while u remic outpatients had serum p cresol levels of 6.702.11 mg/L (31) Reduction of p cresol generation and excretion has been demonstrated using fermentable fibers. In a study of healthy volunteers, decreased, urinary p cresol excretion was observed by ingestion of a 50/50 v/v mixture of inulin and FOS (3) A 4 week trial in HD patients using a total of 20 g daily (10 g the first week) of the inulin and FOS reduced p cresol sulfate concentrations in HD patients by 17 % (7) Uremic Symptoms Uremic molecules such urea and p cresols accumulation lead to uremic symptoms (1, 123) As such, uremic symptoms can range in both type and severity in individuals and is not necessarily present similarly among individuals. Symptoms of uremia include fatigue, inten se itching, slowed thinking, anorexia (loss of appetite) fishy taste, constipation, impaired sleep, and restless leg syndrome (1, 61, 124 126) Excessive buildup of metabolic wastes causes acidosis induced anemia, damaged nerves and muscle cells, shortness of breath, nausea, vomiting, and malnutrition (61) Uremic symptoms are usually not detected until at le ast half of the kidney function capacity is lost compared to normal healthy individuals (1) Patients with 50% decline in kidney function are said to have a moderate decline in kid ney function as classified by KD Q O I with uremic symptoms being more moderate as compared to more severe stages A re view of the existing evidence by the KD Q O I panel shows that eGFR of less than 60 mL/min/1.73m 2 is associated with reduced well being (21) and cognitive impairments (127) Current treatment of uremia has poor outcomes compared to intervention of

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48 other complications associated with chronic diseases such as diabetes because uremia symptoms cannot be traced to single molecule (1) In the MDRD study, subjects with eGFR less than 55 mL/min/1.73m 2 had a correlated fatigue and reduced stamina (128) which may be attributed to muscle energy failure and neural defects (129) Simple nitrogen containing uremic molecules such a s amines may be responsible for impaired brain function as seen in human and animal studies (130 132) while indoles and phenols may interfere with central nervous system function due to structural similarity with neurotransmitters (1) Health Related Quality of Life and Symptoms Health related quality of life is a self reported multidimensional measure usually of physical and mental health and how the disease interferes with day to day activit ies (133) Typical ly, patients suffering from chronic diseases see a reduction in quality of life as the disease progresses. In CKD, patients may experience a reduction in quality of life due to the number of neural, muscular, endocri ne or metabolic symptoms. A valid qualit ative tool to assess quality of life is the Kidney Disease Quality of Life ( KDQOL 36 A ). The self assessment questionnai re is a kidney disease specific measure and is divided into five categories. The first two are the Physical Component Summary subscale and Mental Component Summary subscale spanning the first 12 questions. These items cover general health, activity limits, ability to accomplish desired t asks depression and anxiety, energy levels, and social activities Burden of Kidney Disease subscale (questions 13 16) contain s questions about how much kidney disease causes frustration, or makes the respondent feel like a burden interferes with daily life, or takes up time The Symptoms/Problems List subscale (17 28 th questions) contai ns items about how bothered a respondent feels by various uremic symptoms. These

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49 symptoms are faintness/dizziness, lack of appetite, sore muscles, itchy or dry skin, chest pain, cramps, or shortness of breath, feeling wash out or drained, numbness in the h ands or feet, and nausea. The final subscale items (questions 29 36) refer to the Effects of Kidney Disease on daily life. They contain questions about how bo thered respondent feels being dependent on doctors and other medical staff, by fluid limits, diet restrictions, stress or worries, ability to work around the house or travel, sex life, and personal appearance. Chronic kidney disease patients in stage 4 or 5 score significantly lower on the Physical Component Summary subscale of the KDQOL 36 compared to patients with hypertension, diabetes mellitus, asthma, chronic obstructive lung disease, or liver failure suggesting a significantly reduced quality of life (23 25). C hronic kidney disease patients with creatinine clearance <60 mL/min/1.73 m 2 report s lower physical function independent of age, sex, and other confounding factors (134) In dialysis patients both the Physical and Mental C omponents were consistent predictors of hospitalizations and mortality rates with each point increase in either component reducing mortality relative risk by 2% and hospitalization relative risk by 2% and 1% respectively (135) Gorodetskaya et al reported that each one mL/min/1.73 m 2 decline in eGFR per year was associated with 5.0 points changes in Burden of Kidney Disease subscale score in stage 4 and 5 CKD patients (136) Gastrointestinal Health and Well being Gut health may be measured as the absence or presence of gut disorders or disease and severity. Complex f actors including neural, and chemical, nutritional can alter the well being percept ions originating from the gut. The area of well being and functional gastrointestinal health is not well understood, and there is remarkably little

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50 known about it. The autonom ic nervous system governs gut function and thus regulatory reflexes and digestion processes are not consciously perceived. However, there is evidence that under certain circumstances sensory dysfunction of the gut may induce conscious perception (symptoms) (137) Therefore it is possible that one well being is affect ed by gastrointestinal changes. One of the symptoms of uremia is constipation (138) Constipation can be particularly bothersome. The prolonged time between evacuations and difficulty emptying and associated straining can introduce a feeling of anxiety or un wellness. Increased transit time in itself can be d etrimental as proteins and amino acids can be exposed to increased bacterial activities that are thought to negatively impact the host cell due to the increase d production of uremic molecules. Improved gut transit time may therefore produce desirable effec ts and may contribute to enhanced quality of life. Lower fiber intakes in the diet leads to reduced bulking. Bulking is necessary for regular defecation. Thus, reduced bowel frequency may be attributed to lower fiber intake in CKD. In addition, decreased m ovement of wastes along the colon will increase the time in which substances come in contact with epithelial cells and/or bacteria that may lead to increased protein fermentation when carbohydrate substrates are depleted in the colon, particularly in the d istal colon. This will lead to increased generation and absorption of molecules that may otherwise be excreted in feces adding to the uremic load. Elevated levels of uremic toxins in the blood can impair neural and muscular functions (1) which in turn may affect transit time as well. The self administrated version of the validated Gastrointestinal Symptom Rating Scale (GSRS ) (Appendix B ) can be used to assess gastrointestinal symptoms presence and severity in patients with

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51 gastrointestinal disorders and patients who suffer from gastrointestinal related side effect secondary to non gastrointestinal complaint (139, 140) The questionnaire includes 15 items representing five syndromes and uses a seven point grade Lik ert like represents severe discomfort (maximum). The reflux syndrome (heartburn and acid regurgitation), abdominal pain syndrome (abdominal pain, hunger pains, and nausea), ind igestion syndrome (rumbling, bloating, burping gas, and passing gas), diarrhea syndrome (diarrhea, loose stools, and urgent need for defecation), and constipation syndrome (constipation, hard stools, and feeling of incomplete evacuation) (141, 142) Appetite and Sleepiness Anorexia and weight loss are also symptoms of uremia. The simplified nutritional appetite questionnaire (SNAQ) (Appendix C ) as sessment tool is a s hort 4 item, validated clinical tool that objectively quantifies appetite in people ( >20 and older) at risk for weight loss (143) It is a reliable and efficient tool with singular construct of appetite with a view to prevent weight loss A score of <14 may identify a person with anorexia and significa nt risk of weight loss. The Epworth S leepiness Scale (ESS) questi onnair e is used to assess parti c i pants likel i hood of daytime sleepiness (144) The tes t measure s the aver a ge sleep propensity in the day time situations such as sitting and reading, sitting in active in public enviro n ments, as a passenger in a ca r for an hour without a break, in a car while stopped for a few minu t es in the traffic, and watch ing TV among others (Append i x D ). point scale. These options rank from 0 for the lowest scale option to 3 for the

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52 le score is 24 and the lowest possible is 0. Scores from 0 10 are within the normal range, 10 12 Borderline, 12 24 is abnormal.

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53 CHAPTER 3 MATERIALS AND METHOD S Study 1 : Overview This study was designed as a pilot study to provide information on the effect of adequate fiber intake on blood markers, quality of life and tolerance in patients with CKD. Future directions would be to conduct studies with larger sample size, longer durat ion, and additional blood markers. The following section provides a description of the design and methods used in this pilot study. The Institutional Review Board 1 (IRB 1) at the University of Florida approved the study. Clinical visits were arranged at t he Food Science and Human Nutrition Department (FSHN), University of Florida. The Division of Nephrology, Hypertension, and Renal Transplantation within the Department of Medicine at the University of Florida in Gainesville, FL carried out the initial scr eening of patient medical records for patient referrals. Study Design A single blind rolling admission intervention study was carried out for six weeks Seventeen CKD patients were consented, 16 enrolled, and 15 completed the study (Figure 3 1). Participants were in various stages of the disease ranging from stage 3 to 5, but not on dialysis. Participants started a control period, which lasted two weeks. During this period, participants consumed control foods containing a small amount of fiber (1. 6 g/day). After two weeks, participants started the second period in which they consumed closely matched foods with higher fiber content (23 g/d). Participants were seen in the clinical lab at the FSHN Department on days 1, 14, 28 and 42 (Figure 3 2). T wo blood draws were collected during the control period The intervention period lasted four weeks and two blood dra ws were made during that period. Visits consisted

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54 of collecting blood samples and questionnaires data. Participants also received coaching abou t how to record dietary information using food records. During each visit participants picked up study related questionnaires, food records and study foods. Screening, Recruitments and Obtaining Consent Eligible participants were screened by clinical nurse s from the Division of Nephrology, Hypertension, and Renal Transplantation within the Department of Medicine at the University of Florida in Gainesville, FL. Initial screening by the clinical nurses was done according to the following criteria i) age 18 an d older ii) eGFR of 50 mL/min/1.73 m 2 (mid stage 3, 4 and 5 but not on dialysis) iii) not diagnosed with acute kidney injury glomerulonephritis or lupus disease iii) not prescribed immunosuppressant medications) able to understand, verbalize and sign the informed consent in English. The clinical nurses of the nephrology department carried out the screening process and provided potential participants contact info rmation after obtaining initial verbal consent as per IRB protocol number (16 2010). Participants meeting the initial screening criteria and expressing interest in the study were contacted by the study coordinator by phone to further assess eligibility. U pon calling, a telephone script was used to provide potential participants with more details about the study and check for inclusion/exclusion criteria (Appendix E ). Participants self excluded if they had a history of kidney transplant, liver disease, or d iagnosed with active gastrointestinal bleeding, were breastfeeding, or scheduled to be on dialysis or undergo transplantation within three months of study initiation, had changes in medication over the past four weeks prior to study initiation, were taking probiotic supplement and ref used to discontinue, or were on immunosuppressant or

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55 steroid medications. Potential participants who indicated that they met all inclusion/exclusion criteria according to protocol, and expressed willingness to participate were scheduled to come to the clinical lab in the FSHN building to review the informed consent. The informed consent was signed in person after allowing participants the chance to reflect on it. Study Foods The s tudy protocol required participants to consume control foods for 14 days (control period) followed by intervention foods for 28 days ( intervention period) according to study flow. For the control period, individually packaged, commercially available Publix chocolate chip cookies (1 serving/day), Kello participants. Control foods were consumed from day 1 to day 1 4 providing a total of 1.6 g of dietary f iber a day (Table 3 1). For the interventio n foods, individually packaged Weight Watchers chocolate chip cookies (1 serving/day), Fiber One bars (1 vings/day) were provided unchanged Total fiber contents from these foods was 23 g /d mostly of fermentable fiber sources (chicory root fiber and resistant corn dextrin) and smaller amount of insoluble fiber source (pea hull) (Table 3 1). U nused foods were collected during clinic visits to determine fiber consumption and compliance to the protocol.

