CLINICO- DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS

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1 CLINICO- DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS By Dr. MOHANA RAO TIVANANA B.V.Sc. & A.H. I.D.No: GVM/ THESIS SUBMITTED TO THE SRI VENKATESWARA VETERINARY UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF VETERINARY SCIENCE (VETERINARY MEDICINE) IN THE FACULTY OF VETERINARY SCIENCE DEPARTMENT OF VETERINARY MEDICINE NTR COLLEGE OF VETERINARY SCIENCE, GANNAVARAM SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI NOVEMBER, 2015 i

2 CERTIFICATE Dr. MOHANA RAO TIVANANA, I.D.No: GVM/ has satisfactorily prosecuted the course of research and that the thesis entitled CLINICO- DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS submitted is the result of original research work and is of sufficiently high standard to warrant its presentation to the examination. I also certify that the thesis or part thereof has not been previously submitted by him for a degree of any University. Date: (Dr. V. VAIKUNTA RAO) Major Advisor Professor& Head Department of Veterinary Medicine NTR College of Veterinary Science Gannavaram Krishna (Dt), A.P ii

3 CERTIFICATE This is to certify that the thesis entitled CLINICO- DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS submitted in partial fulfillment of the requirements for the degree of MASTER OF VETERINARY SCIENCE for Sri Venkateswara Veterinary University, Tirupati, is a record of the bonafied research work carried out by Dr. MOHANA RAO TIVANANA, I.D.No: GVM/ under our guidance and supervision. The subject of the thesis has been approved by the Student s Advisory Committee. No part of the thesis has been submitted by the student for any other degree or diploma. The published part has been fully acknowledged. All the assistance and help received during the course of investigations have been duly acknowledged by the author of the thesis. (Dr. V. VAIKUNTA RAO) Chairman of the advisory Committee Thesis approved by the Student Advisory Committee Chairman: Dr. V. Vaikunta Rao Professor& Head Department of Veterinary Medicine N.T.R. College of Veterinary Science Gannavaram , Krishna (Dt), A.P Member: Dr. N. Lakshmi Rani Associate Professor Department of Veterinary Medicine N.T.R. College of Veterinary Science Gannavaram , Krishna (Dt), A.P Member: Dr. G. Srinivasa Rao Professor& Head Department of Veterinary pharmacology & Toxicology N.T.R. College of Veterinary Science Gannavaram , Krishna (Dt), A.P iii

4 DECLARATION I, Dr. MOHANA RAO TIVANANA, I.D.No: GVM/ hereby declare that the thesis entitled CLINICO-DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS submitted to SRI VENKATESWARA VETERINARY UNIVERSITY for the Degree of MASTER OF VETERINARY SCIENCE is a result of original research work done by me. It is further declared that the thesis or any part thereof has not been published earlier in any manner. Date: (Dr. MOHANA RAO TIVANANA) iv

5 TABLE OF CONTENTS Chapter No. Title Page No I 1 INTRODUCTION 1 II 2 REVIEW OF LITERATURE CHRONIC KIDNEY DISEASE OCCURRENCE Age Breed Gender DIAGNOSIS Clinical signs URINALYSIS Physical examination Urine specific gravity and protein Urine protein creatinine ratio (UP/C) Urinary enzymes HAEMATOLOGY Haemoglobin Packed cell volume Total erythrocyte count Total leukocyte count Differential leukocyte count SERUM BIOCHEMISTRY Serum creatinine Blood urea nitrogen (BUN) Total protein Serum albumin Serum sodium Serum potassium Serum phosphorus Serum calcium BLOOD PRESSURE RADIOGRAPHY ULTRASONOGRAPHY TREATMENT STRATAGIES ACE inhibitors 32 v

6 Conservative therapy Punarnawadi mandur Rubenal III 3 MATERIALS AND METHODS 3.1 DESIGN OF STUDY Control animals Clinical cases Selection criteria for inclusion in the treatment trials Treatment trials General considerations in the treatment 3.2 CHARACTERS UNDER STUDY EVALUATION OF PARAMETERS Clinical examination Haematology Serum biochemistry Serum creatinine Blood urea nitrogen Total protein Albumin Sodium Potassium Phosphorous Calcium URINALYSIS Physical examination ph, specific gravity and urine protein Urine protein and creatinine (UP/C) ratio Urinary enzymes NEPHROSONOGRAPHY Instrumentation Preparation and restraint of the patient Nephrosonographic technique Interpretation of Nephrosonographs STAGING OF THE DISEASED ANIMALS Classification of CKD cases vi

7 EVALUATION OF THE TREATMENT STATISTICAL ANALYSIS IV 4 RESULTS 4.1 OCCURRENCE Breed Age Gender Clinical signs Urinalysis Haematology Serum biochemistry Blood pressure Radiography Nephrosonography Classification of CKD dogs according to IRiS classification based on serum creatinine values 4.10 Survival rate of dogs with CKD in different groups during the treatment period V 5 DISCUSSION 93 VI 6 SUMMARY 102 LITERATURE CITED 106 APPENDIX vii

8 LIST OF FIGURES Plate No. Particulars 1 Breed-wise occurrence of chronic kidney disease in dogs 62 2 Age wise occurrence of chronic kidney disease in dogs 62 3 Gender wise occurrence of chronic kidney disease in dogs 64 4 UP/C ratio values in treatment groups 64 5 Values of Haemoglobin (g/dl) in treatment groups 84 6 Values of PCV (%) in treatment groups 84 7 Values of TEC (/µl) in treatment groups 85 8 Serum BUN profile in treatment groups 85 9 Serum creatinine profile in treatment groups 86 Page No. 10 Classification of CKD dogs according to International Renal Interest Society (IRiS) standards based on serum creatinine levels Survival rate between treatment groups 91 viii

9 LIST OF PLATES Plate No. Particulars 1 Bleeding oral ulcers in a dog with CKD 65 2 Pallor mucous membrane in a dog with CKD 65 3 Oral ulcers in a dog with CKD 66 4 Salivation in a dog with CKD 66 5 CKD dog with chronic anorexia and emaciation 67 6 CKD dog with chronic vomitions 67 7 Association of periodontitis and CKD in a dog 68 8 Urine collection procedure by catheterization procedure 68 9 Blood pressure recording in a dog with CKD Radiograph showing normal size kidneys in a dog with CKD Radiograph showing round kidneys in a dog with CKD 88 Page No. 12 Nephrosonogram showing indistinct corticomedullary junction Nephrosonogram showing sunken kidneys Nephrosonogram showing renal cyst 89 ix

10 LIST OF TABLES Table No. Particulars 1 Breed-wise occurrence of CKD in dogs 61 2 Age-wise occurrence of CKD in dogs 61 3 Gender wise occurrence of CKD in dogs 63 4 Clinical findings exhibited by dogs with CKD 63 5 Urinalysis in control and CKD dogs 69 6 Haematological values in control and CKD dogs 69 7 Serum biochemical profile in control and CKD dogs 70 8 Blood pressure values in control and CKD dogs 70 9 Urinalysis in treatment groups (0 day) Urinalysis in treatment groups (15 th day) Urinalysis in treatment groups (30 th day) Urinalysis in treatment groups (45 th day) Haematological findings in treatment groups (0 Day) 75 Page No. 14 Haematological findings in treatment groups (15 th Day) Haematological findings in treatment groups (30 th Day) Haematological findings in treatment groups (45 th Day) Serum biochemical profile in treatment groups (0 Day) Serum biochemical profile in treatment groups (15 th Day) Serum biochemical profile in treatment groups (30 th Day) Serum biochemical profile in treatment groups (45 th Day) Blood pressure values in treatment groups (0 Day) Blood pressure values in treatment groups (15 th Day) Blood pressure values in treatment groups (30 th Day) x

11 Blood pressure values in treatment groups (45 th Day) Radiographic findings in CKD dogs Nephrosonographic findings in CKD dogs Classification of CKD dogs according to International Renal Interest Society (IRiS) standards based on serum creatinine levels The survival rate between treatment groups xi

12 List of symbols and abbreviations % - per cent or percentage et al. - and others g/dl - grams per deciliter mg/dl - meq/l - milligram per deciliter milli equivalents per liter ± - plus or - at the rate of µg - microgram < - less than > - greater than SE - standard error viz., - namely i.e. - that is / - per ACE - Angiotensin Converting Enzyme b. wt - body weight BID - Twice in a day BP - Blood Pressure BUN - CKD - CRD - Blood Urea Nitrogen Chronic Kidney Disease Chronic Renal Disease CRF - Chronic Renal Failure DAP - Diastolic Arterial Pressure DLC - Differential Leukocyte Count EDTA - ESRD - Ethylene Diamine Tetra Acetic acid End Stage Renal Disease GFR - Glomerular Filtration Rate

13 GN - Glomerular Nephropathy Hb - Haemoglobin IRiS - International Renal Interest Society IV - Intravenous KDIGO- MHz - Kidney Disease Improving Global Outcomes Mega hertz No. - number PCV - Packed Cell Volume PO - Per os RAAS - Renin Angiotensin- Aldosteron system SAP - Systolic Arterial Pressure SCr - Serum creatinine SID - Once in a day SPSS - Statistical Package for Social Sciences TEC - Total Erythrocyte Count TLC - Total Leukocyte count UP/c - Urine protein/ creatinine ratio USG - Urine Specific Gravity

14 ACKNOWLEDGEMENT I gratefully acknowledge to my major advisor Dr. V. VAIKUNTA RAO, Professor and Head, Department of Veterinary Medicine, NTR College of Veterinary Science, Gannavaram for his guidance, valuable suggestions, constant encouragement, criticism and meticulous planning during the course of my research and I ever remain grateful to his support and personal attention in planning execution of my research and in writing every step of my thesis. I was blessed and I deem it as my privilege to work under his guidance. I am immensely pleased to place on record my profound gratitude and heartfelt thanks to Dr. N. LAKSHMI RANI, Associate Professor, Department of Veterinary Medicine, NTR College of Veterinary Science, Gannavaram for her motivation, timely cooperation, attention to minute details, involvement, affectionate encouragement, precious suggestions and support during my course work. I am very much thankful to the member of Advisory committee Dr. G. SRINIVASA RAO, Professor and Head, Department of Veterinary Pharmacology and Toxicology, NTR College of Veterinary Science, Gannavaram for his support, valuable suggestions and timely cooperation during the course of my work. I also express my sincere gratitude to Dr. K. Rajesh, Assistant Professor, Department of Veterinary Medicine, NTR College of Veterinary Science, Gannavaram for extending his valuable suggestions, unconditional cooperation, critical evaluation while planning and executing this research work. I am taking this opportunity to express my profound gratitude to Dr. Y. Chaitanya, Assistant Professor, Department of TVCC, NTR College of Veterinary Science, Gannavaram for her valuable suggestions, affectionate encouragement and support during the period of my research work. It is my pleasure and privilege to express my sincere thanks and gratitude to Dr. K. S. Sai Krishna, Assistant Professor, Department of Veterinary Medicine and Dr. V. Srinivasulu Assistant Professor, Department of TVCC for their valuable advice and constant help throughout the course of my study. My special thanks to Dr. P. Ramesh, Dr. K. Basava Reddy and Dr. Usha Sree for their cooperation for completion of my research work. xiv

15 I heartily express my sincere thanks to Dr. R. Sridhar, VAS, SIAH, Tanuku for his timely and wise counsel, generous help, unconditional cooperation, critical evaluation and encouragement during the course of study. My special thanks to Dr. T.V. Suresh, VAS, Panduru, EG Dist. for his cooperation and wise counsel during my research work. My sincere thanks to Dr. K. Krishna murthy, Dr. E. Prameela and Dr. Sandeep Reddy, VPC, Kakinada and Dr. K. Lavanya, Dr. K. Karunakar and Dr. L. Krishnaveni, V.H, Visakhapatnam for their co-operation and helping me to find and refer relevant cases for this research work. My special thanks to Dr.M. Muralidhar, Assistant Professor in Animal Genetics and Breeding for his excellence in statistical analysis of my work. I express my special thanks to Dr. CH. Sudha Rani Chowdary, Assistant Professor, Dept of TVCSC, NTR College of Veterinary Science, Gannavaram and Dr. Kalyani, Assistant Professor, Dept of Biochemistry, NTR College of Veterinary Science, Gannavaram for their passion and support extended while documenting this investigation. My heartfelt thanks to my fellow colleagues Dr.V.Madhu Babu, Dr.S. Roopakala, Vinaya, Dr. Bharghavi, Dr. Srinivas, Dr. Praveen Raj, Dr. Chaitanya Sankar, Dr. Ramesh Chandra, Dr. Sridhar, Dr. Murali and my beloved juniors Hema, Deepa, Dr. Kameswara Rao, Dr. Durga Jyothi and Vijay who created healthy environment during my course work. I can t imagine my current position without the love and support from my family. I thank my parents Sri T.Satyam and Satyavathi, for striving hard to provide good education for me. I always fall short of words and felt impossible to describe the support of my brothers Sasikumar and Vijay from my childhood. This is an opportunity to express inexpressible amiability to my friends K. Pradeep, Chaitanya, Satish, Mahesh, L.Pradeep, Praveen, Subbarayudu, Durga Prasad, Ramu, Prasanna, Srinivas, Hema, Dr. Srinivasa reddy, Nanda kishore, Chanu, Nithin and Manthru Naik for their valuable support, making every movement memorable and joyful during my course of study. xv

