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Downregulation of vasopressin receptor and aquaporin 2 in the inner medullary collecting duct of chronic… Lee, Andrew B. H. 2001

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DOWNREGULATION OF VASOPRESSIN RECEPTOR AND AQUAPORIN 2 IN THE INNER MEDULLARY COLLECTING DUCT OF CHRONIC RENAL FAILURE KIDNEY: SIGNIFICANCE OF ANGIOTENSIN II AND ENDOTHELIN REGULATION by Andrew B.H. Lee M.D., China Medical College, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Experimental Medicine Program We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A S e p t e m b e r 2001 © A n d r e w B . H . L e e , 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T In chronic renal failure (CRF) , inability to concentrate urine is, in part, due to downregulat ion of aquaporin 2 (AQP2) in the renal collecting duct. In the present study; we investigated the interrelationship of endothel in (ET), vasopress in , and A Q P 2 in the inner medullary collecting duct (IMCD) of k idneys obtained from C R F (5/6 nephrectomized) and control rats (sham-operated). Two ser ies of experiments were performed. In the first series, c learance studies showed that serum B U N increased in the C R F rats by 53.2% (from 23.27 ± 1.08 to 49.75 ±3 .13 mg%, p < 0.01). G F R decreased by 61.5% (from 0.89 ± 0.04 to 2.31 ± 0.09 ml/min, p < 0.01), and F E H 2 o increased by 81.0% (from 0.68 ± 0.05 to 3.57 ± 0.27 %, p < 0.01). ET-1 m R N A in the C R F rat IMCD increased by 56.3% (from 0.31 ± 0.04 to 0.71 ±0 .10 amol/u.g, P < 0.01). E T B m R N A decreased by 55.7% (from 14.00 ± 0.70 to 6.20 ± 0.43 amol/ng, p < 0.01). A Q P 2 m R N A decreased by 31.9% (from 13.90 ± 0.73 to 9.74 ± 0.83 amol/ug, p < 0.01). Changes of the E T B m R N A correlated with the changes in A Q P 2 mRNA , whereas changes in ET-1 m R N A inversely correlated with the changes in A Q P 2 mRNA . In the second series of experiment, an A C E inhibitor, enalapril, was administered to control and C R F rats. An ima ls were divided to 2 subgroups: group 1 consisted of control and C R F rats without any treatment; group 2 compr ised of control and C R F rats treated with enalapri l . Rena l c learance studies showed no significant renal functional improvement after enalapri l treatment. In C R F treated rats, serum B U N increased by 55.6% and G F R decreased by 58.0% compared to the control treated rats. In group one ii (without treatment), ET-1 m R N A increased by 69.9% (from 0.31 ± 0.03 to 1.03 ± 0. 26 amol/uo,, p < 0.01). E T B m R N A decreased by 65.8% (from 15.50 + 0.38 to 5.30 ± 0.36 amol/ug, p < 0.01). V 2 R m R N A decreased by 50.9% (from 1350.13 ± 222.92 to 662.68 ± 162.47 amol/ug, p < 0.01). A Q P 2 m R N A decreased by 29.7% (from 13.45 ± 1.34 to 9.46 ± 0.60 amol/ug, p < 0.01) in the C R F rats. In group 2 (enalapril treated), ET-1 m R N A increased by 41 .1% (from 0.33 ± 0.07 to 0.56 ± 0.05 amol/u,g, p < 0.01). E T B decreased by 43.9% (from 16.18 ± 1.09 to 9.07 ± 0.53 amol/ug, p < 0.01). V 2 R decreased by 40.4% (from 1558.76 ± 129.64 to 929.40 ± 211.83 amol/ng, p < 0.01). A Q P 2 decreased by 12.5% (from 20.68 ± 0.72 to 18.10 ± 2.17 amol/ng, p < 0.01) in the C R F rats. In situ hybridization images confirmed these results. Compared to the comparab le control rats, immunoblotting showed that V 2 R protein decreased by 52.4% and 42.9% in the untreated C R F and treated C R F rats, and A Q P 2 protein level dec reased by 29.0% and 11.8% respectively. Although enalapri l partially restored V 2 R and A Q P 2 m R N A level in the C R F rats, there was no significant change in the FE H 2o after treatment. These data indicate that: 1. ET-1 m R N A increased, whereas ET B , V2R, and A Q P 2 m R N A decreased in the IMCD of C R F rats. 2. A C E inhibitor enalapril partially normal ized ET-1, ET B , V2R , and A Q P 2 m R N A changes in the IMCD of the C R F rats. 3. Enalapri l partially restored V 2 R and A Q P 2 protein levels in the IMCD of C R F rats. 4. Enalapri l did not improve