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56 Study Food Packaging Portions of breakfast c ereal were weighed using electronic precision scales (Adam Equipment PGW 1502e Precision Balance). After weighing, portions of breakfast cereal were sealed in airtight vacuum sealed bags. Cookies and bars were removed from their original wrapping and were sealed in airtight vacuum sealed bags. All packaged foods were placed in large zip lock bags labeled with participant study number and dated according to study period. Clinic Visits Consented partici pants visited the clinical lab four times throughout the study to collect blood samples, fill out questionnaires, check for adherence, and collect daily dairy and food records. Prior to each visit, participants were asked to fast overnight. Once participan ts arrived, a licensed phlebotomist drew a 10 mL blood sample in the s and height s were measured using conventional methods described below. After complet ing the KDQOL GSRS and th e ESS questionnaires and after questions and concerns were addressed, each participant was given a two week supply of the study foods according to the period of the study. In addition, a food journal was given during visits 1, and 3 and the GSRS questionna ire was given on visit s 2 and 3 in a dated envelop to be filled out the following week. Qualitative Questionnaires Gastrointestinal Symptoms and Daily Diary A self administrated tool to assess changes in gastrointestinal symptoms was administered to parti cipants every week of the study. The GSRS was administered during clinic visits at days 1, 14, 28, and 42. Additionally, participants were given the

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57 GSRS questionnaire in dated envelops during clinic visits 2 and 3 to be filled out at home the following we ek (weeks 3, and 5). A daily diary workbook (Appendix F) was given to participants during the first visit. The daily diary contained 42 tear out pages for each day of the study in which participants were asked to bring back at each visit. During visits, da ted pages were removed from the workbook. Quality of Life, Appetite and Risk of Weight Loss R ela ted Quality of L ife was assessed using the self assessment KDQOL 36 questionnaire. The questionnaire was administered to participants every two weeks during scheduled clinic visits. Higher scores for the KDQOL 36 indicates better results. Appetite and risk of weight loss were assessed during clinic visits using the SNAQ. Anthropometric Measurements Patients' weight s and height s were measured using conventional methods with shoes removed. Height was measured the first visit using Ayrton 226 Hite Rite stadiometer, and weight was monitored during each clinical visit using Seca 874 portable flat platform scale. Both devices were calibrated prior to use. Food Intake Assessment Participants were given a food record (Appendix G ) to log intak es of foods and beverages for three days during each period. Food j ournal workbooks (Appendix H ) containing a sample page of food record, tips to follow for a co nsistent and successful logging of food entries, and visual examples of food servings were also provided. The first 3 day food record was collected for the second week of the control period. The second 3 day food journal was collected for the last week of the intervention period. To ensure compliance and consistency, participants were briefly coached about the use of

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58 the food journal, and how to log related information. In addition, measuring bowls given to participants to use throughout the study for consistency in reporting portion quantities. The coaching sessions were administered during clinic visits 1 and 3. Nutrient Assessment Three day food records were analyzed using (Food Processor versio n 10.4.0.0 ESHA Researc h Inc., Salem, OR) for nutrient and energy intake. Foods equivalent or similar to those eaten by the participants during the study period were chosen from the database. For unique foods, such as combination of salads and desserts ind ividual ingredients were entered into the database. Blood Samples Collection Blood samples were collected in two 5 mL tubes and were placed in upright position unstirred for 30 minutes to allow specimen clotting. All samples were centrifuged and then placed in stoppered containers with ice at 2 8 C All samples were processed with in 12 hours of centrifugation. Aurora Diagnostics Clinical Services, a Florida based clinical laboratory, provided blood assay services. Assays All Assays were performed using SIEMENS ADVIA Clinical Chemistry System Blood Urea Nitrogen Blood urea nitroge n was assayed using the Urea Nitrogen Concentrated Reagent method based on Roch Ramel enzymatic reaction using urease and glutamate dehydrogenase (145) Urea is hydrolyzed in the presence of water and urease to produce ammonia and carbon dioxide. The ammonia reacts with oxoglutarate in the

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59 presenc e of glutamate dehydrogenase and NADH. The oxidation of NADH to NAD is measured as an inverse rate reaction at 340/410 nm. Serum Creat i nine Serum creatinine reacts with picric acid in an alkaline medium to produce a red colored creatinine picrate complex. The rate of complex formation is measured at 505/571 nm and is proportional to the creatinine concentration. Serum creatinine is analyzed by modification of the Jaff method (146) The method is modified by using rate blanking and intercept s correction to minimize bilirubin interference and serum correct for the non specific serum protein interactions with reagent which produces a positive bias of 0.3 mg/dl Serum Triglycerides (TG) The triglycerides are hydrolyz ed to glycerol and free fatty acids by lipase. The glycerol was then converted to glycerol 3 phosphate by glycerol kinase followed by its conversion by glycerol 3 phosphat e oxi dase to hydrogen peroxide and dihydroxyacetone phosphate A colored complex is formed from hydrogen peroxide, 4 aminophenazone and 4 cholrophenol under the catalytic influence of peroxidase. The absorbance of the complex is measured as an endpoint reaction at 505/694 nm.

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60 Total Serum Cholest er ol Cholesterol esters are hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. The cholesterol is converted to choletenone by cholestero l oxidase in the presence of oxygen to form hydrogen peroxide A colored complex is formed from hydrogen peroxide, 4 aminoantipyrine and phenol under the catalyt ic influence of peroxidase The absorbance of the complex is measured as an endpoint reaction at 505/694 nm. High Density Lipoprotein (HDL) Cholesterol esters in serum were hydrolyzed by cholesterol esterase The free cholesterol produced is oxidized by cholesterol oxidase to cholestenone with the simultaneous production of hydrogen peroxide which oxidatively couples with 4 aminoantipyrine and phenol in the pr esence of peroxidase to yield a chromophore. The red quinoneimine dye formed is measured spectrophotometrically at 596 nm as an increase in absorbance. Low Density Lipoprotein (LDL) LDL cholesterol is assayed in two steps. Step 1: Release of cholesterol from non LDL particles followed by degradation of the cholesterol by cholesterol esterase and cholesterol oxidase and elimination by catalase of the resulting hydrogen peroxide. Step 2: Release of cholesterol from LDL cholesterol by a surfactant in reagent 2, followed by degradation of the cholesterol by cholesterol esterase and cholesterol oxidase in the same manner as in the first step, except that the catalase in reagent 1 is inhibited by sodium azid in reag ent 2. The intensity of the quinoneimine dye produced in the reaction is directly proportional to the LDL cholesterol concentration when measured at 596 nm.

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61 Glucose Glucose is ph osphorylated by a denosine triphosphate in the presence of hexokinase. The gluc ose 6 phosphate that forms is oxidized in the presence of glucose 6 phosphate dehydrogenase causing the reduction of NAD to NADH. The absorbance of NADH is measured as an endpoint reaction at 340 410 nm. eGFR Calculation Estimated Glomerular Filtration Rat e was calculated based on the non standardized original MDRD study equation (147) as follows: eGFR= 186 SCr 1.154 age 0.203 1.212 (if African American) 0.742 (if female) General Statistical Approach All data are presented as me an S E (standard error). Correlations were det ermined using Pe a aired t test was used for all comparisons of parameters for each test between control and intervention periods Significance was concluded when p<0.05. Study 2 : Overview This study was designed to provide further information about the effect of adequate fiber intake on uremic blood markers, eGFR, symptoms and quality of life. The study was conducted to determine the effect of insoluble fiber alone and in combination with inulin/FOS supplement on uremic blood markers, eGFR and quality of life. This section provides a description of the design and methods used. The IRB 1 at the University of Florida approved the study (IRB protocol 580 2011). The Division of Nephrology, Hypertension, and Renal Transplantation within the Department of

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62 Medicine at the Universi ty of Florida in Gainesville, FL carried out the initial screening of Study Design A twelve week, single blind, intervention study with escalating fiber regimen. Study was carried out with CKD stages 3 to 5, non dialysis. The study required participants to consume control muffins and 5.5 g of sucrose for two weeks (control period ) followed by consumption of pea hull fiber containing muffins and sucrose for four weeks (pea hull fiber period) an d finally, pea hull fiber containing muffins and Frutafit HD in ulin supplement for six weeks (pea hull and inulin fiber period) (Figure 3 3). The 5.5 g of sucrose was chosen as a control for inulin and the amount was selected to match inulin energy conten t Two blood samples were collected during each period. During the control period at 0 and 2 weeks During the pea hull fiber period blood samples were collected at 4 and 6 weeks. For the pea hull and inulin fiber period, b lood samples were collected at 9 and 12 weeks. A dditionally two single blood samples were collected during the study for p cresol analys is. One sample was collected during first week of the control period and another sample was collected at week 12 Questionnaires and 3 day food records were delivered (dropped off or mailed) to participants prior to the day in which they were required. Participants were asked to bring any leftover bags of sucrose and inulin at the end of the study. Screening and Recruitments and Obtaining Consent Eligib le participants were screened for by clinical nurses from the Division of Nephrology, Hypertension, and Renal Transplantation within the Department of Medicine at the University of Florida in Gainesville, FL (Figure 3 4) Initial screening by