16 I have been fortunate enough to have a number of genuine friends who remain an infinite source of encouragement and immaculate fun during my stay at this institute. I would like thank to my beloved juniors Polinaidu, Suresh, Naveen, Nanda kishore and Vinod for their encouragement during college days. I am grateful to sub staff P. Subba Rao, B. Adinarayana and Ch. Yeshaiah of Dept of Veterinary Medicine, Verraju sub staff of Dept of TVCC and Kalyan, Suman of VH, Visakhapatnam. I would like to acknowledge all the teachers I learnt from since my childhood, I would not have been here without their guidance, blessing and support. I wish to extend my thanks to one and all who have contributed even in a small way in completion of this study. I thank College of veterinary science, korutla and my KV friends for first three years of my B.V.Sc course and for molding my personality and for building up my career in the profession. I solicit my gratitude towards N.T.R College of Veterinary Science, Gannavaram and Sri Venkateswara Veterinary University, Tirupathi for providing all facilities and financial support for conducting this research work. I finally thank to GOD almighty. Dr. Mohan Rao Tivanana. xvi

17 Name of the author ID No Title of the thesis : MOHANA RAO TIVANANA : GVM/ : CLINICO- DIAGNOSTIC AND THERAPEUTIC STUDIES ON CHRONIC KIDNEY DISEASE IN DOGS Degree to which it is Submitted Faculty Department Major advisor : Master of Veterinary Science : Faculty of Veterinary Science : Department of Veterinary Medicine, N.T.R College of Veterinary Science, Gannavaram. : Dr.V. Vaikunta Rao, Professor and Head, Department of Veterinary Medicine, N.T.R College of Veterinary Science, Gannavaram. University : Sri Venkateswara Veterinary University, Tirupati. Year : ABSTRACT The aim of the study was to study the clinical signs and haematobiochemical, radiographic and ultrasonographic changes in dogs with CKD and to assess the efficacy of the therapy adopted in stage II and III of chronic kidney disease in dogs. The study revealed higher occurrence of CKD in Spitz/ Pomeranian breeds with male predominance and in age group of years. Clinical examination of CKD cases revealed anorexia, weakness, weight loss, pallor mucous membrane, vomiting, melena, polyurea/ polydipsia, halitosis, oral ulcers, emaciation, nervous signs, diarrhoea, recumbent, congested mucous membrane and blindness. Blood pressure measurement showed significant elevation of systolic pressure in CKD dogs. Haemotological values showed the results of anemia. In serum biochemical xvii

18 profile, elevated blood urea nitrogen, creatinine, phosphorus, sodium and normal serum potassium and calcium levels were observed. However decreased total protein and albumin levels were observed in CKD dogs as compared to apparently healthy dogs. Urinalysis showed normal ph, decreased specific gravity and elevated urine protein and creatinine ratio (UP/c ratio) and urinary enzymes (ALP and GGT) excretion. The radiographic changes in dogs with CKD were normal in size and round kidneys. Nephrosonography revealed indistinct corticomedullary junction, sunken kidneys, hyperechoic cortex and renal cyst in CKD dogs. Combination of Rhubarb supplementation (Rubenal ) and conservative treatment was found to be effective in the therapeutic management of Stage II and III CKD dogs. xviii

19 CHAPTER-I INTRODUCTION Chronic kidney disease (CKD) is the most commonly recognized form of kidney disease in dogs and causes an irreversible and progressive loss of kidney function. This leads to a decreased ability to remove waste products from the body and perform homeostatic functions. It is most common in older dogs, but can occur at any age with significant morbidity and mortality. The prevalence of CKD has been estimated to range between 0.5% to 7% in dogs. Nephron damage associated with CKD is usually irreversible and the cause is often difficult to determine. Patient history, results of physical examination, urinalysis, haematology, serum biochemistry and nephrosonography provide a practical means of diagnosing CKD in dogs. Conservative medical management of CKD is designed to minimize the clinical and pathophysiological consequences of reduced kidney function. The CKD is a progressive disease with increasing incidence, having very little success rate in current conventional therapies once CKD reaches stage IV. Clinical intervention at stage II and III is best to decrease or stop further development of the disease. Even though the disease is progressive, appropriate treatment helps many dogs live comfortably for several months to years. Recent studies suggest that, dietary supplementation with specific antioxidants is an important consideration for limiting renal oxidative stress and progression of CKD (Polzin, 2011). Hence the study is undertaken with the following objectives. 1

20 2 Objectives: 1) To study the clinical signs and haemato-biochemical changes in dogs with chronic kidney disease. 2) To study the radiographic and ultrasonographic changes of kidney in dogs with chronic kidney disease. 3) To assess the efficacy of the therapy adopted in stage II and III of chronic kidney disease in dogs.

21 CHAPTER-II REVIEW OF LITERATURE 2.1 CHRONIC KIDNEY DISEASE Keeping in view of the fact that the terms kidney disease, kidney insufficiency, kidney failure, azotemia and uremia can use synonymously, as it was brought out by many descriptors in their documents and even they have not been defined kidney insufficiency and kidney failure uniformly and adequately; therefore these terms have been replaced by a chronic kidney disease (CKD) staging system by the International Renal Interest Society (IRiS). It is recommended that the term kidney be used in preference to the term renal, because clients understand the kidney. So the present review of literature is mainly done by taking the above fact into consideration or by considering both CKD and CRF as synonyms. Chronic kidney disease (CKD) is the most commonly recognized form of kidney disease in dogs and cats. It is defined as any structural and/or functional abnormality of one or both kidneys that has been continuously present for three months or longer (Polzin, 2011). 3

22 4 2.2 OCCURRENCE Age David et al. (1995) stated that chronic renal failure is the most common form of renal failure in dogs, although commonly considered as the disease of older animals, CRF occurs with varying frequency in dogs of all ages. Rubin (1997) reported that CRF occurs in dogs and cats of all ages, commonly considered a disease of older animals, and the incidence increasing with age. Shelly et al. (1997) in a study on dogs with renal failure recorded that the age of acute renal failure ranged from 11 months to 15 years. Age of CRF patients ranged from 6 months to 16 years. Squires et al. (1997) stated that the incidence of chronic renal disease is high in middle aged to older dogs. Polzin et al. (2000) following a study on chronic renal failure in dogs, observed that the incidence of CRF in adult dogs was 20 per cent and 45 per cent in above 10 years of age group. Watson (2001) opined that prevalence of kidney disease has been estimated to range between 0.5% and 7 % in dogs and between 1.6% and 20% in cats. Muralikrishna (2003) observed the incidence of CRF in adult dogs as 17 per cent and 38.1 per cent in the above 13 years of age dogs.

23 5 Adams (2004) reported that the mean age group of dogs affected with CRF was seven years years. Mrudula et al. (2005) reported CRF in 60 dogs with the mean age of 7.8 ± Brown (2007) stated that the incidence of chronic kidney disease in the general population of dogs and cats was 0.5% to 1.5% and 1 to 3 % respectively. Girishkumar et al. (2011) reported that chronic renal failure was more common in dogs aged above six years Breed Osborne (1995) reported familial and congenital renal disease in more than thirty breeds of dogs like German shepherd, Doberman, Great Dane, Lhasa apso and Boxer. Muralikrishna (2003) reported higher incidence of chronic renal failure in German shepherd (33.33%) followed by Pomeranians (19.05%), Mongrels (14.29%), Boxer ( 9.50%), Labrador Retriever (9.52%), Dachshund ( 4.76%) and Lhasa apso (4.76%) respectively. Adams (2004) opined that incidence of CRF was common in German shepherd, Doberman pinscher, Lhasa apso, English cocker spanial, Golden retriever, Rottweiler and Samoyed breeds of dogs. Mrudala et al. (2005) stated a higher incidence of CRF in German shepherd (26.66%) and Spitz (21.66%) breeds.

24 6 Eriksen and Grondalen (2008) reported CRF in two litters of soft coated Wheaten Terriers, having the same parents. Kavitha (2010) in one study conducted at Veterinary College Hospital, Bangalore stated that occurrence of renal failure in different breeds of dogs ranged from 3.22 to per cent. The incidence was highest in the GSD and Non- descript (22.58%) followed by Labrador Retriever (19.35%), Pomeranian (19.35), Dachshund (6.45%), Doberman Pinscher (6.45%) and Dalmatian (3.2%) Gender Dougall et al. (1986) following a study on renal diseases in dogs suggested that there was lack of evidence of any predisposition of renal disease related to gender or breed. Mary (1992) opined that there is no age, breed or gender predilection for renal failure. Acute renal failure occurs more frequently than is generally recognized and is often misdiagnosed as chronic renal failure. Squires et al. (1997) reported that dogs of both gender were susceptible to chronic renal failure. Muralikrishna (2003) observed that the incidence of chronic renal failure was higher in male (66.67%) than female (33.33%) dogs. Adams (2004) stated that there was no gender predilection in chronic renal failure in dogs. Mrudala et al. (2005) stated that the incidence of renal insufficiency was more in males (60%) when compared to females (40%).

25 7 Girishkumar et al. (2011) reported that higher incidence of chronic renal failure in males could be attributed to the fact that males formed the higher percentage of canine population. 2.3 DIAGNOSIS Recognizing CKD requires consideration of evidence from multiple sources, including renal function tests, serum electrolyte concentration and acid base status, urinalysis and renal imaging studies. The CKD is usually suspected on the basis of reduced kidney function or markers of kidney disease. Markers of kidney disease may be recognized from haematological or serum biochemical evaluations, urinalysis, imaging or pathological studies. Findings suggestive of kidney disease may also be found by physical examination or from the medical history (e.g. Changes in kidney size or shape, changes in urine volume). Brown (2007) reported that the diagnostic tests that are routinely used to establish a diagnosis in a patient with kidney disease includs urinalysis, urine culture, urine protein to creatinine ratio, serum biochemical panel, complete blood count and sequential evaluation of serum creatinine concentration and these are useful in assessing the rate of change of kidney function over time Clinical signs Lucre et al. (1980) studied chronic renal failure in dogs and recorded the signs like anorexia, unthriftiness, stunting, lethargy, polydipsia, weight loss and occasional vomiting. Chew et al. (1983) documented the signs of CRF as anorexia, lethargy, polydipsia, polyuria, weight loss, vomiting and dehydration in dogs.

26 8 Di Bartola et al. (1983) observed the clinical signs like polydipsia, polyuria, anorexia, lethargy, vomiting and bone deformities in dogs affected with chronic renal failure. Robbinson et al. (1989) studied chronic renal failure in Bull Terrier dogs and observed signs like lethargy, anorexia, polydipsia, polyuria and weight loss. Brown et al. (1990) recorded signs like vomitings, polydipsia, polyuria and weight loss in dogs affected with renal diseases. Hoppe et al. (1990) stated the common clinical signs of canine kidney disease to be depression, polydipsia and vomiting. Polzin et al. (2000) stated up to 67% loss of renal function occurs as clinically asymptomatic condition and that with a per cent of loss of renal function polyuria and polydipsia may be manifested and they further stated that loss of per cent of renal function would be manifested as vomiting, diarrhea, apathy and when less than ten per cent of renal function is present. It was accompanied with the signs of uremic encephalopathy, indicating terminal stages of illness. Raskin (2001) mentioned that uremic encephalopathy may accompany acute or chronic renal failure. Muralikrishna (2003) observed symptoms like vomiting, melena and oral ulcers in dogs suffering from renal failure. Pugliese et al. (2005) stated that with a loss of 67-75% of the filtration rate, severe polydipsia and polyuria occurred, when renal failure increased (75-90%) the accumulation of blood nitrogen catabolic products determined the occurrence of systemic signs such as anorexia, weight loss and specific signs such as vomiting and

27 9 diarrhea. When the residual renal function was found to be less than 10 per cent uremia was present associated with the neurological signs i.e. uremic encephalopathy that indicated terminal stage of illness. Reine and Langston (2005) opined that the presence of isosthenuria might be suggestive of primary renal failure with renal disease. Isosthenuria occurs with greater than 66 per cent of damage to the kidney where as azotemia occurs after more than 75 per cent of renal damage has been sustained. Robertson and Seguin (2006) opined that many cases of chronic renal disease are asymptomatic (other than PU/PD) until dehydration leads to decompression, leading to more acute history from the owner perspective. Eriksen and Grondalen (2008) reported that the clinical signs of canine CRF were inappetence, weight loss, vomiting and depression and in two cases polydipsia and polyuria. Lucre et al. (2008) observed chronic renal failure in thirteen dogs with advanced kidney disease. Signs reported were anorexia, lethargy and weight loss. Mc Grotty (2008) reported that the dogs with CRF had polyuria, polydipsia, anorexia, lethargy, weight loss, vomiting, oral ulcers, halitosis and acute blindness as common clinical signs. Ross (2008) stated that a history of signs such as polyuria, polydypsia, weight loss, selective appetite, deteriorating hair coat occurring over several months is a strong evidence of chronic kidney disease. Grauer (2009) stated that decreased production of erythropoietin contributes to the non-regenerative anemia of chronic kidney disease and decreased metabolism

28 10 and excretion of parathyroid hormone and gastrin contribute to osteodystrophy and gastritis respectively. Pradhan and Roy (2012) in their study on CRF in dogs found that which are in advanced stage of renal disease showed nervous signs like ataxia, tremors, in coordination, seizures, syncope and progressive deterioration of health. Shaw and Ihle (2013) noticed polyuria, polydipsia, anorexia, lethargy, weight loss, vomiting, oral ulcers and dehydration as symptoms of chronic renal failure in dogs URINALYSIS Physical examination Macroscopic examination of urine is an integral part of urinalysis, and blood and bile pigments are a common cause of abnormal coloration. The dipstick test for ph, blood, glucose, ketone bodies and bilirubin in urine can be used. Microscopic examination of urine sediment must be interpreted in combination with the physical and chemical composition of urine, but excessive number of cells, casts, crystals and bacteria may provide evidence of disease (Brobst, 1989). Albasan et al. (2003) reported that urine sample should be analyzed within sixty minutes of collection to minimize temperature and time dependent effects on in vitro crystal formation. Presence of crystals observed in stored samples should be validated by reevaluation of fresh urine. Reine and Langston (2005) stated that urinalysis assessment includes evaluation of physical characteristics, biochemical parameters and microscopic sediment evaluation.