overall renal function in the C R F rats. Enalapri l partially corrected A Q P 2 m R N A levels, but failed to normal ize F E H 2 O in the C R F rats, suggest ing that collecting duct A Q P 2 level is partially responsible for the urine concentrating defect in this renal segment. iv T A B L E OF CONTENTS A B S T R A C T ii T A B L E OF CONTENTS v LIST OF FIGURES ix LIST OF T A B L E S xi LIST OF ABBREVIATIONS xii A C K N O W L E G E M E N T S xiv 1.0 INTRODUCTION 1 1.1 Urine Concentration 1 1.2 Short-term Regulation by Vasopressin 2 1.3 Long-term Regulation by Vasopressin 2 1.4 Vasopressin V2 Receptor (V2R) 3 1.5 Kidney Aquaporins and Urine Concentration 6 1.6 Downregulation of AQP2 in the C R F Rats 6 1.7 Basic Concepts of ETs 9 1.8 Endothelins in the Kidney 13 1.9 E T and Chronic Renal Failure 14 1.10 ET Receptors 15 1.11 ET Receptor Distribution in the IMCD 15 1.12 Endothelin Signaling Transduction Pathways in the Renal Cells - Role of PKC 16 1.13 Renal Ang II and Some Other Modulators 17 1.14 Interactions between ET-1 and the Renin-Angiotensin-Aldosterone System 18 v 1.15 ET-1 Effects on cAMP Accumulation and Collecting Duct Permeability 19 1.16 PKC might be an Inhibitor; PKA might be an Activator for V2R Expression 20 1.17 Hypothesis 21 1.18 Objectives and Rationale 22 2.0 MATERIAL AND METHOD 25 2.1 Materials 25 2.1.1 Animal Model for C R F 25 2.2 Methods 25 2.2.1 24-Hour Clearance Studies 25 2.2.2 Enalapril Treatment for the Normal and C R F Rats 26 2.3 mRNA Measurement-Competitive Reverse-Transcription Polymerase Chain Reaction (RT-PCR) 26 2.3.1 Total RNA Extraction 26 2.3.2 Total RNA Level Determination 27 2.3.3 cDNA Synthesis 27 2.3.4 Generation of Standard Curve for the Competitive R T - P C R 27 2.3.5 Competitive RT -PCR 30 2.3.6 DNA Signal Quantification 33 2.4 In Situ Hybridization 35 2.4.1 Controls 36 2.5 Western blotting 37 2.5.1 Cell Membrane Preparation 37 2.5.2 Sample Total Protein Level Determination 38 2.5.3 Protein Molecular Weight Standard 38 2.5.4 Electrophoresis and Protein Transfer 38 vi 2.5.5 Blocking Procedure for the Westernblotting 41 2.5.6 Antibody Incubation 41 2.5.6.1 Primary Antibody 41 2.5.6.2 Secondary Antibody 41 2.5.7 Signal Detection 42 2.5.8 Semi-Quantification on the Immunoblotting Signals 42 2.6 Data Analysis 48 3.0 RESULTS 49 3.1 Clearance Studies 49 3.1.1 Series One: Rats without Enalapril Treatment 49 3.1.2 Series Two: Rats with or without Enalapril Treatment 49 3.2 ET-1, ET B , and AQP2 mRNA Level in the Series One Animals 55 3.3 ET-1, ET B , V2R, and AQP2 mRNA Level in Series Two Animals 62 3.3.1 Rats with or without Enalapril Treatment 62 3.4 In Situ Hybridization mRNA Staining for ET-1, ETB, V2R, and AQP2 73 3.4.1 ET-1 mRNA 73 3.4.2 E T B mRNA 73 3.4.3 V2R mRNA 73 3.4.4 AQP2 mRNA 74 3.5 Westernblotting Protein Semi-Quantification 79 3.5.1 V2R 79 3.5.2 A Q P 2 79 4.0 DISCUSSION 85 vii 4.1 5/6 Nephrectomy as an Animal Model for C R F 87 4.2 V2R Expression is Downregulated in C R F 87 4.3 AQP2 is Downregulated in the C R F Rats 88 4.4 ET-1 is Increased in the Chronic Renal Failure IMCD 89 4.5 Possible Mechanisms for Controlling V2R in C R F 90 4.6 V2R Expression Rescue Experiments with Enalapril in C R F Rats 90 5.0 S U M M A R Y A N D C O N C L U S I O N 94 R E F E R E N C E S 96 viii LIST OF FIGURES Figure 1 3 Possible models for short-term and long-term AQP2 regulation by 5 vasopressin Figure 1 7.1 Primary structures of endothelins 11 Figure 1 7.2 Maturation of human ET 12 Figure 1 17 Possible mechanisms for Ang II, AVP, and ET-1 in regulation of AQP2 expression in C R F 24 Figure 2 3.4 An example for showing competitive RT -PCR standard curve 29 Figure 2 3.6 Representative gel of competitive RT-PCR standard curve of rat E T B 34 Figure 2 5.2 Westernblotting sample protein concentration standard curve 39 Figure 2 5.7 Principals for E C L system protein detection 43 Figure 3 1.1 Relationship between G F R & FE H2o in experiment series one rats 52 Figure 3 1.2 Relationship between G F R & FE H2o in experiment