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63 the clinical nurses was done according to the following criteria: i) age 18 and older ii) have an eGFR of 50 mL/min/1.73 m 2 (stage 3, 4 and 5 but not on dialysis) iii) not diagnosed with acute kidney injury, glomerulonephritis or lupus disease i) not prescribed immu nosuppressant medications iiii) able to understand, verbalize and sign the informed consent in English. Participants meeting the initial screening criteria and expressing interest in the study were contacted by the study coordinator by phone to further ass ess eligibility (Appendix I ). Participants were excluded if they had a history of kidney transplant, liver disease, active gastrointestinal bleeding, lactating, or scheduled to be on dialysis or undergo transplantation during study duration, had celiac d isease, food allergies or were not willing/could not discontinue the use of fiber/prebiotic/probiotics containing products for two weeks prior to first blood draw, or were scheduled to have a major surgical procedure during the duration of the study. Poten tial participants who were eligible and expressed willingness to participate were then scheduled to come to the clinical lab in the FSHN building to review the informed consent. Potential participants who were unable to come to the lab were offered the opt ion to be visited at home for consenting according to IRB protocol. The informed consent signed in person after allowing participants the chance to reflect on it. Consented participants received all study supply in mail or in person according to study cale ndar. Study Food During the control period participants consumed control muffins with 1 g fiber (Orange Blueberry Muffin with No Pea Fiber, AC125 72 1b, Penford Ingredients, Centennial, CO. USA) (Appendix J ) and approximately 5.5 g sucrose. Immediately af ter,

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64 participants started the pea hull fiber period that lasted four weeks During this period, participants consumed muffins containing 10 g/d of pea hull fiber (Orange Blueberry Muffin with Pea Fiber, AC125 72 2d, Penford Ingredients, Centennial, CO. USA ) (Appendix J ) and approximately 5.5 g/d of sucrose to serve as a control After six weeks of the c ontrol and pea hull fiber period s, participants started the pea hull and inulin fiber period which lasted six weeks consuming a total of 23.5 g/d of fiber ev eryday ( 10 g/ d of pea hull in muffins and 13.5 g/d of fiber from a supplement (inulin/FOS) (see product sheet Appendix K ) (FrutafitHD, SENSUS, The Netherlands) (Nutrient Composition Table 3 2). Muffins were delivered every two weeks by overnight UPS carr ier or dropped off by study coordinator. Sucrose and inulin were delivered by mail or dropped off to participants prior to the day in which they were needed. Penford Ingredients ( Centennial, CO ) formulated control and fiber containing muffins Study Food Packaging Prior to packaging, sucrose and inulin were measured using the precision scales (Adam Equipment PGW 1502e Precision Balance). Packaged products were placed in corresponding bins during packaging periods to avoid mixing. Control products were weighed and packaged on separate days. Once all control products were prepared, intervention products were prepared in a similar fashion. Muffins were placed in airtight vacuum sealed bags, while sucrose and inulin were placed in small 2x2 inch clear, doub le sealed bags. Demographic and Baseline Data Collection After the informed consent was obtained, participants provided information about age, race, medical history, medications, antibiotics use, supplement use, nutritional foods and beverages using the ba seline questionnaire (Appendix L ).

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65 Food Intake and Nutrient Assessment Three day food records (Appendix M ) were mailed to participants in pre paid envelop e s for each period of the study. Food records were explained during consenting process. Food intake an d nutrient assessment analysis was conducted using (Food Processor, version 10.6.0.0 ESHA Research Inc., Salem, OR). Foods equivalent or similar to those eaten by the participants during the study period were chosen from the database. For unique foods, su ch as combination salads and desserts individual ingredients were entered into the database. Qualitative Questionnaires The self administered KDQOL questionnaire (Appendix N ) were administered to participants three t imes during the st udy. Questionnaires were mailed for each period. Mailed envelopes contained returning envelop with pre paid postages. Clinic Visits Participants visited the FSHN clinical lab during the first and the last week of the study to provide anthropometric measurements, and additional blood samples to determine changes in p cresol concentrations Patients' weight s and height s were measured using conventional methods with shoes removed. Weight was measured using portable flat platform scale (Seca 874). For height, portable s tadiometer (Seca 213) was used. A licensed phlebotomist drew a maximum of 10 mL blood during each visit using 10mL plasma EDTA tubes in which they were centrifuged. Samples were then transferred into two 3 mL vials and stored within 45 minutes in 80 C until analysis of total plasma p cresol

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66 Quest Diagnostics Quest Diagnostics Inc. provides the diagnosti c testing and services with several facilities around the state of Florida. Quest Diagnostics Inc. was contracted to provide blood draw and assays services according to protocol. Blood draw requisitions were mailed to participants prior to the day in which they were required. Participants were requested to visit nearby Quest Diagnostics facilities during the specified dates according to study calendar for total of six visits. Assays BUN and Serum C reatinine Serum urea was determined by a kinetic method usin g urease and glutamate dehydrogenase (145) Serum creatinin e was analysed by a kinetic method based on the reaction (146) both methods were described earlier. Assays were performed using Olympus AU5400 Chemistry System. Ammonia Enzymatic method with glutamate dehydrogenase was applied to derive and measure ammonia (148, 149) Glutamate dehydrogenase catalyzes the reductive amination of 2 oxoglutarate with NH 4 + and NADPH to form glutamate and NADP + The concentration of the NADP + formed is directly proportional to the ammonia concentration. It is determined by measuring the decrease in absorbance at 3 40 nm. Assay was performed using COBAS INTEGRA 800. CRP Particle enhanced turbidimetric assay was used to measure CRP (150, 151) Human CRP agglutinates with latex particles coated with monoclonal anti CRP

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67 antibodies. The precipitate is determined turbidimetrically at 552 nm. Assay was performed using COBAS INTEGRA 800. Cystatin C C ystatin C was measured by the N Latex cystatin C assay on the SIEMENS BN II nephelometer system using a particle enhanced immunonephelometric assay. I n this assay, polystyrene beads coated with rabbit antibodies to c ystatin C agglutinate when mixed with samples containing c ystatin C The intensity of the scattered light in the nephelometer depends on the concentration of c ystatin C (antigen) in the sample. This concentration is determined by comparison with dilutions of a cali brator (152) GC MS Analysis of T otal p Cresol in P lasma Concentrations of total p cresol in plasma were measured using a gas chromatography mass spec trometry (GC MS) method on plasma samples stored at 80 C prior to analysis. After combined acid and heat deproteinization and deconjugation (hydrolysis of conjugates), p cresol was extracted in ethyl acetate and injected into the Thermo Finnigan Trace DS Q Single Quadrupole GC MS instrument. For use as standard, p cresol was purchased from Acros Organics (part of Thermo Fisher Scientific). The p cresol d 7 was obtained from C/D/N ISOTOPES and used as internal standard. For the heat acid intervention of the plasma samples and subsequent cresol extraction, a previously described method (114) was used, with minor modifications. eGFR Calculations Estimated Glo merular Filtration Rate was calculated based on the CKI EPI creatinine equation adjusted for age, sex, and race (76) : eGFR = 141 x min(Scr/k, 1) a x max(Scr/k, 1) 1.209 x 0.993 age [ x 1.018 if female ] [ x 1.159 if black ] Scr is serum creatinine, k is 0.7 for females and 0.9 for males

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68 a is 0.329 for females and 0.411 for males The CKD EPI cystatin C equation by Inker et al (78) : eGFR = 133 x min(Scys/0.8, 1) 0.499 x max(Scys /0.8, 1) 1.328 x 0.996age [ x 0.932 if female ] Scys is serum cystatin C. The CKD EPI creatinine cystatin C equation adjusted for age, sex, and race by Inker et al. (78) : eGFR = 135 x min(Scr/k, 1) a x max(Scr/k, 1) 0.601 x min(Scys/0.8, 1) 0.375 x max(Scys/0.8, 1) 0.711 x 0.995age [ x 0.969 if female ] [ x 1.08 if black ] Scr is serum creatinine and Scys is serum cystatin C. k is 0.7 for females and 0.9 for males a is 0.248 for females and 0.207 for males General Statistical Approach A ll data are presented as mean SE (standard error) To determine significance ANOVA was used to compare each mean and overall model significance was determined when p< 0.05. For bowel movement frequency, the KDQOL 36 subscales, and the individual symptoms list in the Symptoms/Problems L ist subscale of the KDQOL 36 multiple paire d t test s were calculated independent of the ANOVA significance P value of <0.05 is considered for unadjusted significance level. For significant after adjustment, Bonferonni correction was used and significance was set at 0.01667 The natural logarithmic transformation was applied to meet the a ssumptions of normality when comparing the two transformed means of total p cresol.

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69 Table 3 1 Nutrient composition of foods provided in the control and fiber intervention p eriods Weight (g) Quantities Energy (kcal) Fiber (g) Fat (g) Protein (g) Carbohydrate s (g) Source of Added Fiber Control foods Corn Pops (Battle Creek, MI) 58 2 oz 220 < 1 0 2 52 None Publix Chocol ate Chip Cookies (Lakeland, FL) 24 2 cookies 120 < 1 6 1 15 None Creek, MI) 23 1 bar 90 < 1 1.5 1 17 None Fiber intervention Corn Pops with Fiber (Battle Creek, MI) 64 2 oz 240 6 0 2 58 Resistant c orn dextrin Weight Watchers Chocolate Chip Cookies (Jerico, NY) 50 2 cookies 180 8 5 2 36 Inulin, pea hull fiber General Mills FiberOne Bar (Minneapolis, MN) 40 1 bar 140 9 4 2 29 Chicory root fiber (inulin)

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70 Table 3 2. Nutrient composition of foods and supplements p rovided Muffins (Control) Muffins ( Intervention ) Frutafit HD Sucrose Fiber g 1 10 13.5 0 Energy kcal 180 170 0 22 Carb g 30 26 0 5.5 Fat g 4.5 6 0 0 Protein g 3 3 0 0 Figure 3 1 Renal Study 1 : s creening and recruitment

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71 Figure 3 2 Renal Study 1 : s tudy design Figure 3 3 Renal Study 2 : s tudy design

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72 Figure 3 4. Renal Study 2 : s creening and recruitment