29 Urine specific gravity and protein Larkin et al. (1972) recorded proteinuria with low urine specific gravity (1.018) in a dog with nephritic syndrome. English (1973) reported that measurement of urine specific gravity and osmolality are used to determine the kidney ability to concentrate urine. The ability to concentrate urine in the chronically diseased kidney depends upon adequate secretion of anti diuretic hormone and the presence of enough nephrons. Schepper et al. (1974) observed that proteinuria and low urine specific gravity in a dog with nephritic syndrome and glomerulonephritis. Lucre et al. (1980) stated that low urine specific gravity (USG) in a dogs suffering with CRF as against a normal value ranging between Robbinson et al. (1989) documented proteinuria and low urine specific gravity ranging from 1.01 to in dogs affected with CRF. Booth (1990) observed proteinuria and low urine specific gravity of in a dog with nephropathy. Kowalwich and Hawkins (1992) stated that isosthenuria (USG ) in a bitch with chronic renal failure. Carl et al. (1995) reported that dogs with primary renal failure, azotemia usually follows loss of ability to concentrate urine to a specific gravity of at least Osborne et al. (1995) opined that the specific gravity cannot be taken as early indicator of renal damage as the kidneys have a tremendous reserve capacity.

30 12 Impairment of the kidneys ability to concentrate or dilute urine may not be detected until at least two third of the total population of nephrons has been damaged. Rubin (1997) stated that isosthenuria (USG to 1.015) in a dog suffering with chronic renal failure. Borku et al. (2000) stated low USG in dogs exhibiting signs of CRF. Haller (2002) opined that determination of USG was very important in assessment of renal disease. It should always be measured before any treatment is initiated because fluids, glucocorticoids or diuretics may result in artificially diluted urine. Hyposthenuric urine ( ) indicated active dilution, isosthenuria ( ) indicated unchanged excretion and hypersthenuric urine indicates active concentration of the glomerular filtrate. Sato et al. (2002) observed USG in a range of in all dogs suffering with CRF. Lees (2004) concluded that detection and treatment of animals with persistent renal proteinuria, which is one of many possible manifestations of CKD in dogs and cats that are important to evaluate and treat appropriately. Camacho et al. (2005) stated that USG below threshold of in all dogs exhibiting azotemia of renal origin. Reine and Langston (2005) reported that the presence of isosthenuria (USG to 1.012) might be suggestive of primary renal failure with renal disease. Isosthenuria occurs with greater than 66 per cent damage to the kidneys where as azotemia does not occur until more than 75 per cent damage has been sustained.

31 13 Shaw and Ihle (2013) suggested that the urine was frequently isosthenuric (USG < 1.030) and USG ranged from to in patients with chronic renal failure Urine protein creatinine ratio (UP/C) Schwedes and Grunbaum (1994) reported that UPC was of prognostic value in chronic renal failure dogs. Finco et al. (1997) suggested that the progressive increase in UPC might be a marker of an accelerated rate of renal injury. Tanyel (2000) stated that significantly higher UPC (1.71 ± 0.19) occurred in dogs exhibiting signs of renal dysfunction than the healthy dogs. Jacob et al. (2005) reported that initial high UPC determination i.e. more than 1.0 in dogs with CRF was associated with greater risk of developing uremic crisis and death, compared to dogs with UPC less than 1.0 and thus concluded that UPC determination in dogs with CRF could be prognostic value. Welles et al. (2006) reported that the Multistix PRO, with manual calculation of UP/UC ratio may be a good alternative for the diagnosis of clinically significant proteinuria in dogs, but not in cats. Dipstick creatinine should be considered as an estimate. Yathiraj (2006) concluded that UPC determination was one of the useful adjunctive procedures to evaluate the glomerular function in case of patients with renal failure.

32 14 Buranakarl et al. (2007) stated that UPC was a good indicator of renal disease regarldless of disease progression. Wehner et al. (2008) observed that proteinuria and systemic hypertension are well recognized risk factors in CRF. Some of the dogs with CRF were proteinuric almost all were hypertensive. Neoplasia was commonly associated with proteinuria in the dogs with a normal ECPC. Grauer (2009) reported that protenuria in dogs could indicate the presence of CKD before onset of azotemia or the presence of more severe CKD after the onset of azotemia Urinary enzymes Price (1982) concluded that urinary enzymes, viz., N- acetyl B- glucosamidase (NAG), alanine amino peptidase (AAP), lactate dehydrogenase (LDH), lysozyme, kallikrein, carbolic anhydrase C anylsulphatase. Alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT) provided very sensitive indicators of renal damage in renal disease. Dash (1987) reported that certain kidney specific enzymes, viz. NAG, LDH and GGT were increased in urine during renal disease. Farshid et al. (1993) calculated the mean urinary gamma- glutamyl transpeptidase (GGT) level as ± U/L in experimental tubular damage in dogs and thus emphasized importance of urinary GGT estimation in the diagnosis of renal damage in dogs. Uechi et al. (1994 a ) stated that urinary N- acetyi-b-d- glucosaminidase (SAG), gamma- glutamyt transpeptidase (Gamma- GTP) and glycyl propyl

33 15 dipeptidyl amino peptidase (GP-DAP) index were significantly increased in renal failure dogs. According to Uechi et al. (1994 b ) in a study on circadian variation of urinary enzymes in dogs concluded that enzyme activity in non periodically collected urine was diagnostically useful. Aguiar Heinemann et al. (1997) observed an increase in urinary gamma glutamyl transpeptidase (GGT) activity in dogs with induced nephrotoxicity and suggested that urinary GGT activity might be a sensitive indicator of renal damage. Finco (1997) reported that appearance of enzymes of renal origin in urine useful for detection of renal injury. Tanyel (2000) documented a marked increase in urinary gamma- glutamyl transpeptidase (GGT) in dogs exhibiting signs of renal dysfunction. Heiene et al. (2001) stated that high urinary enzyme, viz ALP, GGT, NAG values often reflected extensive lesions in renal proximal tubular cells and sometimes reduced glomerular filtration rate. Sato et al. (2002) observed that the NAG index in dogs with chronic renal disease as 15.7 to USG and that the urinary NAG index increase before elevation of BUN and creatinine. Therefore, they suggested use of NAG activity as a sensitive indicator of the progression of renal disease. Braun and Lefebvre (2005) concluded that in case of kidney damage, the enzyme activity in the urine increases, before any observable alteration in kidney function.

34 16 Mrudula et al. (2005) stated that the mean ALP and GGT in renal failure in dogs at ± 2.05 and ± 2.16 against a normal level of 3.67 ± 1.27 and 2.5 ± 0.36 U/m mol creatinine respectively. Shilpa and Yathiraj (2006) opined that urinary NAG values above U/g of creatinine could be considered as indicators of renal failure. Yathiraj (2006) documented elevated activity of renal enzymes, viz. NAG, LDH, ALP and GGT as a sensitive indicator of renal damage. Haque (2007) reported that urinary GGT was an early and persistent indicator of tubular damage HAEMATOLOGY Haemoglobin CRF. Osborne (1970) stated that anemia is a common clinical finding in dogs with Eschbach and Adamson (1991) reported that a major factor related to anemia appeared to be decreased erythropoietin production by CRF kidneys. King et al. (1992) reported non regenerative normochromic normocytic anemia in 70.6 per cent of dogs with chronic renal failure and were of opinion that many factors including decreased erythropoietin, haemolysis and blood loss might influence the development of anemia in CRF. Lulich et al. (1992) stated that erythropoietin is produced primarily in the peritubular interstitial cells of the inner renal cortex and outer medulla in the kidney,

35 17 and as kidney disease progresses, there are fewer erythropoietin- producing cells within the kidneys. Erslev and Besarab (1995) in their study on renal failure observed that uremia had been associated with decreased RBC survival, but the pathophysiology of that was unclear and most likely multifactorial. CRF. Pecherau et al. (1997) reported anemia condition in 37 dogs suffering with Cowgill et al. (1998) stated that the severity and progression of the anemia and clinical signs correlate with the degree of CRF in dogs. Mrudula et al. (2005) observed that 90 per cent dogs showed anemia with chronic renal failure. Robertson and Seguin (2006) opined that chronic renal failure could be associated with lymphopenia, which reflected the effects of endogenous glucocorticoids or stress of chronic disease. Rusenov et al. (2009) observed severe erythropenia and hypochromia in dogs with CRF and stated that the main cause for hypo proliferative anemia in animals with CRF was erythropoietin deficiency. Chalhoub et al. (2011) stated that acute and chronic inflammation contributed to anemia of renal disease by the production of inflammatory cytokines and substances such as hepcidin that will decrease erythropoietin function, red cell survival and available iron.

36 18 Bradea et al. (2013) concluded that complete blood count in CKD provided useful information about the progress of the disease as well as appreciation of type of anemia offering additional information for therapeutic protocol adjustment for amending induced hematological consequences. Nonregenerative anemia represented a common finding in chronic kidney disease pathology in dogs Packed cell volume Coles (1986) reported mean PCV in normal dogs as 45%. per cent. King et al. (1992) documented PCV in dogs with CRF ranging from Cowgill et al. (1998) mentioned that PCV as 27 ± 6.60 per cent in dogs with protein losing glomerular disease. from CRF. Cowgill et al. (2000) estimated mean PCV as 17.6 ± 5.2 % in dogs suffering Total erythrocyte count Coles (1986) reported mean TEC in healthy dogs as /µl. Cowgill et al. (1998) estimated mean RBC count as 4.20 ± /µl in dogs with protein loosing glomerular disease. Cowgill et al. (2000) estimated RBC count as 2.50 ± /µl in six dogs suffering from chronic renal failure.

37 Total leukocyte count Larkin et al. (1972) observed WBC count as /µl in a dog with nephritic syndrome. Coles (1986) reported mean TLC in normal canines as 11300/µl. King et al. (1992) mentioned TLC in dogs with chronic renal failure ranging from /µl. CRF. Cowgill et al. (1998) observed mean TLC as ± 696 /µl in dogs with Girishkumar et al. (2011) reported that TLC was not much significant in dogs with chronic renal failure Differential leukocyte count Sasthry and Rao (2007) stated that the average DLC as 20%, 70%, 4% and 5% lymphocytes, neutrophils, eosinophils and monocytes respectively in healthy dogs. He also observed that basophils were rare. neutrophilia. King et al. (2008) recorded that 7 of 17 dogs with renal failure had mature Girishkumar et al. (2011) reported that DLC was not significant in dogs with chronic renal failure.

38 SERUM BIOCHEMISTRY Serum creatinine Creatinine is a substance that the body produces during normal metabolism. The body eliminates creatinine almost exclusively through the kidney s filtration process, so measurement of creatinine is an accurate estimation of how well the kidney filtration processes are working. Anything that alters the ability of the kidneys to filter efficiently can cause changes in the level of creatinine in the blood (Wyss and Kaddurah-Daouk, 2000). Schepper et al. (1974) recorded the serum creatinine level as 2.36 mg/dl in a dog with nephritic syndrome. Finco (1976) recorded the mean serum creatinine level as 1.22 ± 0.6 mg/dl in dogs with familial renal disease. Taboada and Palmer (1989) studied 4 cases of dogs with renal failure associated with bacterial endocarditis and observed increased serum creatinine level in all cases. Srinivasan et al. (1993) mentioned that the mean creatinine level in dogs affected with chronic renal failure was 4.56± 0.72 mg/dl. Brown et al. (1998) reported that azotemia is the presence of elevated serum concentrations of creatinine and further observed that CRF the presence of azotemia of renal origin for a minimum duration of 2 weeks. Cowgill et al. (1998) calculated mean serum creatinine level as 6.7± 2.5 mg/dl in dogs with renal failure.