series two rats 54 Figure 3 2.1 Relationship between serum BUN & ET-1 in experiment series one rats 57 Figure 3 2.2 Relationship between G F R & ET-1 in experiment series one rats 58 Figure 3 2.3 Relationship between ET-1 & FE H2o in experiment series one rats 59 Figure 3 2.4 Relationship between FE H2o & AQP2 in experiment series one rats 60 Figure 3 2.5 Relationship between ET-1 & AQP2 in experiment series one rats 61 Figure 3 3.1.1 Relationship between G F R & ET-1 in experiment series two rats 64 Figure 3 3.1.2 Relationship between G F R & V2R in experiment series two rats 65 Figure 3 3.1.3 Relationship between G F R & AQP2 in experiment series two rats 66 Figure 3 3.1.4 Relationship between FE H2o & ET-1 in experiment series two rats 67 Figure 3 3.1.5 Relationship between FE H2o & V2R in experiment series two rats 68 Figure 3 3.1.6 Relationship between FE H2o & AQP2 in experiment series two rats 69 Figure 3 3.1.7 Relationship between V2R & AQP2 in experiment series two rats 70 Figure 3 3.1.8 Relationship between ET-1 & V2R in experiment series two rats 71 Figure 3 3.1.9 Relationship between ET-1 & AQP2 in experiment series two rats 72 Figure 3 4.1 In situ hybridization-ET-1 mRNA levels in IMCD 75 Figure 3.4.2 In situ hybridization-ETB mRNA levels in IMCD 76 Figure 3.4.3 In situ hybridization-V2R mRNA levels in IMCD 77 Figure 3.4.4 In situ hybridization-AQP2 mRNA levels in IMCD 78 Figure 3.5.1.1 Westernblotting-V2R protein level before enalapril treatment 81 Figure 3.5.1.2 Westernblotting-V2R protein level after enalpril treatment 82 Figure 3.5.2.1 Westernblotting-AQP2 protein level before enalpril treatment 83 Figure 3.5.2.2 Westernblotting-AQP2 protein level after enalapril treatment 84 x LIST OF TABLES Table 1.6 Classification of the causes of chronic renal failure 8 Table 2.3.5 Primers for competitive RT -PCR 31 Table 2.5.4 Components for Westernblotting mini-gels 40 Table 3.1.1 Clearance studies for the experiment series one animals 51 Table 3.1.2 Clearance studies for the experiment series two animals 53 Table 3.2 ET-1, E T B , & AQP2 mRNA level in the series one rats • 56 Table 3.3.1 ET-1, E T B l & AQP2 mRNA level in series two rats 63 Table 3.4 V2R & AQP2 in Westernblotting 80 XI LIST OF ABBREVIATIONS A C E Angiotensin converting enzyme Ang II Angiotensin II A N P Atrial natriuretic peptide AT1 Angiotensin II receptor subtype 1 A T 2 Angiotensin II receptor subtype 2 A Q P Aquapor in A Q P 2 Aquapor in 2 A V P Arginine vasopress in B U N Blood urea nitrogen c A M P Adenos ine 3', 5'-cyclic monophosphate C C T Cortical collecting tubules C R E c A M P responsive element C R E B 3', 5'-cyclic monophosphate responsive element binding protein C R F Chronic renal failure D A G Diacylglycerol D D A V P (1 -desamino-[8-D-arginine]) vasopress in ET Endothel in E T A Endothel in receptor subtype A E T B Endothel in receptor subtype B E T C Endothel in receptor subtype C FEfH20 Fractional excretion of water G F R Glomerular filtration rate Xll IL-1 lnterleukin-1 IMCD Inner medullary collecting duct IPs inositol 1, 4, 5-triphosphate M A P Mitogen activated protein m R N A Messenger ribonucleic acid NDI Nephrogenic diabetes insipidus Ox -LDL Oxidized low-density lipoprotein P B S Phosphate buffer sal ine P C - P L C Phosphatidylchol ine-specif ic phosphol ipase C P C - P L D Phosphatidylchol ine-specif ic phosphol ipase D P G E 2 Prostaglandin E 2 PI -PLC Phosphatidyl inositol-specif ic phosphol ipase C P K A Protein k inase A P K C Protein k inase C P L C Phospho l ipase C ppET-1 Preproendothelin-1 R A S Renin-angiotensin system R B F Rena l blood flow R T - P C R Reverse-transcription polymerase chain reaction S E M Standard error of mean S N G F R Single nephron glomerular filtration rate T N F - a Tumor necrosis factor-a V 2 R Vasopress in receptor V2 xi i i ACKNOWLEDGEMENTS I am grateful to my supervisor Dr. Norman W o n g for the opportunity to work in his laboratory and for his continual patience and support. I would also like to thank Dr. Eric Wong , Dr. Dirk Kerstan for their encouragement. I a lso appreciate A l ice Fok, Edward Mak, and Edward Roberts for their technical ass is tance. X I V 1.0 I N T R O D U C T I O N 1.1 U r i ne C o n c e n t r a t i o n Water from the glomerular filtrate is reabsorbed in three main regions of the renal tubule. In the first region, the proximal renal tubule, water reabsorption is assoc iated with pass ive sodium reabsorption. In the second region, the thin segments of the loop of Henle, the countercurrent multiplier system functions (74). In the third region, the thick ascending limb of the loop of Henle actively pumps sodium chloride out of the lumen to the medullary interstitial fluid. This segment is impermeable to water. Accordingly, it generates and maintains the hypertonic condition in the renal medul la. It is this hypertonic environment in the medullary interstitium that causes the withdrawal of water from the lumen of this descend ing limb of Henle's loop. The permeabil ity to water varies from segment to segment but is not physiologically regulated until the filtrate reaches the collecting duct, the last portion of the renal tubule. The regulator of water permeabil ity in collecting ducts is antidiuretic hormone (ADH) or vasopress in, re leased from the posterior lobe of the pituitary gland. A D H binds to the vasopress in V 2 receptor (V2R) to recruit specif ic water channels (such as aquaporin-2) onto the apical cell membrane of the collecting duct epithelial cells, thereby, increasing the water permeabil ity to this segment of the nephron. Vasopress in is crucial in urine concentration in that defective secret ion of this hormone can lead to urinary water loss (termed diabetes insipidus). Actually, the osmolal ity of filtrate is lower in the lumen of thick ascending limb of Hele 's loop relative to the p lasma. Vasopress in acts in the collecting duct to l concentrate urine. It regulates water reabsorption of the collecting ducts by two separated modes: short-term and long-term. 1.2 Short-term Regulation by Vasopressin The short-term regulation of water reabsorption by vasopress in is known as the shuttle hypothesis (127). This hypothesis proposes that binding of the vasopress in to the V 2 R increases the intracellular c A M P concentrat ion that in turn causes the fusion of cytoplasmic aquaporin-2 (AQP-2) containing ves ic les to the apical cell membrane. Thus, the permeabil ity to water increases in the collecting ducts. This mechan ism has been shown to be activated within minutes (90). Aquapor ins (AQPs) are specif ic membranous water channels and A Q P 2 is thought to be the main target for vasopress in regulation (57). 1.3 Long-term Regulation by Vasopressin Vasopress in not only increases the amounts of A Q P 2 in the apical membranes of the collecting duct cells, but also increases the transcriptional express ion of A Q P 2 genes. Incubation of renal collecting duct cells with A D H for 24 hours or more, increased the water permeabil ity because A D H enhances the transcription of A Q P 2 genes (30). Yasu i et al found that vasopress in activated transcriptional factors by phosphorylating adenos ine 3',5'-cyclic monophosphate responsive element binding protein ( CREB ) or by increasing the express ion of c-Fos. Therefore, binding of transcriptional factors to the c A M P responsive element (CRE) and AP1 activated the A Q P 2 promoter to increase the express ion of A Q P 2 m R N A (139). Thus, vasopress in exerts its 2 long-term regulatory effects on urine concentration by increasing the express ion of A Q P 2 in the collecting duct. Figure 1.3 shows the possible mechan i sms for vasopress in in regulating A Q P 2 . Vasopressin V2 Receptor (V2R) Hayash i et al. descr ibed the relationship between V 2 R and A Q P 2 levels in the collecting ducts. They concluded that the distribution and amount of A Q P 2 were regulated by the amount of vasopress in V 2 receptor (42). Diuretic animals induced by water overloading were found to have less A Q P 2 m R N