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73 CHAPTER 4 RESULTS STUDY 1 Demographics D ata Sixteen participants were enrolled in Study 1 Average age was 66 4 years (mean SE ). Nine females and six males completed the study with eight participants diagnosed with type 2 diabetes. Eleven participants were white and four were African American Four patents were categorize with stage 3 CKD, ten with stage 4, and one with stage 5. Weight BMI was calculated based on recorded measurements of height and weight. Weight was 89 6 during control and 90 6 during intervention which was not significantly different. Nutrient and Fiber I ntake There was no difference in energy, protein potassium or phosphorous intake s during the control and the intervention period (Table 4 1). Fat intake decreased from 57 6 g/d to 47 6 g/d (p< 0.05). Mean total fiber intake increased from 10.70.8 g/d (9.3 g usual intake and 1.4 g from control foods) during the control period to 26.52.2 g/d (9.7 g usual intake and 16.8 g from intervention foods) du ring the intervention period (p< 0.00 0 1 ). Compliance D ata During the control period, 4 servings of study foods provided 1.6 g of fiber a day. Compliance was determined by amount of food consumed compared to food distributed. Study foods that were returned were weighed and counted after each pe riod. Study compliance was consistent throughout the study. During the

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74 control period, participants were 85% compliant with the control foods and the mean fiber intake was 1.4 0.1 g/d from these foods. Compliance during the 28 days of the intervention peri od was 73% with an estimated fiber intake of 16.8 1.5 g/d from intervention study foods (12.5 of which came from fermentable fibers ( 8.5 g inulin 4 g resistant corn dextrin ) and 4 g/d from pea hull fiber Compliance was not significantly different between control and intervention periods. Compliance was highest with cereal bars and cookies (82% and 81% respectively) and lowest for cereal (68%). Bowel Movement F requency A daily journal was used to assess bowel movement frequency and changes in patients' m edications. Figure 4 1 represents the average number of bowel movements before and with fiber intervention Daily bowel movement frequency increased from 1.3 0.2 to 1.6 0.2, p< 0.05. Participants did not report any changes in medication use. Two participants reported antibiotic use during the study. Lipid and B lood G lucose There were no changes in fasting blood glucose HDL or triglycerides between control and intervention periods. Total cholesterol was significantly lower between the two periods declining from 175 12 mg/dL to 167 11 mg/dL (p<0.05) (Table 4 2 ). LDL decreased from 100 8 mg/dL during control to 93 7 mg/dL a t intervention (p= 0.05 3). Total cholesterol/HDL ratio declined significantly from 4. 0 0.3 during control to 3. 7 0. 2 during interv ention (p<0.05).

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75 Total Cholesterol Dietary Fat and Fiber C orrelations Total Cholesterol correlated with total fat intake during control (r=0.62, p<0.05) and intervention (r=0.6, p<0.05) but not total fiber during these two periods. BUN, Serum C reatinine a nd eGFR All pair wise comparisons between each day for BUN, serum creatinine, and eGFR are shown in (Table 4 2 ). BUN decreased by 10.5 % from 3 8 6 mg/dL during control to 34 5 mg/dL during intervention period ( p= 0.05 8 ) Serum creatinine decreased by 8 % from 2.40 0.29 mg/dL during control to 2.20 0.26 mg/dL ( p<0.0 0 5 ) eGFR was significantly higher increasing by 10 % from 30 3 mL/min/1.73m 2 during control to 33 4 mL/min/1.73m 2 during intervention ( p<0.0 0 5 ) Qualitative R esults SNAQ Participants r eported an average score of 15 1 points during control SNAQ significant risk of at least 5 % weight loss within six months. There were no significant changes in the overall score mean during intervention (14 1) Epworth Sleepiness Scale (ESS) M ean scores for the sl eepiness scale decreased from 10 1 during control to 8 1 during intervention (p <0.05). T here were five participants with score s of 10 or higher, 33% of the sample during control compared to three p articipants during intervention or 20% of the sample The scale score range of 10 12

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76 indicat e s boarderline risk of day time sleep propensity while a score range of 12 24 indicat e s an abnormal tende n cy. GSRS Mean GSRS score s did not change between the two periods with a mean of 23 1 during control compared to a score of 22 1 during intervention There were no differences in the sub scales for each of the syndromes. Health Related Quality of Life There were no changes in KDQOL 36 overall mean score. In add ition, there were no significant differences in the Symptom/Problem List Effects of Kidney Disease and Burden of Kidney Disease subscales mean scores between the periods However, Physical Component Summary subscale mean score was signific antly higher in creasing from 3 1 2 during control to 35 3 during intervention (p<0.05). Mean score for the Mental Component Summary subscale decreased significantly from 53 2 during control to 48 2 during intervention ( p< 0.05 ) Table 4 2

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77 Table 4 1. Energy, fiber, and macronutrients intakes Control Period Intervention Period Energy Kcal 166571 157692 Carbohydrate g 21410 22713 Total Fiber g Usual Fiber Intake g Control Foods Fiber g Intervention Foods Fiber g 10.70.8 9.3 1.4 26.52.2 a 9.7 16.8 Protein g 597 566 Fat g 576 476 b Values are means SE, n =12.Three participants did not complete/provide the daily records. a s ignificance indicated at p<0. 0 001. b s ignificance indicated at p<0.05.

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78 Table 4 2. Participants weight, clinical markers, qual ity of life and symptoms scores Control Period (no fiber added) Intervention Period with added fiber P value Weight (kg) Clinical Markers (reference range) 89 6 906 NS BUN mg/dL (7 25 mg/dL) 38 6 34 5 P=0.058 Serum Creatinine mg/dL (0.60 1.30 mg/dL) 2.40 0.29 2.20 0.26 P<0.005 eGFR mL/min/1.73m 2 ( 60 mL/min/1.73m 2 ) 30 3 33 4 P<0.005 Glucose mg/dL (65 106 mg/dL) 13629 14625 NS LDL mg/dL (<130 mg/dL) 100 8 93 7 P=0.053 HDL mg/dL (40 50 mg/dL) 47 4 47 4 NS Total CHOL mg/dL (0 200 mg/dL) 17512 16711 P=0.015 TG mg/dL (30 150 mg/dL) 16521 15421 NS CHOL/HDL Ratio 4.00.3 3.70.2 P=0.02 KDQOL Symptom/Problem List (17 28) 783 803 NS Effects of Kidney Disease (29 36) 844 873 NS Burden of Kidney Disease (13 16) 676 736 NS Physical Component Summary (1 12) 312 353 P=0.02 Mental Component Summary (1 12) 532 482 P=0.01 Overall Mean Scores 633 643 NS Symptoms SNAQ 141 141 NS GSRS 231 221 NS ESS 101 81 P=0.04 Data are mean SE. Means are compared using paired t test with significance set at p<0.05, n=15. Means are reported for n =13.

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79 Figure 4 1. Average bowel movement per day Data presented as mean SE, n=12, p < 0.05. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Control Period (1.3 0.2) Treatment Period (1.6 0.2) Stools per day Fiber Intake g/d

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80 CHAPTER 5 RESULTS STUDY 2 Demographic Characteristics Thirteen participants were enrolled in Renal Study 2 with a mean age of 65 3 years (meanS E ). Seven females and six males completed the study Most participants had hypertension ( 12, 92%), while only 3 ( 23% ) had type 2 diabetes There was no changes in body weight (Table 5 1). Nutrient and Fiber Intake Mean fiber intake increased from 16.61.7 (15.6 g from usual intake and 1 g from control muffins) to 26.52.4 g/d (17.3 g from background diet and 9.2 g from intervention muffins) (p<0.0001) in the pea hull fiber period and 34.52.2 g/d (14.9 g from background diet, 9.2 g from intervention muffins and 10.4 g from inulin/FOS supplement) during the pea hull and in ulin fiber period compared to control (p<0.0001) and pea hull fiber period (p=0.003) (Table 5 2). No changes in energy, fat, protein, or carbohydrate intakes were observed. Compliance Compliance was 97% for muffins during control period. For intervention muffins, compliance was 92% providing 9.20.2 g/day from pea hull fiber during the intervention periods. Compliance was 91% for sucrose supplement during the first six weeks of the study and before inulin intervention. Inulin compliant was at 84% (57% 10 0%) or 11.30.6 g/day (7.7 g/d 13.5 g/d). Reported Sym p toms There were no significant side effects reported prior to inulin supplement introduction. Upon supplement introduction, participants complained of flatulence (mild

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81 n=5) moderate (n=1) severe (n=2 ), feeling bloated (n=5), frequent bowel movement, watery stool or lose stool (n=3), rumbling (n=1), nausea (n=1). Three participants did not report any symptoms throughout the study. After being advised to divide the supplement in to smaller portions durin g the day, symptoms subsided or improved significantly (n=8). Two participants reduced their supplement intake by 25 to 50% and reported improved symptoms. Other complaints were related to taste and inability to dissolve inulin in cold beverages. No advers e events were reported during the study. Bowel Movement Frequency A 5 day bowel movement frequency journal was used during first week of control period (control muffin and sucrose), last week of the pea hull fiber period and last week of the pea hull and inulin fiber period Mean bowel movement frequency increased from 1. 40.2 to 1.90.3 (n=13, p=0.026) during pea hull fiber period and remained significantly higher during the pea hull and inulin fiber period (1.9 0.3 p =0.044 Figure 5 1). BUN, Ammonia S erum Creatinine, Cystatin C CRP and eGFR BUN, ammonia, creatinine cystatin C, CRP, and eGFR were not significantly different between periods (Table 5 3). p Cresol The mean difference for total plasma p cresol between control and post intervention measure was 20% changing from 7.25 1.74 mg/L (LN 1.73) to 5.821.72 ( N 1.27) mg/L (significance based on natural log mean difference, p<0.05) Table 5 3 Percentage change averaged 24% (+54% to 83%, median 49%) Figure 5 2 When excluding non compl iant patients (defined by < 70% consumption of inulin), mean difference for total plasma p cresol between control and post intervention measure was

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82 37% (n=10) changing from 6.71 1.98 (LN 1.63) mg/L to 4.221.16 (1.04) mg/L (significance based on natural log mean difference, p<0.05). Figure 5 2. Results are GC MS output adjusted for method of extraction by adding 10% to the GC MS output to account for the difference between the 90% efficiency of the extraction method and the absolute total in the blood (114) KDQOL Higher KDQOL 36 scores indicate better results. Overall mean score for KDQOL 36 icantly different between periods. There were no changes in the Symptom/Problem List, Effects of Kidney Disease and Mental Component Summary subscale scores between periods However, Burden of Kidney Disease subscale mean score increased from 65 8 at baseline to 77 7 (p= 0.014 ) during the pea hull fiber period ( Table 5 3 ). Mean score for the Physical Component Summary subscale increased from 37 3 at baseline to 41 3 during the pea hull fiber period (p=0.046 ) ( Table 5 3). Using the Symptom/Problem List (Questions 17 27) of the KDQOL 36 to assess a host of uremic symptoms. During the pea hull and inulin fiber period, participants reported a scores of 8 6 9 for the dry skin compared to control 6911 (P=0.04). During the pea hull and inulin fiber period participants also reported a score of 83 6 for the numbness in hand or feet compared to control 65 9 (P=0.02 ) (Table 5 4). Higher scores indicate that participants were less bothered by these reported symptoms. Responses to how bothered b y other symptoms did not differ between periods