39 21 Finco et al. (1999) observed that clinically plasma creatinine concentration was commonly measured to assess renal function as a crude of glomerular filtration rate. Muralikrishna (2003) observed the mean serum creatinine level as 4.06± 0.65 mg/dl in dogs suffering with CRF. Mrudula et al. (2005) estimated the mean serum creatinine level in dogs that were apparently healthy and suffering with nephritis as 6.56 ± 0.09 mg/dl and 5.59± 0.45 mg/dl respectively. Eriksen and Grondalen (2008) opined that the laboratory data in two litters of soft coated Wheaten terriers revealing isosthenuria, non regenerative anemia, extremely high serum urea nitrogen and creatinine values. Mc Grotty (2008) stated that plasma creatinine levels are typically used as biochemical marker of chronic kidney disease. Arulmozhi et al. (2010) observed systemic uremia and encephalopathy in the dogs with progressive unresponsive renal failure. The haemogram showed anemia and serum biochemistry revealed severe uremia with 18.2 mg/dl serum creatinine level. Kavitha (2010) in a study conducted on early diagnosis of renal failure in dogs reported that the creatinine level was studied to assess the kidney function, normal levels of creatinine in blood indicated ability to eliminate nitrogenous waste products successfully.

40 22 Lefebvre (2011) opined that serum creatinine concentration is currently considered as the best indirect marker of GFR and is also used by the IRiS to stage canine and feline chronic kidney disease. Polzin (2011) reported the relationship between serum creatinine and glomerular filtration rate (GFR), is such that every time GFR declines by half, the serum creatinine concentration doubles Blood urea nitrogen (BUN) BUN is the measurement that represents the level of urine in the blood. Urea is considered one of the body s waste products. It is usually eliminated from the body by both the kidneys. Therefore, both the liver and kidneys must function properly to maintain a normal level of urea in the body (Wyss and Kaddurah-Daouk, 2000). Schepper et al. (1974) observed BUN values as 73 mg/dl in German shepherd dog suffering with renal disease. Watson and Canfield (1979) recorded increased BUN level (80.4 mmol/l) in a dog affected with renal failure and hyperparathyroidism. mg/dl. Weller et al. (1985) documented BUN value in a dog with renal failure as 450 Booth (1990) reported the BUN level in a dog with chronic renal failure as 48.6 m mol/l against the normal level of 2.8 to 8.3 m mol/l. Srinivasan et al. (1993) mentioned that the mean BUN level in dogs affected with chronic renal failure was 78.18± 7.06 mg/dl.

41 23 Brown et al. (1998) reported that azotemia is due to elevated serum concentrations of BUN in dogs with CRF. Cowgill et al., (1998) reported that mean BUN value as ± 31.3 mg/dl in dogs with renal failure. Borku et al. (2000) observed the mean BUN levels in dogs that were apparently healthy and suffering with nephritis as ± 1.33 mg/dl and ± mg/dl respectively. Lucre et al. (2008) reported elevated levels of BUN in all dogs affected with chronic renal disease. Mc Grotty (2008) stated that plasma urea levels are typically used as biochemical marker of chronic kidney disease. Arulmozhi et al. (2010) observed systemic uremia and encephalopathy in the dogs with progressive unresponsive renal failure. The haemogram showed anemia and serum biochemistry revealed severe uremia with 276 mg/dl urea nitrogen level. Kavitha (2010) in a study conducted on early diagnosis of renal failure in dogs reported that the BUN level was studied to assess the kidney function, normal levels of urea nitrogen in blood indicated ability to eliminate nitrogenous waste products successfully Total protein Schepper et al. (1974) stated that a case of nephritic syndrome in a three and half year old German shepherd dog and noticed proteinuria, hypoproteinemia, edema and ascites.

42 24 dysfunctions. Dash (1987) reported that serum protein level reduced (<5g) in various renal CRF. Markwell et al. (1996) observed hypoproteinaemia in dogs suffering from Camacho et al. (2005) stated that dogs presenting azotemia of renal origin had significantly lower concentration of total proteins. Mrudula et al. (2005) mentioned the mean total protein levels in canine nephritis as 6.08 ± 0.13 g/dl. Hasan et al. (2007) reported that level of total protein in dogs with renal failure ranged between 5.9 to 6.9 g/dl Serum albumin Hurley and Vaden (1995) reported that hypoalbuminemia occurred in patients affected with glomerular disease. Markwell et al. (1996) observed that hypoalbuminemia was found in dogs suffering with chronic renal failure. Laurence et al. (1999) reported that hypoalbuminemia occurred in chronic renal failure in dogs. Camacho et al. (2005) stated that dogs presenting azotemia of renal origin have significantly lower levels of serum albumin. Mrudula et al. (2005) observed that serum albumin levels in dogs with nephritis as 2.5 ± 0.10 against healthy control as 3.33 ± 0.09 g/dl.

43 25 Hasan et al. (2007) reported serum albumin levels in dogs with renal disease ranging between 2.9 to 3.8 mg/dl. Girishkumar et al. (2011) stated that hypoalbuminemia in CRF dogs attributed to urinary loss of albumin and anorexia. Shaw and Ihle (2013) documented that hypoalbuminemia in CRF resulted from urine protein loss associated with glomerular disease Serum sodium Schepper et al. (1974) observed that serum sodium concentration in a dog with nephritic syndrome as 144 m Eq/L. Finco (1976) reported that the mean sodium concentration as 147 ± 1.74 meq/l in a dog with familiar renal disease. renal failure. Pak (2000) stated that the serum sodium levels as144 meq/l in dog with Kaneko et al. (2008) stated that normal serum sodium concentration as m mol/ L and reported that there was deficiency of sodium ions due to polyuria in generalized chronic renal disease Serum potassium Schepper et al. (1974) observed that serum potassium concentration in a dog with nephritic syndrome and glomerulonephritis as4.85 m Eq/L. Finco (1976) reported that the mean serum potassium concentration as 5.1 ± 0.6 meq/l in a dog with familiar renal disease.

44 26 Pak (2000) observed that the serum potassium levels as 5.7 meq/l in dog with renal failure. Polzin et al. (2000) reported that decreased renal function precedes the development of hypokalemia in dogs. Adams (2004) reported hyperkalemia as one of the clinical finding in dogs suffering with chronic renal failure. Mrudula et al. (2005) stated that the dogs with renal insufficiency showed azotemia, hypo proteinemia, hypoalbuminemia and hyperphosphatemia. Chander et al. (2007) observed 37 boxer dogs with clinicopathological findings of azotemia, hyperphoaphatemia, anemia, isosthenuria and proteinuria. Kaneko et al. (2008) stated that normal range of serum potassium concentration as m mol/ L in dogs. Girishkumar et al. (2011) reported that there was a significant increase of potassium level seen in dogs suffering with both acute and chronic renal failure Serum phosphorus Watson and Canfield (1979) observed increased serum phosphorus level in a dog with end stage of renal disease. failure. Lucre et al. (1980) stated that hyperphosphatemia in 13 dogs with renal Brown et al. (1985) observed that hyperphosphatemia was the common clinical finding in all cases of renal failure.

45 27 De Morais et al. (1996) reported that increased levels of serum phosphorus in dogs with chronic renal disease. Cowgill et al. (1998) recorded mean serum phosphorus values as 6.8 ± 2.2 mg/dl in dogs suffering with CRF. Muralikrishna (2003) reported the mean serum phosphorus level as 8.45 ± 1.3 mg/dl in dogs with CRF. Mrudula et al. (2005) observed increased mean serum phosphorus level ( 5.16 ± 0.2 mg/dl) in dog with nephritis as compared to apparently healthy dog ( 3.93 ± 1.7 mg/dl). Girishkumar et al. (2011) reported that there was a significant increase of phosphorus value seen in dogs suffering with both acute and chronic renal failure Serum calcium Schepper et al. (1974) reported that serum calcium concentration in a dog with nephritic syndrome and glomerulonephritis as 8.2 mg/dl. Watson and Canfield (1979) observed decreased serum calcium level in a dog with end stage of renal disease and hyperparathyroidism. King et al. (1992) recorded serum calcium levels in dogs with chronic renal failure and these levels ranged from 6.9 to 11.2 mg/dl. Kaneko et al. (1997) reported that normal range of serum calcium levels as 9.0 ± 1.3 mg/dl in normal dogs. Cowgill et al. (1998) recorded mean serum calcium values as 10.7 ± 0.08 mg/dl in six dogs suffering with CRF.

46 28 Muralikrishna (2003) reported the mean serum calcium level as 9.43 ± 0.55 mg/dl in dogs with renal failure. Shaw and Ihle (2013) documented both hypocalcaemia and hypercalcaemia in dogs suffering from CRF associated with glomerular disease BLOOD PRESSURE Although direct blood pressure measurement remained the gold standard, indirect measurements techniques (Doppler or Oscillometric methods) were routinely used in clinical settings and were considered reliable in dogs despite under estimation of acute values (Bodey and Michell, 1996). Gwin et al. (1978) explained hypertensive retinopathy in dogs with chronic renal failure associated with high blood pressure. Cowgill (1986) reported that arterial hypertension was one of the most common complications of CRF in 50 to 93 per cent of dogs. Bartges et al. (1996) stated that 50 to 93 per cent of dogs suffering with CRF were affected with elevation in blood pressure. Henik (1997) described in detail the technique of measuring blood pressure in normal dogs using Doppler and oscillometric techniques. Polzin et al. (2000) opined that 50 to 90 per cent of dogs with CRF had hypertension with systolic pressure exceeding 200 mm Hg. Jacob et al. (2003) observed high systolic pressure in 45 dogs with chronic renal failure, leading to decreased renal function and increased serum creatinine levels.

47 29 Systemic hypertension has been commonly associated with immune mediated glomerulonephritis, glomerular sclerosis and amyloidosis. Hypertension occurs because of a combination of activation of the RAAS and decreased renal production of vasodilators coupled with increased responsiveness to normal vasopressor mechanisms (Grauer, 2005). Uzlu and kalnbacak (2005) recorded increased arterial pressure (196/144 mm Hg) in dogs with CRF. Burankarl et al. (2007) in a study of blood pressure measurement in 31 renal azotemic dogs only 19% were hypertensive, where as all had increased urinary protein and electrolyte excretion. The results suggested that dogs with renal azotemia need not have hypertension RADIOGRAPHY Temizoylu et al. (2006) studied the use of radiography and ultrasonography in the diagnosis of renal diseases, concluded that ultrasonography was more sensitive than radiography and survey radiographs had little value in the diagnosis of renal diseases. Kealy et al. (2010) reported that small and irregular shaped kidneys in chronic kidney disease can be better detected by survey radiographs ULTRASONOGRAPHY Ultrasound examination is an integral tool in the thorough evaluation of the urinary system. Ultrasound is a non invasive, non painful and economical procedure that provides valuable information concerning morphology, vascular status and luminal contents usually with little or no sedation.

48 30 Cartee et al. (1980) reported the usefulness of ultrasonography in the diagnosis of renal diseases and found to be useful in diagnosis of hydronephrosis, renal calculi and renal neoplasia. Konde et al. (1986) observed in his study of fourteen dogs with renal lesions opined that ultrasonography is more sensitive than radiography in differentiating the renal lesions. Walter et al. (1987) reported ultrasonographic changes in 32 dogs with renal parenchymal disease. Twenty six dogs were diagnosed precisely with interpretation errors in six dogs. Wood and Carthy (1990) in his study on 26 dogs with both ultrasonography and anatomical observations recorded precise correlation of anatomical studies with ultrasonographic images. Felkai et al. (1996) in his study of eight young Cocker Spaniels evaluated the ultrasonographic findings to histologically confirmed renal dysplasia. Based on the ultrasound findings alone, renal dysplasia can be suspected when small kidneys with thin echogenic cortex are present in young dogs, but cannot differentiate chronic inflammatory disease from end stage kidneys. Vaden et al. (1997) stated that the ultrasonography can be used to characterize the renal shape and size. It provided information about renal parenchyma, increased overall renal echogenicity and decreased corticomedullary distinction can be seen but this cannot be diagnostic for CRF. Churchill et al. (1999) opined that, in normal sonographic anatomy of kidney, the cortex is outer rim of tissue and is normally hyperechoic to the more central

49 31 hypoechoic medulla. The cortex is typically isoechoic to hypoechoic to the liver and hypoechoic to the spleen. Chandler et al. (2007) documented ultrasonographic changes in 37 boxer dogs. Ultrasonographic findings included hyper echoic renal cortices, loss of corticomedullary junction definition, dilated pelvis and irregularly shaped small kidneys. Tripathi and Mehta (2010) reported that renal failure in 7 dogs out of 72 dogs. Out of that 4 dogs showed ultrasonographically loss of architecture detail of renal parenchyma, indistinct contours of renal cortex, hyperechoic periphery and small sized kidneys, lack of demarcation of corticomedullary junction and rest 3 dogs showed small sized kidneys, loss of architecture detail of renal parenchyma with defined irregular border in ultrasonography. Girishkumar et al. (2011) stated that ultrasonography was useful in evaluating the diffuse lesions of the parenchyma and differentiating acute and chronic renal failure. 2.4 TREATMENT STRATAGIES A clinical action plan should be developed for each patient based on the diagnosis, stage of CKD, existing complications, co- morbid conditions and risk factors for progression of kidneys. In general treatment of chronic kidney disease includes specific therapy, prevention and treatment of complications of decreased kidney function, management of co-morbid conditions and therapy designed to slower loss of kidney function.

50 32 Kavitha et al. (2013) reported in their study in 102 dogs based on chronic renal failure concluded that nearly fifty per cent of dogs were in stage I, II, III of renal failure, where pharmacological strategies could be employed to delay the progression of the disease ACE inhibitors De Lellis and Kittleson (1992) observed that angiotension converting enzyme inhibitor 0.5 mg/kg bid was effective in treating dogs with hypertension in chronic renal failure. Jensen et al. (1997) stated that administration of angiotension converting enzyme inhibitor lowered the intraglomerular pressure in dogs with kidney disease. Squires et al. (1997) advocated the use of amlodipine, the dihydropyridine calcium antagonist as antihypertensive drug in dogs with CRF. Brown (1998) reported the use of diuretics, viz. 1 mg/ kg PO every hrs and 1-2 mg/kg PO every hrs antihypertensive agent for use in CRF in dogs. Jacob et al. (2003) stated that retinopathy and hypertensive encephalopathy in three of fourteen dogs with systolic blood pressure (SBP) 180 mm Hg. SBP remained high in 10 of 11 dogs treated with hypertensive drugs. Lefebvre and Toutain (2004) recommended the angiotensin converting enzyme inhibitor (ACE1) therapy in dogs suffering from renal failure.

51 33 Grauer (2005) reported that ACE1 had shown to reduce proteinuria and lower disease progression in case of idiopathic glomerulonephritis, a potential cause of CRF in dogs. Uzlu and Kalnbacak (2005) opined that birbesartan, an angiotensin II receptor antagonist, was effective and safe in treatment of CRF associated hypertension. Brown et al. (2007) stated that ACE I have the potential to produce symptomatic hypotension typically observed when ACE I are used in conjunction with other off loading therapies, such as vasodilators, diuretics and sodium restriction. Hypotension is reversed by altering drug therapies but may be problematic in producing azotemia, inappetance, weakness, lassitude and precipitating digitalis intoxication by reducing elimination. Lefebvre et al. (2007) reported the use of angiotensin converting enzyme inhibitor. Viz. enalapril in dogs with CRF as in reduced glomerular capillary pressure, had antiproteinuric effect, tended to delay the progression of CRF and limit the extent of renal lesions. Rang et al. (2007) reported that the use of potassium sparing diuretic, spiranolactone to prevent potassium loss in cases with hypokalemia which could be caused by the administration of furosemide and CRF. Mishina and Watanabe (2008) stated that the hypertension develops in dogs with CRF through mechanisms involving the Renin-Angiotensin Aldosteron System (RAAS) and demonstrated that Benazapril hydrochloride improves renal hypertension in dogs.

52 Conservative therapy Schepper et al. (1974) reported that daily administration of frusemide was successful in controlling the edema and ascites in dogs affected with nephritic syndrome. According to Polzin et al. (1984) it is necessary to emphasize that the reduction of protein ingestion is a fundamental step when the critical level of renal function is reached. The reduction in intake of non- essential dietary proteins should decrease the quantity of protein metabolic products, limiting the effects of uremic toxins and improving the nutritional status of the patient. Such protein restriction should not modify food palatability and it should not induce a negative balance of proteins as this causes loss of muscular protein and very severe clinical condition. Weller et al. (1985) documented that clinical improvement lasted for 14 days in dogs with chronic disease, following treatment with intravenously administered sterile ringer s lactate solution and sterile 0.9 per cent normal saline solution. Bovee et al. (1989) observed that a dog with hypertension was responding with sodium restriction and standard pharmacological drugs that are used for treatment of hypertension in human beings. Michell et al. (1989) stated that a rough estimation of volume replacement may be made from the PCV alone. A common formula, assuming the extracellular deficit was to allow 10 ml/kg body weight for each one per cent rise in PCV above normal level. Mikicuik and Thornhill (1989) reported that sucralfate was effective in binding phosphorus in intestine.

53 35 King et al. (1992) reported the therapeutic usefulness of erythropoietin in management of the anemia observed in chronic renal failure dogs. Polzin et al. (1992) stated that H2 receptor antagonist 3-4 mg/kg body wt. was effective in the treatment of uremic gastritis and also he advocated the use of mg/ kg TID PO as anti-emetic to block the uremic toxin stimulation of chemoreceptor trigger zone. Pugh et al. (1995) advocated the use of spiranolactone in cases of hypokalemia and frusemide in cases of impaired renal functions, anuria and all types of edema and hypertension in dogs. Grauer (1998) suggested that polyionic maintenance fluid like ringer s lactate solution should be used in the dog with chronic renal failure to rehydrate and facilitate diuresis and also advocated the supplementation of water soluble vitamin B and C, since polyurea resulted in increased loss of water soluble vitamins. Cowgill et al. (2000) documented the use of ranitidine to stop vomiting in dogs with renal failure. Martarena et al. (2000) stated that supplementation with recombinant human erythropoietin was suitable therapy for anemia in dogs with CRF. Polzin et al. (2000) reported the use of sucralfate for gastrointestinal ulceration and to bind the phosphorus in the intestine of dogs with chronic renal failure. Grauer (2003) reported that metoclopramide blocks chemo receptor trigger zone (CTZ), increases gastric motility and emptying without increasing gastric acid

54 36 secretion and is the drug of choice for the management of vomiting associated with renal failure. Jacob et al. (2003) in a study in thirty eight dogs with spontaneous chronic renal failure used the H2 blockers and metoclopramide orally in those dogs which were suffering from vomiting and present anorexia. Adams (2004) stated that chronic administration of subcutaneous balanced electrolyte solutions has been advocated to prevent dehydration, maintain renal blood flow and GFR, increase urine output and ameliorate clinical manifestation of uremia. Burkholder et al. (2004) observed that renal diets appear to reduce the magnitude of proteinuria in proteinuric dogs and are advocated for all dogs with proteinuric kidney disease. Ross et al. (2006) reported that the effectiveness of diet therapy in stage 2 and stage 3 chronic kidney disease dogs and they found that the risk of developing uremic crisis was reduced by approximately 75 per cent for dogs consuming the renal diet as compared to dogs consuming an adult maintenance diet. Brown (2007) stated that hypokalemia is frequent in some cases of chronic kidney disease, in fact, muscle weakness may be the principle presenting sign; in these cases much higher doses of potassium may be needed. Haque (2007) emphasized the use of fluid therapy along with diuretics for initiating intensive dieresis to establish and maintain adequate renal perfusion and glomerular filtration in dogs with renal failure.

55 37 Polzin (2009) reported that treatment of uremic gastritis includes, use of H2 blockers like ranitidine, to limit the gastric acidity, use of antiemetics to suppress the nausea and vomiting, and use of sucralfate for providing mucosal protection. Roudebush et al. (2009) reported that conservative medical management of chronic kidney disease consists of supportive and symptomatic therapy designed to correct deficits and excess in fluids, electrolyte, acid, base, endocrine and nutritional balance thereby minimizing the clinical and pathophysiological consequences of reduced kidney function. Maddison and Syme (2010) opined that fluid therapy is an essential component of the management of chronic kidney disease and the fluid rates should be approximately twice normal of maintenance rates. The fluid required for maintenance is approximately equal to 50 ml/ kg/ day Punarnawadi mandur Pharmacological studies have demonstrated that root of Boerhaavia diffusa possess punarnavocide which exhibits wide range of properties- diuretic (Gaitonde et al., 1974), anti-inflammatory (Bhalla et al., 1968). Antifibrinolytic (Jain and Khanna, 1989), antibacterial (Olukoya et al., 1993) and antihypertensive activity (Gaitonde et al., 1974). B.diffusa root possesses diuretic, anti-inflammatory and antihypertensive activities and due to the combination of these activities B.diffusa is regarded therapeutically as highly efficacious for the treatment of inflammatory renal disease and nephrotic syndrome in human beings (Nadakarni, 1976; Singh and Udapa, 1972; Anand, 1995 and Anjaria et al., 2002).

56 38 Chopra et al. (1923) observed that the root of plant Boerhaavia diffusa contain large quantities of potassium nitrate besides punarnavine. Dhar et al. (1968) reported that B.diffusa is a herbivorous plant of the family Nyctaginaceae. It grows well on waste lands after the rainy season and ascending up to 1 meter with spreading branches. The root of B.diffusa has been used in India s native ayurvedic medicine for long time primarily as a renoprotective drug. Singh and Udupa (1972) stated that the whole plant of B.diffusa is a very useful source of the drug punarnava, which is documented in Indian Pharmacopoeia as a diuretic and was used in the treatment of nephritic syndrome and urinary disorders in human patients. Mishra (1980) treated mice suffering from renal failure with B.diffusa root 500mg/rat once in a day and recorded 75 per cent survival. Seth et al. (1986) isolated a new anti fibrolytic compound punarnavocide from the roots of B.diffusa. Singh et al. (1991) reported that punarnava, a ayurvedic medicine showed equivalent diuretic effect to furosemide. B.diffusa increases the protein level and reduces the urinary protein excretion. It is clinically useful and safe drug in nephritic syndrome. B.diffusa Linn., a herbaceous member of family Nyctaginaceae, is also known as Punarnava, Shothaghni, Kathillaka, Kshudra, Varshabhu, Raktapushpa, Varshaketu and Shilatika in India (Yelne et al., 2000). Dey et al. (2004) treated dogs with renal disorders with B.diffusa in combination with other herbs in capsular form (Nephtone dose of 2 capsules per

57 39 adult dog once daily for 20 to 30 days. Out of 98 cases 74 responded to Nephtone therapy. Sathyapriya et al. (2009) reported that, in a study on antioxidant status in polycystic end stage renal disease (ESRD) in human patients, observed that chronic kidney disease induces the anemia by shortening the lifespan of erythrocytes, due to an increase in oxidative stress, which is considered to be one of the major risk factors in the CKD patients and reported that use of aqueous extract of B.diffusa showed significant antihemolytic activity on the erythrocytes of the polycystic ESRD patients. Pareta et al. (2011) documented that B.diffusa Linn. is a plant widely used in India, as a traditional medicine for the treatment of renal disorders including urolithiasis as mentioned in Ayurveda, Charakasamhita and Sushruta Samhita. Rajpoot and Mishra (2011) reported that punarnava is regarded therapeutically efficacious for the treatment of inflammatory renal disease and common clinical problems such as nephritic syndrome, edema and ascites developing at the early onset of liver cirrhosis and chronic peritonitis. According to Surendra et al. (2011) the combination of diuretic, antioxidants and anti-inflammatory activities of B.diffusa is regarded as therapeutically highly efficacious for the treatment of inflammatory kidney disease, nephrotic syndrome, edema and ascites. Bhowmik et al. (2012) reported that punarnava has been reported to increase serum protein level and reduce urinary protein excretion in clinical trials in human patients suffering with nephritic syndrome.

58 40 Mahesh et al. (2012) opined that B.diffusa extracts possessed significant levels of enzymatic and non-enzymatic antioxidants. So, it possess preventive and productive role to maintain the cell survival, cellular interaction and maintenance of cell membrane architecture. Mohana lakshmi et al. (2012) stated that medicinal plants may serve as a vital source of potentially useful new compounds for the development of effective therapy to combat a variety of kidney problems and elucidated the list of nephroprotective medicinal plants including B.diffusa which are scientifically proved in treating renal disorders. Pradhan and Roy (2012) observed that the treatment of CRF in dogs, calcitrol, sucralfate and sharkoferrol are useful in the treatment of renal failure along with the herbal therapy of Nephtone Rubenal Rhubarb has been used for a variety of conditions, including GI problems, cancer, hyperlipidemia and renal disease. Most of the research into Rhubarb s clinical benefits has been conducted in China. Studies into the effects of Rhubarb on both clinical and experimentally induced kidney diseases have found benefits in showing progression of chronic kidney disease, effects similar to those found with angiotensin converting enzyme inhibitors such as enalapril and captopril. Rhubarb improves creatinine, reduces proteinuria and renal fibrosis. According to Natori et al. (1981), Rhubarb root originates from China and is now cultivated all around the world, the cooked root which decreases the

59 41 anthraquinone levels and thus reduces the cathartic nature of plant, and has long been used as a kidney tonic. According to Yu et al. (1995), in a human clinical trial, 151 chronic kidney disease patients with elevated serum creatinine levels were divided into three groups. Among those groups, one group received Rhubarb extract, another group received catopril, and other group received both treatments. All patients were commonly given a low protein and low phosphorus diet. Follow up over an average of 32.5 months revealed a significant reduction in symptoms of uremic nausea and anorexia. The frequency of end stage kidney disease was 54.3 per cent for the ACE inhibitor group, 25.9 per cent for the Rhubarb treated group and 13.1 per cent for the Rhubarb and ACE inhibitor group. Sanada (1996) reported that 38 patients with chronic kidney disease treated with Rhubarb root 1 gm per day maintained serum creatinine levels comparable to rising levels seen in controls not treated with Rhubarb. Li (1996) conducted clinical trials on the effect of Rhubarb in patients with chronic kidney disease and observed that Rhubarb was able to reduce proteinuria and improvement in renal function by itself and might also cause further reduction of proteinuria and improvement in renal function when used together with ACE inhibitors. Zhang and Nahas (1996) observed the effect of Rhubarb extracts on the improvement of renal failure in Wistar rats. Rhubarb extracts decreased proteinuria and glomerulosclerosis comparable to the rats of control group (no treatment group).