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83 Table 5 Category Description Gender (M/F) 6/7 (47%/53%) CKD by stage Stage 3 n=10 Stage 4 n=1 Stage 5 n=2 Age y 65 3 Race (White/Black/Other) 7/5/1 (54%/38%/8%) Pre weight kg 86 4 Post weight kg 86 4 Type 2 diabetes 3 (23%) Hypertension 12 (92%) Data presented as percentage and mean SE, n=13. Table 5 2 Macronutrients, energy and fiber intakes compared between each period Control Pea hull fiber period Pea hull and inulin fiber period Calories Kcal 1743 123 1733 145 1523 122 Carbohydrate g 225 19 213 20 192 20 Protein g 71 7 68 60 66 6 Fat g 65 6 69 6 55 5 Total Fiber g Background Fiber Intake g Control Muffins Fiber g Intervention Muffins Fiber g Inulin/FOS g 16.6 1.7 15.6 1 26.5 2.4 a b 17.3 9.2 34.5 2.2 c 14.9 9.2 10.4 Macronutrients, fiber and energy intakes are compared between each period and are reported as mean SE, n=13. ANOVA model p value < 0.05 for total fiber. a significantly higher than control (p<0.0001). b significantly lower than the pea hull and inulin fiber period (p=0.003). c significantly higher than the control (p<0.0001).

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84 Table 5 3 inical markers and quality of life scores Control Pea hull fiber period Pea hull and inulin fiber period P value Clinical Markers (reference ranges) BUN mg/dL (7 25 mg/dL) 304 304 294 NS Ammonia mol/L 434 433 505 NS Cystatin C mg/L (0.5 1.0 mg/L) 1.610.24 1.680.26 1.620.23 NS Creatinine mg/dL (0.5 1.52 mg/dL) 2.140.38 2.170.39 2.150.40 NS CRP mg/dL (<0.80 mg/dL) 0.330.13 0.390.12 0.450.21 NS Total plasma p cresol mg/L 7.251.74 -5.821.72 P=0.039 b Kidney Function Estimating Equations eGFR mL/min/1.73 m 2 ( creatinine) 374 364 374 NS eGFR mL/min/1.73 m 2 (creatinine cystatin C) 435 435 435 NS eGFR mL/min/1.73 m 2 (cystatin C) 516 507 506 NS KDQOL Symptom/problem list (17 28) 775 804 813 NS Effects of Kidney Disease (29 36) 815 815 865 NS Burden of Kidney Disease (13 16) 658 777 727 P=0.014 a Physical Component Summary (1 12) 373 413 403 P=0.046 a Mental Component Summary (1 12) 543 552 533 NS Overall Mean Score 634 673 663 NS Overall ANOVA model p value > 0.05 (NS), values are mean SE, n=13 a P value i s for paired t test comparisons between control and pea hull fiber period. b Significance is based on transformed mean for total plasma p cresol from control period ( LN 1.73) 7.25 1.74 mg/L to pea hull and inulin fiber period (LN 1.27) 5.821.72 mg/L n=12.

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85 Table 5 4 Individual responses for uremic symptoms listed in the Symptom/Problem List subscale of the KDQOL Control Pea hull fiber period Pea hull and inulin fiber period P value Soreness in your muscle 658 695 676 NS Chest pain 885 943 906 NS Cramps 6310 679 639 NS Itchy skin 816 738 797 NS Dry Skin 6911 6911 b 886 P= 0.04 a Shortness of breath 796 855 837 NS Faintness or dizziness 856 818 856 NS Lack of appetite 944 982 885 NS Washed out or drained 797 737 796 NS Numbness hand/feet 659 798 836 P =0.02 a Nausea or upset stomach 837 885 816 NS Overall ANOVA model p value > 0.05 (NS) values are mean SE, n=13 a P value is for paired t test comparisons between control and pea hull and inulin fiber period. b one value is missing, n =12.

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86 Figure 5 1 Average bowel movement frequencies Bars are re ported as meansSE n=13 Overall ANOVA p value > 0.05 (NS). P aired t test is used to compare means a p =0.026 compared to control. b p =0.04 4 compared to control. Figure 5 2. Total p cresol comparisons before and after intervention. Data presented as mean SE (natural log mean) a 7.2 1.74 mg/L (LN 1.73) to 5.82 1.71 (LN 1.27) (p< 0.05) n=12, p ercentage change averaged 24% (54% to 83%, median 49%). b 6.71 1.98 (LN 1.63) mg/L to 4.22 1.16 (1.04) m g/L (p<0.05) n=10, p ercentage change averaged 35% (24% 83%, median 62%). 0 0.5 1 1.5 2 2.5 Fiber Intake g/d Stools per day Control Pea hull fiber Pea hull and inulin 0 1 2 3 4 5 6 7 8 9 1 2 3 4 Total Plasma P cresol mg/L All participants Participants with > 70% Pre: .......... Post: _____ a b a b

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87 CHAPTER 6 DISCUSSION AND CONCL USIONS There are limited studies of patients diagnosed with CKD showing changes in BUN after supplementing the diet of these patients with various fiber sources (2, 7, 50, 51) These studies had been carried out in patients with s tage 5 CKD only, both HD and not on HD Further, i nvestigators have not assessed the impact of fiber on symptoms of uremia or the potent ial impact on quality of life. In addition, these studies have no t demonstrated cha nges in serum creatinine and thus, corr esponding eGFR. To address some of these limitations, and to determine whether consumption of added fiber can impact uremic molecules beyond BUN in patients with more moderate decline in kidney funct ion we conducted two clinical studies with insoluble and fermentable fibers. Furthermore, the objective was to assess the changes that may result from pea hull fiber (less fermentable) alone and in combination with inulin (fermentable) on these changes an d the potential impact on quality of life and a host of uremic symptoms. Quality of L i fe and S ymptoms Quality of life was assessed by utilizing the KDQOL 36 questionnaire. The subscales varies in their correlation wi 5 T he Physical Component Summary and Burden of Kidney Disease correlate with eGFR while the Mental Component Summary does not (136) In Study 1 t he Physical Component Summary increased by 4 points after intervention compared to control (P=0.02) while eGFR increased by about 10% (P=0.005). Lowrie et al. reported that for each point improvement in this scale, a 2% reduction in the rate of hospitalization and mortality was observed in HD patients (135) Improved functional health leads to

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88 improved engagement in daily activities and independence, which ultimately can improve the quality of life. In addition, potential reduction in the rate of hospitalization due to improved physical health has great f inancial savings impact. The study was short to assess any impact and the total scores for the overall changes in quality of life were not different. This is mainly because unexpectedly the Me n tal Component Summary scores were significantly lower (P=0.01) leading to unchanged overall quality of life scores. The lower Mental Component Summary scores is unlikely to be due to improved eGFR or treatment effect. Out of the fifteen participants, seven reported lower scores five of which were non responsive to tr eatment It is reported that quality of life is significantly impacted by constipation (153) improved stool frequency may thus partial ly contribute to feeling well. In Study 1 stool frequency was higher after intervention compared to control (Figure 4 1). It is unclear how much the improvement in stool frequency impacted this observed improvement in the reported improvement in physical health. In Study 2 results for all ANOVA tests were not significant and thus results are highly open to criticism when using multiple pairwise comparisons We used t test comparisons for the qualitative data and stool frequency as a way to cautiously explore the data to derive hypothesis for future studies because of lack of power to carry out ANOVA on the qualitative data P articipants reported a score of 41 3 for physical health during the pea hull fiber period (P=0.046) compared to control 37 3 (Ta ble 5 3) Since stool f requency during control was 1.4 0.2 compare d to pea hull fiber period (1.9 0.3 P=0.026) this may explain this improvement in the perceived physical health. However, although the stool frequency increased during the pea hull and i nul in fiber

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89 period (1.90.3 ) compared to control (P=0.044), the Physical Component Summary was not statistically different. The score during the pea hull and inulin fiber period was (40 3) during which participants (n=8 ) experienced undesirable effects such as flatulence and bloating which may have impacted gut health and ultimately the reported scores This is because some of the reported side effects (i.e. flatulence) impact s socialization significantly. In Study 1 stool frequency was higher along with eGF R which may lead to the observed improvement in reported scores for the physical health aspect of quality of life. If gut related function such as bowel movement s can affect perceived health this may exp lain the higher scores (better results, reduced burden) for the Burden of Kidney Disease during the pea hull fiber period (77 7, P =0.01 4 ) compared to the reported score during control (65 8). As discussed, increase flatulence and reported symptoms from fiber supplementation may have impacted the reported score during the pea hull and inulin fiber period (72 7) Bowel Movement Frequency and Fiber Intake Impact and R ecommendation s Intake of i nsoluble fibers are known to increase bulking and reduce symptoms of constipation. In stud y 1, t here was a significant improvement in bowel frequency when participants consumed an additional 4 g/d of supplemental insoluble fiber similar to previously reported finding in elderly people using similar pea hull fiber (154) along with 12.5 g/d of supplemental soluble fermentable fibers (p< 0.05) It is unlikely that the fermentable fibers were the primary contributors to such improvement but likely to be due to the known effect of insoluble fibers o n bulking and transit time. In Study 2 bowel frequency improved significantly when 10 g/d of pea hull fiber was ad ded to the diet for four weeks resulting increased total fiber intake from 16.6 g/d to 26.5 g/d The addition