60 42 According to Hadjzadeh et al. (2013), in a study of effect of aqueous extract of Rheum on cisplatin induced nephrotoxicity in rats, found that daily dose rate of 150 mg/kg, had little effect on serum biochemical parameters. Zhong et al. (2013) conducted a study on lab animals and showed the use of Rheum (Rhubarb) and its components promotes waste product excretion, reduction in proteinuria and improvement in kidney function. Rheum and Emodin were found to have antioxidant property.

61 43

62 44

63 CHAPTER-III MATERIALS AND METHODS Dogs presented to the Teaching Veterinary Clinical Complex, NTR College of Veterinary Science, Gannavaram and surrounding Veterinary Hospitals during the period from October, 2014 to June, 2015 were considered for the present study. 3.1 DESIGN OF STUDY The study comprised of apparently healthy dogs and clinical cases Control animals Ten apparently healthy dogs of different breeds aged between four to seven years were selected as control group for obtaining normal data for comparison of parameters under study Clinical cases The study was conducted on seventy two clinical cases brought to the small animal out-patient ward of Teaching Veterinary Clinical Complex, N.T.R College of Veterinary Science, Gannavram. Dogs with clinical signs suggestive of chronic kidney disease (CKD) were screened by using specially designed nephrology data sheet and subjected to detailed physical examination, haematology, serum biochemical profile, urinalysis and nephrosonography to confirm the diagnosis of CKD Selection criteria for inclusion in the treatment trials Out of total 42 dogs diagnosed as suffering from CKD based on history, clinical signs, haematology and biochemistry, 30 dogs were classified according to 43

64 44 International Renal Interest Society (IRiS) and dogs under stage II and III which were selected and subjected to different therapeutic regimens. Those 30 dogs were randomly allotted to three different groups i.e. Group I, II and III each having 10 dogs Treatment trials Group I The dogs in Group I were treated with 0.5 mg/ kg bd. Wt once daily for a period of 45 days. Group II Animals of Group II received conventional therapy along with one specific therapy i.e herbal nephroprotectant Punarnavawadi Mandur (Root extract of plant Boerhaavia diffusa). The tablets were administered for duration of 45 days at the dose rate of 1 tablet twice a day for large breeds of dogs (Body wt. > 20kg) and one tablet once a day for small and medium breeds of dogs (< 20 kg). Group III The dogs of group III were received conventional therapy along with one specific therapy i.e., Rubenal 300 (a medicinal rhubarb derived from the rhizome and roots of the rhubarb plant, Rheum officinale), Marketed by Vetoquinol USA, Inc. These tablets are contain anthraquinone glycosides like rhein, emodin and aloeemodin and tannins like gallotannins, cathechin, procyanidin derivatives and these were administrated orally. This therapy was continued for one month and dose was based on the weight of the dog.

65 45 Body Weight Dose 8-12 kg ½ tablet bid kg 1 tablet bid kg 2 tablets bid >45 kg 3 tablets bid The following supportive therapy was given to all the three groups, duration of the therapy depending upon the severity of the symptoms. 1. Ringer s Lactate 30ml/kg bd.wt I/V mg/ kg bd.wt I/V 3. 2mg/ kg bd.wt I/M 4. 1g /day P.O 5. Iron Supplementation (Fe-Foliate 5ml P.O General considerations in the treatment The pet owners were advised to give low salt, low protein and low phosphorus diet and increase B- complex vitamins and caloric density. 3.2 CHARACTERS UNDER STUDY The following parameters were studied in all the dogs suffering with CKD. i. History ii. iii. iv. Clinical signs Blood pressure Haematology

66 46 v. Serum biochemical profile vi. vii. viii. Urinalysis Radiography Ultrasonography 3.3 EVALUATION OF PARAMETERS Clinical examination The dogs selected for present study were subjected to a detailed clinical examination and the data was recorded in a proforma specially designed for the data collection (Appendix I) Haematology Two milliliters of whole blood was collected from the saphenous or cephalic vein of the dog in vacutainers with EDTA (0.5-2 mg/ml of blood) from those animals with clinical signs suggestive of CKD for evaluation of hematological parameters like Haemoglobin (Hb), Packed cell volume (PCV), Total erythrocyte count (TEC), Total leukocyte count (TLC) and peripheral smears were made for Differential leukocyte count (DLC) as per the methods described by Coles (1986) Serum biochemistry Five milliliters of whole blood was collected into centrifuge tube without anti coagulant for separation of serum and the following estimations were done Serum creatinine Serum creatinine was estimated by the modified Jaffe s alkaline picrate method (Murrey,1984), analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Erba diagnostics Mannheim, Germany. It is expressed in mg/dl.

67 Blood urea nitrogen Blood urea nitrogen was estimated by Diacetyl monozyme method (Murrey, 1984) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Erba diagnostics Mannheim, Germany. It is expressed in mg/dl Total protein Total protein was estimated by modified Biuret and Bromo cresol green dye method (Doumas, 1975) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in g/dl Albumin Albumin was estimated by Bromo cresol green, end point assay method (Doumas, 1975) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in g/dl Sodium Sodium was estimated by end point method (Terri, 1958) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in meq/l Potassium Potassium was estimated by end point method (Moron et al., 1979) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in meq/l.

68 Phosphorous Phosphorous was estimated by end point method (Moron, 1979) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in mg/dl Calcium Calcium was estimated by end point method (Stein, 1957) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. It is expressed in mg/dl URINALYSIS Physical examination The urinalysis was done with urine collected from the animal in sterile vials either from mild steam free collection, by catheterization or by cystocentesis. 5ml of fresh urine sample was centrifuged at 1500 rpm for five minutes and the sediment is examined for casts, pus cells and other sediments (Coles, 1986) ph, specific gravity and urine protein Urine ph, specific gravity and urine protein were estimated with URISCAN urine dip sticks (Berg et al.,1989) Urine protein and creatinine (UP/c) ratio Five milliliters of fresh urine sample was centrifuged at 1500 rpm for 5 minutes to get supernatant solution. Urinary protein was estimated by modified Biuret method and expressed as mg/dl (Doumas, 1975).

69 49 Urine samples were diluted to 1:50 with distilled water for estimation of creatinine and done by Jaffe s method and expressed as mg/dl (Murrey, 1984) UP/C ratio = Urinary protein concentration (mg/dl) Urinary creatinine concentration (mg/dl) Urinary enzymes Alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) were estimated from urine supernatant after centrifugation. ALP was done by IFCC method (Butris and Ashwood, 1996) and analyzed by Eppendorf BioSpectrometer by standard procedures described by the manufacturer using the kits supplied by Coral clinical systems, Goa. ALP and GGT were expressed as mmol of urine creatinine Radiography Abdominal radiography was taken at standard lateral view NEPHROSONOGRAPHY Instrumentation Nephrosonogram was performed by using ALOKA PROSOUND α 6LT ultrasound machine. A linear array 3.5 MHz, 5.0 MHz, and 7.5 MHz probes were used for large, medium and small dogs to perform nephrosonogram Preparation and restraint of the patient The hair over the entire abdomen was clipped including the midway up the body wall over the right and left caudal intercoastal spaces. Nephrosonographic examination was performed by placing the animals in either lateral or dorsal recumbency. A liberal amount of coupling gel was applied to provide sufficient contact (Nyland, 1995).

70 Nephrosonographic technique After patient preparation, the left kidney was imaged caudal to the greater curvature of the stomach, caudodorsal to the spleen, lateral to the aorta and left adrenal gland at the level of L2 to L4 vertebrae. The right kidney was imaged caudal to the right liver lobes, lateral to the caudal venacava and right adrenal gland at the level of L1 to L3 vertebrae (Ambrust, 2001). Standard mid sagittal and mid transverse planes were obtained according to the established protocol Interpretation of Nephrosonographs Sonograms were evaluated for information on kidney architecture, specifically including focal, multifocal and diffuse alterations in renal cortical, medullary, sinusal and peripheralsion, echogenicity. In addition, cortical and medullary echogenicity were compared subjectively with hepatic and splenic parenchymal echogenicity. The echogenicity of the identifiable lesion as seen on the gray scale 2 dimensional images were classified subjectively as normal, increased (hyperechoic), decreased (hypoechoic) or absent (anechoic), when compared with normal echo pattern for canine kidney ( Walter et al., 1987) 3.4 STAGING OF THE DISEASED ANIMALS Staging was undertaken based on the results of serum creatinine levels following diagnosis of Chronic Kidney Disease (CKD) in order to facilitate appropriate treatment and monitoring of the patient Classification of chronic kidney disease cases According to International Renal Interest Society (IRiS) standards based on serum creatinine levels the CKD cases were classified into four stages.

71 51 Serum creatinine (mg/dl) Stage of CKD Azotemia < 1.4 mg/dl Stage I Non azotemic stage mg/dl Stage II Mild azotemia mg/dl Stage III Moderate azotemia > 5 mg/dl Stage IV Severe azotemia 3.5 EVALUATION OF THE TREATMENT The dogs under the therapeutic trials were monitored for a period of 45 days and the three treatment regimens were evaluated at 15 days intervals based on improvement in clinical signs, blood pressure measurement, haematology, serum biochemical profile, urinalysis and nephrosonographic changes. 3.6 STATISTICAL ANALYSIS Statistical analysis was performed by using one way ANOVAs of variance and correlation analysis by using software called SPSS (Software Packages for Social Sciences).

72 CHAPTER IV RESULTS The study entitled Clinico - diagnostic and therapeutic studies on chronic kidney disease in dogsʺ was carried out at the Department of Veterinary Medicine, N.T.R College of Veterinary Science, Gannavaram during the period of October 2014 to June 2015.The study was carried out with a total of 72 dogs. Among them 42 dogs were clinical cases of chronic kidney disease. 4.1 Occurrence In the present study, forty two cases were confirmed as chronic kidney disease based on detailed clinical examination, haematology, serum biochemistry, urinalysis, blood pressure measurement, abdominal radiography and nephrosonography Breed The breed wise occurrence of chronic kidney disease was presented in Table 1 and Fig 1 out of affected breeds in our study, eleven were Spitz/Pomeranian (26.2%), ten Labrador Retriever (23.8%), five German shepherd (11.9%), four Non descript (9.5%), Boxer (7.1%), two Dachshund (4.8%), two Great Dane (4.8%), two Doberman Pinscher (4.8%), one St Bernard (2.4), one Beagle (2.4%) and one Pug (2.4%) Age The age wise occurrence of chronic kidney diseases was presented in Table 2 and Fig 2. In the present study, the age group of dogs affected included 1-5 years 52

73 53 (n=7), 6-10 years (n=9), and years (n=26). The overall occurrence of chronic kidney disease was found to be higher in years age group with an occurrence of per cent (26/42), which was followed by 6-10 years age group with an occurrence of per cent (09/42) and 1-5 years group with an occurrence of per cent (7/42) Gender The gender wise occurrence of chronic kidney disease was presented in Table 3 and Fig 3. In the present study, 25 were male dogs and 17 were female dogs. Male dogs dominated females in overall occurrence of chronic kidney disease with per cent (25/42) and per cent (17/42) respectively. 4.2 Clinical signs Clinical signs of the present study were given in Table 4. Among the 42 dogs, prominent clinical signs of chronic kidney disease were anorexia (85.71%), weakness (80.95%), weight loss (78.57%), pallor mucous membrane (73.81%), vomiting (66.67%), melena (64.29%), polyurea/ polydipsia (54.76%), halitosis (50%), oral ulcers(42.86%), emaciation (26.19%), nervous signs (21.43%), diarrhoea (19.05%), recumbent (16.67%), congested mucous membrane (14.29%) and blindness (9.52%) (Plate no. 1,2,3,4,5,6& 7). 4.3 Urinalysis The mean± SE values of urinalysis of control and chronic kidney disease groups were presented in Table 5. There was no significant difference in mean ± SE values of urine ph in Chronic kidney disease group (6.31 ± 0.12) when compared with control group (6.93 ± 0.17).