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90 of 1 3.5 g/d ( average six weeks intake 11.3 g/d ) of inulin /FOS did not have any added effect on bo wel movement frequency (Figure 5 1 ) although tot al fiber intake increased si gnificantly to 34.5 g/d (Table 5 2 ) Supplementing the diet of CKD patients with insoluble fiber is likely to help with management of constipation by increasing fecal output and/or reducing transit time Accelerated transit time alone is not likely to impact protein fermentation significantly (155, 156) However, when sufficient FO S and/or inulin fibers are consumed the relative proportion of saccharolytic to proteolytic bacterial species (157, 158) is likely to explain the observed suppression of proteolytic enzymes by fermentable fibers reported previously (110, 159) Such desirable effects will impact uremic load and toxins generation significantly, which may benefit uremic patients (especially when considering that uremic patients suffer from increased protein mal digestion) However the diversity of the fermentable substrate is likely to determine the diversity of the impact on both negative and positive bacterial species and their byproducts. Constipation is a serious symptom of uremia Chronic kidney isease patients are likely to consume less than recommended fiber intakes, and intakes are likely to be even lower as disease worsens We hypothesized that patients in advanced stage s of the kidney disease are likely to consume less fiber than is recommended for healthy individuals and that intake is likely to be lower as disease progress. This was supported by our finding that total f iber intake in Study 1 during control period was 1 0.7 g/d when eGFR was 29 .6 mL/min/1.73 m 2 which is lower than the average intake for healthy individuals. All participants fill short of the IOM recommendation during the control period during which low fiber foods replaced a portion of the daily food inta ke. It is

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91 food intake, which may have negatively impacted the usual fiber intake. T otal fiber intake of the partici pants in Study 2 which had higher eGFR, was 16.6 g/d during the control period, which is similar to that of the healthy population (17 g/d) Both of these levels however fall short of the IOM recommendations (16) The higher than anticipated total fiber intake during the control pe riod for participants in Study 2 may suggest that participants were less uremic and had lower dietary re strictions than those in Study 1 These findings also provide additional framework to what an optima l nutritional therapy should target in terms of fiber composition. Younes et al. supplemented the diet of CKD participants with 40 g/d of fiber (25 g/d insoluble fiber, 15g/d soluble and fermentable fibers ) (2) Bliss et al. provided 50 g/d of soluble and fermentable fiber (51) Amounts of 40 and 50 g/d of additional functional and dietary fibers are impract ical and compliance is difficult i n the long term. Additionally, fermentable and less ferm entable fibers have distinctive effects on health parameters. These findings suggest a total supplemental added fiber of abou t 16.5 g/d, 4 g/d of which insoluble, and 12.5 g/d of fermentable fibers will provide benefits to treat constipation as well as imp act uremic molecules which may help preserve kidney function. This total of 1 6.5 g/d should provide, in addition to participants usual fiber intake a total fiber intake that is in line with the IOM recommendations (16) while providing means to increase the important fermentable and soluble fiber s that are typically low in the diet of CKD patients. Uremic S ymptoms There is a diverse characterization of uremic symptoms and no single uremic molecule is responsible for their onset. Therefore, it is not likely that a single therapy

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92 can improve all symptoms. Uremic patients suffer greatly from constipation, and rates in HD patients are as high as 64% (160) compared with 15% in the overall population (153) I mprovement in bowel movement frequency in less constipated patients such as in these two studies is likely to indic ate a greater potential in more constipated patients. Uremic symptoms include sleep disturbances, fatigue and restless leg syndrome, which may result in an increased tendency to fall asleep in unusual daytime situations. Increasing fiber intake by an addit ional 16.8 g (total 26.5 g) was helpful to reduce the ten dency to fall asleep during day time (p< 0.05). T he Epworth Sleepiness Scale score mean was significantly lower after intervention ( 81 P=0.04) compared to control (10 1 ) indicating a reduced risk of daytime sleeping. This drop in the scale score overlapped with about 10% improvement in eGFR. Higher eGFR indicates higher rate of uremic molecules removal from the blood, which can lead to reduced accumulation of molecules that can cause fatigue, restless leg syndrome both of which can affect sleep cycle, as well as those that can hamper alertness and impact the central nervous system In Study 2 sub analysis of the responses to the Symptom/Problem List (Questions 17 27) show a significant 19 point impro vement in dry skin symptom, and a significant 18 point improvement in the numbness in hand or feet during the pea hull and inulin fiber period ( Table 5 4 ) compared to control Uremia is a broad term to describe the symptoms associated with increased uremic molecules accumulation in the blood with no specific underlying molecule or mechanism to explain the se symptoms. Improvement in these two symptoms overlapped with a significant reduction in p cresol. This preliminary finding may point to a potential link between these two symptoms and p

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93 cresol Changes in total p cresol correlated inversely with changes the dry skin score (higher dry skin score means less bothered by the symptom). Impact of Higher Fiber I ntakes Total fiber intake from foods during the control period in Study 2 average d 16.6 g/d while in Study 1 participants consumed on average 10.7 g /d of total fiber during the control period (Table 4 1 ) No negative impact on energy was seen in Study 1 or 2 after fiber supplementation up to a total daily fiber intake of 34.5 g in Study 2 although absolute caloric intake appear lower with this level of intake. Participants reported bothersome symptoms of being bloated, lo o se stools, and flatulence after the introduction of inulin supplements in Study 2 The severity and frequency was highest during the first 3 weeks of the inulin period, and although some symptoms remained the severity and frequency became less bothersome to participants during the last three weeks of the inulin supplementation period. The severity and frequency of these symptoms is likely related to the fact that most participants were consuming the inulin s upplement at once. Participants were advised to separate the dosage into two or t hree portions to try and reduce symptoms occurrence which may help explain the observed improvement. These symptoms are common and are reported with dosages of 10 and 20 g/d (161) It is widely accepted that consumption of fermentable prebiotic supplements should be in smaller increments during the day to avoid most major side effects. In addition, the composition of inulin can play a role in mitigating the symptoms. The supplement used in the present study contained 90% inulin and only 10% FOS so to reduce gas production the proportion of

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94 FOS to inulin in the supplement should be increased which was shown to be effective in reducing gas production (162) Cardiovascular L ipid P anel M arkers CKD patients are at great risk of developing cardiovascular disease, and suffer from diabetes and hypertension in most cases. Low fiber intakes are linked to increased risk of cardiovascular disease, and fiber is shown to improve cardiovascular profile, and improve glycemic control However, CKD pa tients, especially with advanced stage of the disease, consume less fiber in the diet than the IOM recommendations. Increasing fiber intake in the diet of CKD patients may have significant importance to cardiovascular health. Because foods rich in fiber ar e also often rich sources of potassium, and phosphorous, two nutrients that are usually restricted in the management of late stages of CKD, increasing fiber by supplement and/or fiber fortified foods should be considered an effective way of achieving adequ ate fiber intakes and should be promoted more widely as an essential part of the Medical Nutrition Therapy for CKD. I n Study 1 participants consumed about 11 g/d of fiber during control, and the addition of fiber to the diet brought total fiber intake to levels that are typically a ssociated with better outcomes. In Study 1 with this level of intake, may have resulted in improved total cholesterol/HDL ratio (Table 4 2 ). These fi ndings warrant an empha sis on fiber recommendations for CKD patients that stress the importance of fermentable fiber for the management of disease co m orbidities. Increasing intake with inulin fiber can stimulate the proliferation of colonic bacteria, which then can lead to increase d conversion of cholesterol t o coprostanol leading to increase losses of cholesterol in the

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95 stools (163) ultimately leading to less bile acid re cycling and lower blood cholestero l levels However it i s possible that foods provided replaced the us u al intake of fat in the diet as participants reported lower total fat intake compared to control (Table 4 2 ). Uremic M olecules Lowering of uremic molecules may help protect the kidneys by reducing the uremic load and thus lessening the extent of hyperfiltration as well as lowering the uremic toxic molecules (i.e. p cresol) that can trigger oxidative damage and cause glomerular and tubular damage (5, 121) Thus, r educing the uremic load and the toxic oxidative molecules that need to be filtered by the glomerulus and the tubules may eventually lead to improved glomerulus and tubular hemodynamics and filtration. When patients consumed 26.5 g/d of total fiber (16.8 g/d from added fiber) BUN decreased by 10.5 % after intervention compared to control (P= 0.05 8 ). S erum creatinine decreased from 2.40 0.29 mg/L during control to 2.200.26 mg/dL after intervention (P<0.005). The decline in serum creatinine resulted in a significant improvement in kidney function eGFR by 10% increasing from 303 mL/min/1.73m 2 to 334 mL/min/1.73m 2 after intervention (P<0 .005). Reduction in the uremic load, represented here only by these two molecules may be explained by improved filtration of these molecules and/or increased losses from other route s. The reduction in urea can be explained by improved filtration and/or inc reased urea capture and utilization in the large intestine. It is most likely that the loweri ng of BUN was due to increased utilization by incorporation into bacterial nitrogen leading to increase nitrogen los ses in the stools. Several animal studies demon strate a

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96 decline in BUN due to increase d fecal losses (39 41, 44, 46) These animal studies show that feeding fermentable fibers increase urea entry into the colon (39, 40) which increases the nitrogen loses in t he stools (40, 44) with concurrent reduction in urinary losses (41, 44) Human studies also demonstrate that the dec rease in BUN is coupled with an increase in fecal nitrogen losses (2, 50, 51) sometimes coupled with a concurrent reduc tion in urinary output (2) load by urea and potentially several other nitrogenous molecules which may benefit the remaining nephrons Reduction in the contribution to the uremic load by s erum creatinine was also significant as serum creatinine decreased by 9.4% at the end of the study compared to baseline This reduction in serum creatinine is unlikely to be due to increased influx into the large intestine but rather due to improved filtration. The v iew that during kidney failure significant losses of serum creatinine from other route s particularly the colonic route, has not been substantiated. It is well established that no signifi cant quantity of creatinine can be recovered in the stool. In one study, only patients with serum creatinine above 6 mg/dL had an increased gut bacterial creatininase activity in the stool (86) Study 1 participants had an average serum creatinine of 2.44 mg/dL, thus it is unlikely that significant quantities of serum creatinine entered the large intestine and induced creatininase enzymes in the present study. In addition, previous human studies feeding various fermentable substrat es have failed to show changes in serum creatinine in advanced stages of the disease where serum creatinine levels are more elevated than that in the present study Therefore, it is likely that the reduction in serum creatinine observed in this study was d ue to improved

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97 filtration. It is unlikely the results are due to changes in creatinine metabolism, as, both protein intake (Table 4 1) and body weights did not change during the study. Plasma p C resol p Cresol sulfate concentrations, indirectly quantified as p cresol, are independently associated with overall mortality and are an independent predictor of CVD incident in HD patients (118, 120) p Cresol and its conjugated form p cresol sulfate can induce formation of free radicals which can induce damage to tubular and glomerular cells (5, 6, 121) There was a 20% reduction in total p cresol at the end of the study decreasing from 7.26 mg/dL to 5.82 mg/dL (p<0.05). Two plausible mechanisms that are likely to work together and may explain the observed changes i n p cresol are bulking and reduction of protein bacterial fermentation. This is because s lower colonic transit time may induce expansion of proteolytic bacterial species in the colon (26) which may lead to the increase d production and/or retention o f protein fermentation byprod ucts especially in carbohydrate de prived environment. Such delays in colonic contents passage and longer transit times correlate with higher urinary phenols excretion (164) Insoluble fiber can provide bulking, which can stimulate faster passage of colonic contents. By providing more ferm entable fibers, the availability of carbohydrate substrates to the microbiota will lead to an increased sacch a rolytic bacterial proliferation while inhibit ing proteolytic activity (3) leading to less byproducts of protein fermentation. The lack of a p cre sol blood sample during the pea hull fiber only intervention leave s the question of how much bulking alone may a ffect p et al. reported that feeding of 20 g/d of inulin without insoluble fiber resulted in a 17%

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98 reduction in p cresol sulfate levels in HD patients. High p c resol levels can induce inflammatory oxidative stress response and expression of fibrosis related genes such as (5, 6) that may induce tubulointerstitial fibrosis and glomerular sclerosis (8, 95, 97, 98) and thus reduction of this oxidative metabolite and its conjugate is likely to help lessens the progression of renal inflammation and oxidative damage It is important to consi der the dosage at w hich inulin is more effective w hen considering inulin compliance as a factor W hen comparing the mean for patients who total plasma p cresol was lower by 37% compared to baseline, which may indic ate a dose effect Framework When patients consumed on average 12.5 g/d of additional fermentable fibers in Study 1 with total fiber reaching 26.5 g/d BUN decreased by 10.5% (P=0.058) As explained earlier, this decline in BUN is l ikely to be due to the in crease utilization of urea in the large intestine (2, 40 44, 46) It is likely that the lower (normal and near normal) BUN levels are, the less likely reduction via a dietary therapy will be detectable As the mean is normal or near normal, the magnitude of change in those with higher eGFR and lower BUN remains small compared to elevated levels. The normal and near normal levels are typical of patients whose eGFR is close to 50 mL/min/1.73m 2 compared with those with more advanced decline in eGFR. Younes et al. repor ted a 23% decline in BUN from 73 mg/dL to 56 mg/dL, while Bliss et al. reported a change of 12% from 50 mg/dL to 44 mg/dL. This may explain why no changes in uremic molecules profile was seen in Study 2 (Table 5 3). Participants in Study 2 comparisons to participants in previous studies and even by comparisons to Study 1

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99 For example, in Study 1 ten participants were diagnosed with stage 4 CKD, while in Study 2 had ten participants diagnosed with stage 3, thus participants had much mor e residual nephrons and higher tubular secretion capacity. While the inclusion of a residual nephron as seen in Study 2 focusing on CKD stage 5 only may mask any potential b enefit. This is likely why Younes et al. and Bliss et al. did not observe an effect on serum creatinine after supplementing the diet of advanced CKD patients with fiber even though uremic BUN decreased significantly In these studies, patients had a signif icantly mor e advanced decline in eGFR. S erum creatinine levels are very stable even after the onset of kidney disease and until about 50% of the kidney function is lost (165) after which serum creatinine starts rising due to the loss of the adaptive mechanism and secretory tubules are overwhelmed. For example, serum creatinine for an average male patient in stage 3 is around 2 mg/dL, but for a CKD patient (stage 5), serum crea tinine can be significantly higher (i.e. serum creatinine for male 50 year old with eGFR of 8 mL/min is 8 mg/d). Hyperfiltration allows more of the uremic molecules and creatinine to be filtered thus lowering serum creatinine concentrations. In addition, t he proximal tubular secretion of creatinine maintains lower serum creatinine concentrations when the concentration is low. However, as the disease progresses the adaptive hyperfiltration response becomes inadequate to bring down serum creatinine, and the c oncentration rises significantly, the proximal tubular secretion is overwhelmed leading to an accelerated increase in serum creatinine levels. Therefore, it is unlikely that a dietary intervention provides distinguishable relief at s uch late stage of the d isease when measured by concentration of serum creatinine as the range of serum

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100 creatinine rise increase significantly This is especially important considering that unlike urea, the decline in serum creatinine is likely to be due to improvements in filtra tion capacity and thus improvement is likely to be limited depending on the remaining functional nephrons The lack of any improvement in eGFR in the se studies therefore is due to the proportion in which serum creatinine rises at later stages of the disea se in the blood and the relative concentration Serum creatinine levels for a male adult age 50 that rises from 1 mg/dL to 2 mg/dL represents a significant decline in eGFR from about 87 mL/min/1.73m 2 to 38 mL/min/1.73m 2 a decline of 49 mL/min ( 55%) Howe ver, a rising serum creatinine for the same person from 4 mg/dL to 5 mg/dL for example, changes eGFR by only 4 mL/min from 16 to 12 mL/min. Younes et al reported a non significant decline in serum creatinine after feeding (40 g/d fiber, 15 g/d of which wa s fermentable), from 4.0 mg/dl to 3.8 mg/dl, but that decrease was not statistically significant and was not enough to improve eGFR, although the absolute decline in serum creatinine was comparable to the findings presented in Study 1 Furthermore, Rampton et al. reported that fiber intervention reduced the slop of rising serum creatinine to zero after 8 weeks of fiber intervention (50) In his study, patients had a higher baseline serum creatinine levels than that reported by Younes et al. While our second study with most patients in stage 3 did not show any impact on BUN or creatinine, the previous studies with patients in stage 5 did not show any impact on creatinine. When considered in light of the findings from Study 1 in which most pati ents were diagnosed with stage 4 CKD, it is possible to suggest that patients in stage 4 maybe the ideal target population to observe significant changes

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101 Conclusions Increasing the fiber intake in the diet of CKD patients is achievable with minimum side effects using both fortified foods and supplementation. The addition of insoluble and soluble fibers to the diet had positive effects on quality of life, uremic symptoms, and blood parameters including kidney function markers. The current Medical Nutritio n Therapy to manage the underlying pathophysiology of CKD is focused on the late stages of the disease with limited recommendations regarding the impact of fiber. The recommendations regarding fiber mainly pertain to the role fiber plays in protecting agai nst CHD. Moreover, recommendations are unclear on how to obtain the maximum fiber intake without risking undesirable increases in other nutrients. As demonstrated in this preliminary work, increasing the fiber content of the diet without significantly impa cting protein, phosphorus or potassium intakes can be successfully achieved. Supplements and fortified products that are low in these nutrients and hig h in fiber are well tolerated. In addition, this approach can significantly enhance the fiber intake in this population to meet the IOM recommendations. Moreover, current medical nutrition t herapy recommendations are unclear on the benefits various fibers can provide for renal disease patients. No distinction between fermentable and non fermentable fibers has been emphasized regarding these recommendations. These studies provide preliminary frame work for future studies. The consumption of 4 to 9 g/d of insoluble fibers resulted in significant improvement in bowel movement frequency and impacted perceived p hysical health and burden of kidney disease positively. Furthermore, supplementing the diet with additional fermentable fibers resulted in several desirable benefits for stage 4 CKD patients. While no benefit

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102 was observed by adding fiber on kidney function for patients in stage 3 the r eduction of p cresol in this subset of the CKD population is an important findings. This confirms earlier results in HD but for the first time is demonstrated in patients in stage 3 of the disease. These findings pave the way to suggest that early fiber interventions may be useful to affect disease progression by target ing uremic molecules and reduci ng oxidative toxic molecules that otherwise s timulate disease progression. These findings also provide a new insight in the need to focus on interventions with stage 4 of CKD when considering fiber intervention to study the impact on eGFR. These recommendations should include 12 13 g/d of added fermentable fiber sources (inulin, FOS, and resistant starch) while providing at least 4 g/d in the form of insoluble fiber beside usual intake. Future studies should aim to clearly distinguish between classes of fiber and the potential benefits that may be gained with each class to enhanc e future recommendations while finding the maximum practical potential benefit with the minimum effective dose. Long term studies with large sample size s can provide better understanding of the extent of increasing intake of mixed fibers on changes in the uremic profile, renal function markers and quality of life and symptoms.

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103 APPENDIX A KIDNEY DISEASE QUALI TY OF LIFE

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112 APPENDIX B GASTROINTESTINAL RAT ING SCALE Subject ID: Date: Response Scale: (1) No discomfort at all. (2) Slight discomfort. (3) Mild discomfort. (4) Moderate discomfort. (5) Moderately severe discomfort. (6) Severe discomfort. (7) Very severe discomfort. GSRS items: (1) Have you been bothered by stomach ache or pain during the past week? (Stomach ache refers to all kinds of aches or pains in your stomach or belly.) (2) Have you been bothered by heartburn during the past week? (By heartburn we mean burning pain or discomfort behind the breastbone in your chest.) (3) Have you been bothered by acid reflux during the past week? (By acid reflux we mean regurgitation or flow of sour or bitter fluid into your mouth.) (4) Have you been bothered by hunger pains in the stomach or belly during the past week? (This hollow feeling in the stomach is associated with the need to eat between meals.) (5) Have you be en bothered by nausea during the past week? (By nausea we mean a feeling of wanting to be sick.) (6) Have you been bothered by rumbling in your stomach or belly during the past week? (Rumbling refers to vibrations or noise in the stomach.) (7) Has your stomach f elt bloated during the past week? (Feeling bloated refers to swelling in the stomach or belly.) (8) Have you been bothered by burping during the past week? (Burping refers to bringing up air or gas through the mouth.) (9) Have you been bothered by passing gas or flatus during the past week? (Passing gas or flatus refers to the release of air or gas from the bowel.)