74 54 There was highly significant decrease (P 0.01) in mean ± SE values of urine specific gravity in chronic kidney disease group (1.01 ± 0.01) when compared with control group (1.038 ± 0.03) on zero day and highly significant increase in mean ± SE values of urine specific gravity in Group III (1.035 ± 0.02) when compared with Group I&II (1.029 ± 0.01 & ± 0.01) on 45 th day of treatment. There was highly significant increase (P 0.01) in mean ± SE values of urine protein in chronic kidney disease group (100.08±22.78 mg%) when compared with control group (6.7 ± 0.84 mg%) on zero day and highly significant decrease in mean ± SE values of urine protein in Group III (6.65 ± 1.42 mg%) when compared with Group I&II (7.67 ± 1.89 mg% & 6.72 ± 1.50 mg%) on 45 th day of treatment. There was significant increase (P 0.05) in mean ± SE values of urinary ALP in chronic kidney disease group (8.01 ±0.93 m mol/creatinine) when compared with control group (1.54 ± 0.08 m mol/creatinine) on zero day and significant decrease in mean ± SE values of urinary ALP in Group III (1.5 ± 0.74 m mol/creatinine) when compared with Group I&II (1.61 ±0.08 m mol/creatinine& 1.56 ±0.08 m mol/creatinine) on 45 th day of treatment. There was significant increase (P 0.05) in mean ± SE values of Urinary Gamma- GT in chronic kidney disease group (7.08 ± 0.83 m mol/creatinine) when compared with control group (1.47 ± 0.07 m mol/creatinine) on zero day and significant decrease in mean ± SE values of Urinary Gamma- GT in Group III (0.42 ± 0.01 m mol/creatinine) when compared with Group I&II (1.92±0.09m mol/creatinine& 1.51 ± 0.08 m mol/creatinine) on 45 th day of treatment. There was highly significant increase (P 0.01) in mean ± SE values of UP/C Ratio in chronic kidney disease group (2.9 ± 0.25) when compared with control group (0.47 ± 0.02) on zero day and highly significant decrease in mean ± SE values

75 55 of UP/C Ratio in Group III (0.42 ± 0.01) when compared with Group I&II (0.54 ± 0.01 & 0.5 ± 0.01) on 45 th day of treatment (Table 9, 10, 11& 12, Plate No. 8 and Fig 4). 4.4 Haematology The haematology profile for control and chronic kidney disease group were presented in Table 6. There was a significant decrease (P 0.05) in the mean ± SE values of haemoglobin in diseased groups when compared to control group (Control group 14.5±0.4 g/dl and CKD dogs 8.64±0.38 g/dl). The mean ± SE of haemoglobin value in control, Group I, Group II and Group III on zero day were 14.5 ± 0.4 mg/dl, 8.14 ± 0.48 mg/dl, 8.54 ± 0.45 mg/dl and 9.22 ± 0.58 mg/dl respectively. The mean ± SE of haemoglobin value in Group I, Group II and Group III on 45 th day of treatment were 12.6 ± 0.48 mg/dl, ± 0.45 mg/dl and ± 0.58 mg/dl respectively. There was a statistical significant increase for haemoglobin observed in Group III when compared to Group I and Group II. Mean ± SE values of PCV had significantly decreased (P 0.05) in chronic kidney disease groups (Group I 24.5±1.46 per cent, Group II 26 ±1.32 per cent and Group III 28 ± 1.78 per cent) when compared with the control group (43.8 ± 1.12 per cent) on zero day. The Mean ±SE values of PCV had a significant increase (P 0.05) in Group III (41 ±1.78 per cent) when compared with Group I (37.67 ± 1.46 per cent) and Group II (38.12 ± 1.32 per cent) on 45 th day of treatment. Mean ± SE values of TEC in CKD group (4.58 ± 0.21 /µl) had a significant decrease (P 0.05) when compared with the control group (6.91± 0.19/µl) on zero

76 56 and group III (6.42 ± 0.15/µl) had a significant increase (P 0.05) when compared with the Group I (6.27± 0.18/µl) and Group II (6.18 ± 0.16/µl) on 45 th day of treatment. There was no significant difference (P 0.05) in the mean ± SE values of TLC in CKD group (9100±391.17/µl) when compared with the control group ( ± /µl) (Table 13, 14, 15& 16 and Fig 5, 6& 7). 4.5 Serum biochemistry The mean ± SE values of serum biochemistry parameters of control and chronic kidney disease group were presented in Table 7. There was a significant decrease (P 0.05) in mean ± SE values of serum total protein in chronic kidney disease group (4.88 ± 0.16 g/dl) when compared with control group (6.93 ± 0.15 g/dl) on zero day and a significant increase in mean ± SE values of serum total protein in Group III (6.42 ± 0.15 g/dl) when compared with Group I&II (6.10 ± 0.11 g/dl & 6.21 ± 0.05g/dl) on 45 th day of treatment. There was highly significant decrease (P 0.001) in albumin values in chronic kidney disease group (1.92 ± 0.07 g/dl) when compared with control group (2.85 ± 0.09 g/dl) on zero day and highly significant increase in mean ± SE values of albumin in Group III (2.97 ± 0.08 g/dl) when compared with Group I&II (2.78 ± 0.03 g/dl & 2.93 ± 0.06 g/dl) on 45 th day of treatment. There was highly significant increase (P 0.01) in mean ± SE values of BUN in diseased groups (70.70 ± 7.17 mg/dl) when compared with control group (22.37 ± 1.04 mg/dl) on zero day and highly significant decrease in mean ± SE values of BUN in Group III (26.75 ± 0.82 mg/dl) when compared with Group I&II (29.67 ± 0.89 mg/dl & ± 0.70 mg/dl) on 45 th day of treatment.

77 57 There was highly significant increase (P 0.01) in mean ± SE values of creatinine in chronic kidney disease group (3.23 ± 0.41 mg/dl) when compared with control group (0.45 ± 0.07 mg/dl) on zero day and highly significant decrease in mean ± SE values of creatinine in Group III (0.98 ± 0.03 mg/dl) when compared with Group I&II (1.16 ± 0.03 mg/dl & 1.06 ± 0.04 mg/dl) on 45 th day of treatment. There was no significant difference in mean ± SE values of serum SGPT in chronic kidney disease group (53.13 ± 5.56 U/L) when compared with control group (47.87 ± 6.04 U/L). There was a significant increase (P 0.05) in mean ± SE values of serum phosphorus in chronic kidney disease group (9.68 ± 0.65 mg/dl) when compared with control group (4.05 ± 0.27 mg/dl) on zero day and a significant decrease in mean ± SE values of serum phosphorus in Group III (4.97 ± 0.04 mg/dl) when compared with Group I&II (5.28 ± 0.10 mg/dl & 5.19 ± 0.08 mg/dl) on 45 th day of treatment. There was no significant difference in mean ± SE values of serum calcium in chronic kidney disease group (9.87 ± 0.18 mg/dl) when compared with control group (10.36 ± 0.18 mg/dl). There was a significant increase (P 0.05) in mean ± SE values of serum sodium in chronic kidney disease group ( ± 1.56 meq/l) when compared with control group (145.7 ± 0.86 meq/l) on zero day and a significant decrease in mean ± SE values of serum sodium in Group III ( ± 0.41 meq/l) when compared with Group I&II ( ± 0.88 meq/l & ± 0.45 meq/l) on 45 th day of treatment.

78 58 There was no significant difference in mean ± SE values of serum potassium in chronic kidney disease group (4.72 ± 0.14 meq/l) when compared with control group (4.33 ± 0.17 meq/l) (Table 17, 18, 19 & 20 and Fig 8& 9). 4.6 Blood pressure The mean ± SE values for blood pressure of control and CKD dogs were presented in Table 8. The mean of systolic arterial pressure measured in control dogs, Group I, Group II and Group III on zero day were ± 1.77 mmhg, ± 2.28 mmhg, ± 2.45 mmhg and ± 2.28 mmhg respectively. The mean of diastolic arterial pressure measured in control dogs, Group I, Group II and Group III on zero day were ± 1.86 mmhg, 98.5 ± 1.96 mmhg, 102 ± 2.32 mmhg and 99.2 ± 2.08 mmhg respectively. The mean of systolic arterial pressure measured in control dogs, Group I, Group II and Group III on 45 th day were ± 1.77 mmhg, ± 1.78 mmhg, ± 1.77 mmhg and ± 1.69 mmhg respectively. The mean of diastolic arterial pressure measured in control dogs, Group I, Group II and Group III on 45 th day were ± 1.86 mmhg, 84.9 ± 1.86 mmhg, ± 1.8 mmhg and ± 1.78 mmhg respectively. A significant increase (P 0.005) in systolic and diastolic arterial pressure was observed in CKD dogs when compared to control group. There was statistical significant difference for systolic and diastolic arterial pressure observed in Group III (Table 21, 22, 23 & 24 and Plate No. 9). 4.7 Radiography Radiography changes in CKD dogs observed were kidneys in normal in size, round kidneys and hydronephrosis (Table 25 and Plate no. 10& 11).

79 Nephrosonography The nephrosonogram of control dogs showed renal cortical echogenicity of left kidney was less than that of adjacent spleen and right kidney cortical echogenicity was less than adjacent liver echogenicity. The medulla was hypoechoic, round in appearance, with well defined corticomedullary junction. At the center hyperechoic renal pelvis was noticed. The ultrasonographic changes in CKD dogs observed were indistinct corticomedullary junction in per cent dogs, sunken kidneys in per cent, hyperechoic cortex in per cent, renal cyst in 4.76 per cent (Table 26 and Plate no.12, 13& 14). 4.9 Classification of chronic kidney disease (CKD) dogs according to International Renal Interest Society (IRiS) standards based on Serum creatinine level CKD in dogs was classified as per the standards of International Renal Interest Society (IRiS) on the basis of serum creatinine level. In the present study, out of 42 cases, nine cases (21.40 per cent) were found in Stage II (Serum creatinine mg/dl), 21 cases (50 per cent) were found in Stage III (Serum creatinine 2-5 mg/dl), 12 cases (28.60 per cent) were found in Stage IV (Serum creatinine >5 mg/dl) and no cases were found in Stage I. The details are shown in the Table 27 and Fig Survival rate of dogs with CKD in different groups during the treatment period Of the ten animals in Group I which received the conventional treatment, three animals survived where as seven animals were died. Out of the ten animals in

80 60 Group II which received the specific therapy of Punarnawadi Mandur with conventional treatment, six animals survived where as four animals died during the period of treatment. Out of the ten animals in Group III which received the specific therapy of Rubenal with conventional treatment, eight animals survived where as two animals died. The details are shown in the Table 28 and Fig 11.

81 61 Table 1: Breed-wise occurrence of CKD in dogs (n=42) Breed Total Percentage Spitz/ Pomeranian Labrador Retriever German Shepherd Non- Descript Boxer Great Dane Dachshund Doberman Pinscher St Bernard Beagle Pug Total Table 2: Age-wise occurrence of CKD in dogs (n=42) Age No. of dogs Percentage < 1 yr yrs yrs yrs Total

82 no. of dogs no.of dogs 62 Fig 1: Breed-wise occurrence of CKD in dogs (n=42) Fig 2: Age wise occurrence of CKD in dogs yrs 5-10 yrs yrs

83 63 Table 3: Gender-wise occurrence of CKD in dogs (n=42) Gender No. of dogs Percentage Male Female Total Table 4: Clinical findings exhibited by dogs with CKD (n=42) Symptoms No. of Cases Percentage Anorexia Weakness Weight loss Pallor mucous membrane Vomitions Melena Polyurea/ Polydipsia Halitosis Oral Ulcers Emaciation Nervous Signs Diarrhoea Recumbent Congested mucous membrane Blindness

84 UP/c ratio 64 Fig 3: Gender-wise occurrence of CKD in dogs (n=42) 40.5 % 59.5% Male Female Fig 4: UP/c ratio values in treatment groups Control Group I Group II Group III 0 0 Day 15th Day 30th Day 45th Day

85 65

86 66

87 67

88 68

89 69 Table 5: Urinalysis in control and CKD dogs Parameter Control Group CKD Dogs ph 6.93 ± ± 0.12 Specific gravity ± ± 0.01 ** Protein (mg%) 6.7 ± ± ** Urinary ALP (m mol/creatinine) 1.54 ± ±0.93 * Urinary Gamma- GT (m mol/creatinine) 1.47 ± ± 0.83 * UP/c Ratio 0.47 ± ± 0.25 ** ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 6: Haematological values in control and CKD dogs Parameter Control Group CKD Dogs Haemoglobin (g/dl) 14.5 ± ± 0.38 * PCV % 43.8 ± ± 1.56 * TEC / µl 6.91 ± ± 0.21 * TLC / µl 9100 ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

90 70 Table 7: Serum biochemical profile in control and CKD dogs Parameter Control Group CKD Dogs Total Protein (g/dl) 6.93 ± ± 0.16 * Albumin (g/dl) 2.85 ± ± 0.07 ** Blood Urea Nitrogen (mg/dl) ± ± 7.17 ** Creatinine (mg/dl) 0.45 ± ± 0.41 ** Phospharus (mg/dl) 4.05 ± ± 0.65 * Calcium (mg/dl) ± ± 0.18 Sodium (meq/l) ± ± 1.56 * Potassium (meq/l) 4.33 ± ± 0.14 ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 8: Blood pressure values in control and CKD dogs Parameter Control Group CKD Dogs Systolic arterial pressure (mmhg) ± ± 1.78 * Diastolic arterial pressure (mmhg) ± ± 1.86 * ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

91 71 Table 9: Urinalysis in treatment groups (0 day) Parameter Control Group Group I Group II Group III ph 6.93 ± ± ± ± 0.24 Specific gravity ± ± 0.01 ** ± 0.01 ** ± 0.02 ** Protein (mg%) 6.7 ± ± ** ± ** ± ** Urinary ALP (m mol/creatinine) 1.54 ± ±1.03 * 8.06 ± 1.09 * 7.98 ± 1.09 * Urinary Gamma- GT (m mol/creatinine) 1.47 ± ± 0.83 * 7.61 ± 1.01 * 7.52 ± 0.96 * UP/C Ratio 0.47 ± ± 0.25 ** 3.01 ± 0.26 ** 2.77 ± 0.22 ** ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

92 72 Table 10: Urinalysis in treatment groups (15 th day) Parameter Control Group Group I Group II Group III ph 6.93 ± ± ± ± 0.25 Specific gravity ± ± 0.01 ** ± 0.01 ** 1.02 ± 0.02 ** Protein (mg%) 6.7 ± ± ** ± ** ± ** Urinary ALP (m mol/creatinine) 1.54 ± ±0.93 * 7.06 ± 1.01 * 5.98 ± 0.94 * Urinary Gamma- GT (m mol/creatinine) 1.47 ± ± 0.73 * 5.61 ± 0.91 * 5.72 ± 0.96 * UP/C Ratio 0.47 ± ± 0.21 ** 2.32 ± 0.24 ** 2.27 ± 0.22 ** ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

93 73 Table 11: Urinalysis in treatment groups (30 th day) Parameter Control Group Group I Group II Group III ph 6.93 ± ± ± ± 0.21 Specific gravity ± ± 0.01 ** 1.03 ± 0.01 ** ± 0.02 ** Protein (mg%) 6.7 ± ± ** ± ** ± ** Urinary ALP (m mol/creatinine) 1.54 ± ±0.83 * 3.04 ± 0.81 * 2.98 ± 0.8 * Urinary Gamma- GT (m mol/creatinine) 1.47 ± ± 0.82 * 3.11 ± 0.81 * 2.72 ± 0.79 * UP/C Ratio 0.47 ± ± 0.21 ** 1.32 ± 0.14 ** 1.27 ± 0.12 ** ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

94 74 Table 12: Urinalysis in treatment groups (45 th day) Parameter Control Group Group I Group II Group III ph 6.93 ± ± ± ± 0.15 Specific gravity ± ± 0.01 ** ± 0.01 ** ± 0.02 ** Protein (mg %) 6.7 ± ± 1.89 ** 6.72 ± 1.50 ** 6.65 ± 1.42 ** Urinary ALP (m mol/creatinine) 1.54 ± ±0.08 * 1.56 ±0.08 * 1.5 ± 0.74 * Urinary Gamma- GT (m mol/creatinine) 1.47 ± ± 0.09 * 1.51 ± 0.08 * 1.48 ± 0.07 * UP/C Ratio 0.47 ± ± 0.01 ** 0.5 ± 0.01 ** 0.42 ± 0.01 ** ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

95 75 Table 13: Haematological findings in treatment groups ( 0 Day) Parameter Control Group Group I Group II Group III Haemoglobin (g/dl) 14.5 ± ± 0.48 * 8.54 ± 0.45 * 9.22 ± 0.58 * PCV % 43.8 ± ± 1.46 * 26 ± 1.32 * 28 ± 1.78 * TEC / µl 6.91 ± ± 0.18 * 4.41 ± 0.16 * 4.62 ± 0.15 * TLC / µl 9100 ± ± ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 14: Haematological findings in treatment groups ( 15 th Day) Parameter Control Group Group I Group II Group III Haemoglobin (g/dl) 14.5 ± ± 0.39 * 9.08 ± 0.45 * 9.94 ± 0.51 * PCV % 43.8 ± ± 1.18 * 27.5 ± 1.38 * 30.2 ± 1.50 * TEC / µl 6.91 ± ± 0.13 * 4.71 ± 0.15 * 4.98 ± 0.09 * TLC / µl 9100 ± ± ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

96 76 Table 15: Haematological findings in treatment groups ( 30 th Day) Parameter Control Group Group I Group II Group III Haemoglobin (g/dl) 14.5 ± ± 0.52 * ± 0.59 * 11.4 ± 0.49 * PCV % 43.8 ± ± 1.46 * ± 1.75 * ± 1.45 * TEC / µl 6.91 ± ± 0.88 * 5.1 ± 0.23 * 5.48 ± 0.10 * TLC / µl 9100 ± ± ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 16: Haematological findings in treatment groups ( 45 th Day) Parameter Control Group Group I Group II Group III Haemoglobin (g/dl) 14.5 ± ± 0.48 * ± 0.45 * ± 0.58 * PCV % 43.8 ± ± 1.46 * ± 1.32 * 41 ± 1.78 * TEC / µl 6.91 ± ± 0.18 * 6.18 ± 0.16 * 6.42 ± 0.15 * TLC / µl 9100 ± ± ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

97 77 Table 17: Serum biochemical profile in treatment groups ( 0 Day) Parameter Control Group Group I Group II Group III Total Protein (g/dl) 6.93 ± ± 0.16 * 4.79 ± 0.11 * 4.91 ± 0.10 * Albumin (g/dl) 2.85 ± ± 0.08 ** 1.83 ± 0.1 ** 1.97 ± 0.09 ** Blood Urea Nitrogen (mg/dl) ± ± 6.97 ** 72.3 ± 6.41 ** 67.8 ± 5.35 ** Creatinine (mg/dl) 0.45 ± ± 0.42 ** 3.26 ± 0.42 ** 2.82 ± 0.37 ** Phospharus (mg/dl) 4.05 ± ± 0.65 * 9.98 ± 0.65 * 9.6 ± 0.76 * Calcium (mg/dl) ± ± ± ± 0.21 Sodium (meq/l) ± ± 1.56 * ± 2.50 * 166 ± 1.90 * Potassium (meq/l) 4.33 ± ± ± ± 0.13 ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

98 78 Table 18: Serum biochemical profile in treatment groups ( 15 th Day) Parameter Control Group Group I Group II Group III Total Protein (g/dl) 6.93 ± ± 0.11 * 4.98 ± 0.09 * 5.13 ± 0.08 * Albumin (g/dl) 2.85 ± ± 0.08 ** 2.04 ± 0.04 ** 2.21 ± 0.07 ** Blood Urea Nitrogen (mg/dl) ± ± 7.19 ** 63.5 ± 6.23 ** 56.7 ± 5.29 ** Creatinine (mg/dl) 0.45 ± ± 0.41 ** 2.89 ± 0.42 ** 2.44 ± 0.36 ** Phospharus (mg/dl) 4.05 ± ± 0.32 * 8.06 ± 0.48 * 8.44 ± 0.75 * Calcium (mg/dl) ± ± ± ± 0.18 Sodium (meq/l) ± ± 1.69 * ± 1.98 * ± 1.87 * Potassium (meq/l) 4.33 ± ± ± ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

99 79 Table 19: Serum biochemical profile in treatment groups ( 30 th Day) Parameter Control Group Group I Group II Group III Total Protein (g/dl) 6.93 ± ± 0.18 * 5.32 ± 0.11 * 5.58 ± 0.09 * Albumin (g/dl) 2.85 ± ± 0.10 ** 2.20 ± 0.06 ** 2.41 ± 0.07 ** Blood Urea Nitrogen (mg/dl) ± ± 9.20 ** ± 5.94 ** ± 3.14 * Creatinine (mg/dl) 0.45 ± ± 0.51 ** 2.16 ± 0.37 ** 1.76 ± 0.27 ** Phospharus (mg/dl) 4.05 ± ± 0.27 * 6.82 ± 0.52 * 6.27 ± 0.36 * Calcium (mg/dl) ± ± ± ± 0.16 Sodium (meq/l) ± ± 1.69 * ± 1.32 * 155 ± 1.05 * Potassium (meq/l) 4.33 ± ± ± ± 0.08 ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

100 80 Table 20: Serum biochemical profile in treatment groups ( 45 th Day) Parameter Control Group Group I Group II Group III Total Protein (g/dl) 6.93 ± ± 0.11 * 6.21 ± 0.05 * 6.42 ± 0.15 * Albumin (g/dl) 2.85 ± ± 0.03 ** 2.93 ± 0.06 ** 2.97 ± 0.08 ** Blood Urea Nitrogen (mg/dl) ± ± 0.89 ** ± 0.70 ** ± 0.82 ** Creatinine (mg/dl) 0.45 ± ± 0.03 ** 1.06 ± 0.04 ** 0.98 ± 0.03 ** Phospharus (mg/dl) 4.05 ± ± 0.10 * 5.19 ± 0.08 * 4.97 ± 0.04 * Calcium (mg/dl) ± ± ± ± 0.18 Sodium (meq/l) ± ± 0.88 * ± 0.45 * ± 0.41 * Potassium (meq/l) 4.33 ± ± ± ± 0.13 ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

101 81 Table 21: Blood pressure values in treatment groups ( 0 Day) Parameter Control Group Group I Group II Group III Systolic arterial pressure (mmhg) ± ± 2.28 * ± 2.45 * ± 2.28 * Diastolic arterial pressure (mmhg) ± ± 1.96 * 102 ± 2.32 * 99.2 ± 2.08 * ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 22: Blood pressure values in treatment groups ( 15 th Day) Parameter Control Group Group I Group II Group III Systolic arterial pressure (mmhg) ± ± 2.18 * ± 2.15 * ± 2.08 * Diastolic arterial pressure (mmhg) ± ± 1.86 * 92.2 ± 1.8 * ± 1.07 * ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

102 82 Table 23: Blood pressure values in treatment groups ( 30 th Day) Parameter Control Group Group I Group II Group III Systolic arterial pressure (mmhg) ± ± 1.98 * ± 1.92 * ± 1.82 * Diastolic arterial pressure (mmhg) ± ± 1.92 * ± 1.88 * ± 1.86 * ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05) Table 24: Blood pressure values in treatment groups ( 45 th Day) Parameter Control Group Group I Group II Group III Systolic arterial pressure (mmhg) ± ± 1.78 * ± 1.77 * ± 1.69 * Diastolic arterial pressure (mmhg) ± ± 1.86 * ± 1.8 * ± 1.78 * ** - Statistically highly significant (P 0.01) *- Statistically significant (P 0.05)

103 83 Table 25: Radiographic findings in CKD dogs Findings No. of Dogs Percentage Normal in size Round kidneys Hydronephrosis Table 26: Nephrosonographic findings in CKD dogs Findings No. of dogs Percentage Indistinct corticomedullary junction Sunken kidneys Hyperechoic cortex Renal cyst ( Small spherical anechoic structures) Total Table 27: Classification of CKD dogs according to International Renal Interest Society (IRiS) standards based on serum creatinine levels Stage of CKD Stage I Stage II Stage III Stage IV Total No. of dogs Percentage

104 PCV (%) Hemoglobin (g/dl) 84 Fig 5: Values of Hemoglobin (g/dl) in treatment groups Control Group I Group II Group III Day 15th Day 30th Day 45th Day Fig 6: Values of PCV (%) in treatment groups Day 15th Day 30th Day 45th Day Control Group I Group II Group III

105 BUN (mg/dl) TEC (/µl) 85 Fig 7: Values of TEC (/µl) in treatment groups Control Group I Group II Group III 0 0 Day 15th Day 30th Day 45th Day Fig 8: Serum BUN profile in treatment groups Control Group I Group II Group III 0 0 Day 15th Day 30th Day 45th Day

106 creatinine (mg/dl) 86 Fig 9: Serum creatinine profile in treatment groups Control Group I Group II Group III Day 15th Day 30th Day 45th Day Fig 10: Classification of CKD dogs according to International Renal Interest Society (IRiS) standards based on serum creatinine levels 28.6% 21.4% 50% Stage II Stage III Stage IV

107 87

108 88

109 89

110 90 Table 28: The survival rate between treatment groups Group Total no. of cases No. of cases survived Percentage (%) I II III

111 91 Fig 11: Survival rate between treatment groups 30% 80% Group I Group II Group III 60%