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113 (10) Have you been bothered by constipating during the past week? (Constipation refers to a reduced ability to empty the bowels.) (11) Have you been bothered by diarrhea during the past week? (Diarrhea refers to frequent loose of watery stools.) (12) Have you been bothered by loose stools during the past week? (If your stools have been alternately hard and loose. This question only refers to the extent you have bee n bothered by the stool being loose.) (13) Have you been bothered by hard stools during the past week? (If your stool has been alternately hard and loose, this question only refers to the extent you have been bothered by the stools being hard.) (14) Have you been bothered by an urgent need to have a bowel movement during the past week? (This urgent need to open your bowel makes you rush to the toilet.) (15) When going to the toilet during the past week, have you had the feeling of not completely emptying your bowels? ( The feeling that after finishing a bowel movement, there is still more stool that needs to be passed.) Response Scale: (1) No discomfort at all. (2) Slight discomfort. (3) Mild discomfort. (4) Moderate discomfort. (5) Moderately severe discomfort. (6) Severe discomfort. (7) Very severe discomfort

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114 APPENDIX C SIMPLIFIED NUTRITION AL APPETITE QUESTIONNAIRE

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115 APPENDIX D EPWORTH SLEEPINESS SCALE

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116 APPENDIX E TELEPHONE SCRIPT Hello, my name is___________ with the fiber study. (Potential participant indicates they are calling about the study) G reat. I would be happy to give you more information about the study. The purpose of this study is to determine whether the providing adequate fiber to patients wit h CKD will result in improved gastrointestinal function and quality of life. If you qualify and decide to participate, you will be randomly assigned to a intervention or control group, but you will not be told which group you are in until completion of th e study. Both groups will be given cookies, cereal bars and breakfast cereal and to consume daily for a period of 6 weeks (42 days), with the intervention group receiving high fiber food. During the course of the study you would be asked to come in our c linical lab on four separate occasions to have your blood drawn and fill out questionnaires. These appointments should take no more than an hour. The questionnaires will ask questions regarding your quality of life, appetite, and gastrointestinal symptom s. Participants will also be asked to provide three stool samples during the study. Foods provided to participants provide nutrients and energy to all participants. Individuals selected for the fiber fortification group may experience improved gastroin testinal function and quality of life due to the fiber. Does this sound like something you would be interested in doing? (Responds Yes) Great, now I will read the inclusion/exclusion criteria for the study to make sure you qualify. Please wait until I fi nish reading through this list, then you can let me know if you are still interested. (Read inclusion/exclusion criteria without pausing) Does this still sound like something you would like to take part in? (Responds Yes) Great, then we can schedule an in itial appointment for you to receive more detailed information on the study and review a consent form. Is there a date and time that works best for you? (Schedule appointment and obtain best way to contact patient)

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117 Inclusion Criteria Participants Must: Be 18 years of age or older Have GFR of 50 mL/min/1.73 m 2 (stage 3,4 and 5 but who are not on dialysis) Exclusion Criteria Have you been diagnosed with acute kidney injury (AKI) Have you been diagnosed with glumerulonephritis (GN)? Are you on immunosupp ressant/steroid medications? Are you taking a probiotic supplement and refuse to discontinue it? Are you scheduled for dialysis within 3 months of study initiation? Do you have a history of liver disease? Have you been on dialysis? Have you undergone renal transplantation? Are you breastfeeding? Do you have active gastrointestinal bleeding? Have a change in medications over the past 4 weeks? part in the study. Ar e you still interested in taking part in the study?

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118 APPENDIX F DAILY DIARY

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120 APPENDIX G FOOD RECORD Please be as specific as possible when recording foods and beverages. Include types of breads (wheat, rye, etc ), preparations of foods (grilled, raw, canned, etc), any added dressings or condiments, or brand names when applicable. A meal will require more than one entry if multiple foods were eaten. Date Time Type of Meal or Snack Food Eaten (include preparati on, brand names, or specific types of bread where applicable) Amount of Food Eaten (cups, ounces, number of items, etc) Additional Notes or Comments

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121 APPENDIX H FOOD JOURNAL WORKBOO K

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126 APPENDIX I TELEPHONE SCRIPT (study coordinator calling after potential participant indicated preference to be called at specific number and during certain time) Hello, my name is___________ and I am calling from the Food science and Human Nutrition department at University of Flo rida. May I speak with (the name of participant). We were informed by your doctor office that you were interested to receive more information about a fiber study, is that correct? Participant, yes. So do you have few minutes to discuss the study? Participant, yes. G reat. I would be happy to give you more information about the study. The purpose of this study is to determine whether providing adequate fiber to patients with CKD will result in improved gastrointestinal function and quality of life, and clinical markers. If you qualify and decide to participate, you will be randomly assigned to a intervention or control group, but you will not be told which group you are in until completion of the study. Both groups will be given study foods (eg. cr ackers) and fiber powder supplement that can be added to foods and beverages to be consumed daily for a period of 12 weeks (84 days), with the intervention group receiving high fiber food. During the course of the study you would be asked to go to a Quest Diagnostic facility near you to have your blood drawn on six separate occasions. Additionally, you will be asked to come to our clinical lab at University of Florida campus in Gainesville on two separate occasions approximately twelve weeks apart to have blood drawn. These appointments should take no more than 30 minutes. Throughout the study you will be receiving questionnaires in in the mail in a pre paid return envelops. These questionnaires will be asking questions regarding your quality of life, bo wel frequency, and food intake. Foods provided to all participants will provide nutrients and energy. Individuals selected for the fiber fortification group may experience improved gastrointestinal function and quality of life due to the fiber. Does thi s sound like something you would be interested in doing? (Responds Yes) Great, now I will read the inclusion/exclusion criteria for the study to make sure you qualify. Please wait until I finish reading through this list, then you can let me know if you a re still interested. (Read inclusion/exclusion criteria without pausing) Does this still sound like something you would like to take part in? (Responds Yes) Great, then we can schedule an initial appointment for you to receive more detailed information on the study and review a consent form. Is there a date and time that works best for you? (Schedule appointment and obtain best way to contact patient)

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127 Inclusion Criteria Participants Must: Be 18 years of age or older Have GFR of 50 mL/min/1.73 m 2 (sta ges 3,4 and 5 but who are not on dialysis) Exclusion Criteria Have you been diagnosed with acute kidney injury (AKI) Have you been diagnosed with glumerulonephritis (GN)? Are you on immunosuppressant/steroid medications? Are you taking a probiotic supplement and refuse to discontinue it? Are you scheduled for dialysis within 3 months of study initiation? Do you have a history of liver disease? Have you been on dialysis? Have you undergone renal transplantation? Are you breastfeeding? Do you have active gastrointestinal bleeding? part in the study. Are you still interested in taking part in the study?

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128 APPEN DIX J PEA HULL FIBER MUFFINS FORMULATIONS NUTRITION FACTS

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130 APPENDIX K FRUITAFIT HD PRODUCT SHEET

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131 APPENDIX L BASELINE QUESTIONNAIRE Address: Phone number: D.O.B: ____________ Sex (circle one): M or F Please describe your race/ethnicity. Check all that apply. Race and Ethnicity o American Indian or Alaska Native o Black or African American o Asian o White o Native Hawaiian or other Pacific Islanders o Hispanic o Other________________ o Hispanic o Other________________ Please verify health history and medication use Medical history Are you currently or have been diagnosed with any of the following conditions? o Diabetes I o Diabetes II o Hypertension o Wasting disease o Inflammatory bowel disease o Other; please indicate: Medication History Are you currently taking any of the following medications (please indicate by circling ) o Insulin injections o Diabetes medication o Antibiotics or Trimethoprim or similar class medications (e.g. cimetidine, cefoxitin)

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132 o Hypertension medications (Antihypertensive agents, diuretics, beta blockers, or calcium channel blockers) o ACE inhibitors (e.g. Trandolapril, Benazepril (Lotensin), Captopril (Capoten) etc. o Other, please indicate; If you are currently taking an antibiotic or have ta ken antibiotics in the past 2 weeks, please indicate: When did you start? If finished, when did you finish? How long is/was your prescription for (when is/was your last day) ? Name of prescription: _____________ Dosage: ______________________ Have you had a change of medication recently? No Yes, if yes: What medication did you stop using? What medication did you start using? Diet and Supplement Use Are you currently taking any supplements or nutritional foods? Please indicate foods/beverages that are supplemental to the diet (i.e. ensure, special foods, fiber formula, vitamins, minerals etc.) Specifically, indicate any fiber or prebiotics/probiotics supplements or foods you are taking: Yes No if yes: What are you taking? (please specify typ e, name and brand if possible) How long have you been taking it for? How long do you intend to take it for? Name of supplement/nutritional food: ______________________ Dosage/amount: _________________ Are you on a special diet? (I.e. vegetarian, gluten fr ee etc.) Yes No if yes Please list:

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133 APPENDIX M FOOD RECORD 3 day Food Record Study No: Study Phone No: 352 870 9086 Tips for Completing an Accurate Food Record Complete it for three days Your food record should be for three days of intake. Include the day and date at the top of each form. The three days can be consecutive or non consecutive and should include at least one weekend day. Use a separate form Use a separate sheet for each day of the food record. Multiple sheets are included. Carry it with you Carry the food record with you during the day and document your meals and snacks soon after you eat. It is very difficult to recall what you ate days or hours later. Describe combination foods If you are eating combination foods, such as pizza with various toppings, make sure to record these ingredients. Estimate serving size Estimate the serving size to the best of your ability. Use the serving size on the food label if available. If you are uncertain, estimate using familiar objects Write down beverages Make sure to record all beverages that you consume in the food and beverage description. This als o includes no calorie drinks such as diet sodas and unsweetened ice tea. Eating out. Indicate the name of the restaurant or franchise if possible and indicate the food you ate from the menu by writing the exact menu name and amount. Approximate water intake Record your total daily estimated water intake at the bottom of each daily food record. Include other beverages (e.g. juice, soda) in the Food & Beverage Description section. Write clearly and use extra room or sheet. Use the extra sheet or the ba ck of the sheet page if you need more room. Mail back. When done please mail it back as soon as possible using the prepaid mail provided Call Us. have any question at anytime.

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134 Food Record Day # Subject No ___________ Day/Date______________ Serving Size/Food & Beverage Description Breakfast /Snack Lunch /Snack Dinner /Snack

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135 APPENDIX N BOWEL FREQUENCY QUESTIONNAIRE Date: ____________ Study #: _____________ 1. How many bowel movements did you have today? 0 1 2 3 4 5 >6 2. Did you experience diarrhea today? Yes No 3. Did you take laxative today? Yes No 4. Are you currently taking antibiotics? Yes No 5. Did your medication change? Yes No 6. Did you consume a fiber supplement today? If so, what did you take, Yes ___________________________________ No

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150 BIOGRAPHICAL SKETCH Younis Salmean ea rned his Bachelor of Science degree in n utrition from California State University Fresno in 2005. He was the recipient of a highly sought after scholarship awarded by Kuwait University. He went on to join the D epartment of Food Science and Human Nutrition at the University of Florida, where he earned his Master of Science degree in 2008. Younis subseq uently joined the doctoral program at the University of Florida and earned his Doctor of Philosophy degree in August, 2013. Younis Salmean has presented his dissertation research at conferences such as the Food & Nutrition Conference & Expo in San Diego, 2011. As well as Experimental Biology in 2010, and 2013. Additionally, Younis Salmean has published part of his research in t he Journal of Renal Nutrition.