A and protein express ion. Pretreatment with d D A V P before water loading could not restore the A Q P 2 express ion. However, the trafficking of A Q P 2 to the cell membrane was verified to be intact. Therefore, the "escape from vasopress in- induced antidiuresis is attributable, at least in part, to a vasopress in- independent decrease in A Q P 2 water channel express ion in the renal collecting duct" (32). Moreover, the IMCD of the previous exper imental vasopressin-resistant animals was shown to have less intracellular c A M P accumulat ion (31). This reduction in intracellular c A M P may be responsible for the downregulat ion of A Q P 2 . The inability to concentrate urine in C R F animals was not due to a reduction in vasopress in secretion. In fact, blood vasopress in levels were increased in C R F patients (49). It would be of interest to investigate the V 2 R express ion levels for 3 both m R N A and protein in the collecting duct of the C R F rats. Te i te lbaum et al showed that the IMCD taken from 5/6 nephrectomized rats lacked V 2 R m R N A express ion on the cell membranes (115). In their study, they also found that cultured IMCD cells failed to accumulate enough c A M P , which is a crucial secondary signaling messenger for vasopress in-mediated A Q P 2 installation or A Q P 2 synthesis. Therefore, they concluded that the A V P resistance in C R F is due, at least in part, to the selective downregulation of V 2 R m R N A express ion. 4 Vasopressin cell membrane V2R 1 cAMP i PKA activation nucleus c-Jun/c-Fos +- CREB-P synthesis API CRE AQP2 gene - transcription Figure 1.3 Possible models for short-term and long-term AQP2 regulations by vasopressin. Adapted from Aperia et al. 1997. 1.5 Kidney Aquaporins and Urine Concentration Rena l physiology with regard to urine concentration has exper ienced a dramatical ly revolution s ince many membrane transporters and receptor proteins were c loned. Aquapor ins (AQPs) are the most well known members among them. In general, A Q P s are integral membrane proteins containing 200 -300 amino acids. Their major function is to mediate water transport across the cell membrane. The specia l term "water channel" precisely descr ibes their biological functions. At least six A Q P water channels have been found to be expressed in the kidney (92,125,136). They all distribute in the kidney at specif ic sites along the nephron (93). Of the A Q P s that have been d iscovered to be contained in the kidney, A Q P 2 is the one that is found to be abundant in the apica l cell membrane as well as in subapica l ves ic les of the col lecting duct principal cells (91). It was first c loned by Se i et al (38). Vasopress in enhances water reabsorption in the renal collecting ducts by inserting A Q P 2 into the apical membrane or increasing A Q P 2 gene express ion. 1.6 Downregulation of AQP2 in the C R F Rats Chronic renal failure (CRF) is a clinical syndrome of different etiologies (see Table 1.6). Polyuria is one of the symptoms found in C R F . The ability to concentrate urine decreases with progression of renal failure. In exper imental C R F rats induced by 5/6 nephrectomy, polyuria was noted, and total kidney A Q P 2 level was found to be downregulated (76,82). S ince 6 vasopress in regulates A Q P 2 levels, attempts were made to restore A Q P 2 with vasopress in replacement. Surprisingly, d D A V P treatment in this model of C R F rats showed no improvement in A Q P 2 express ion. This result suggested that there was a vasopressin-resistant downregulation of A Q P 2 assoc iated with polyuria of the C R F rats (76). S ince A Q P 2 levels are c losely related to vasopress in regulation, downregulation of A Q P 2 in C R F rats was reasonably thought to be assoc iated with the impairment of vasopress in responses. Whether it is due to downregulation of V 2 R express ion needs to be further investigated. 7 Table 1.6 Classification of the causes of chronic renal failure. (Adapted from CHRONIC RENAL FAILURE, M. Roy First, M.D.) A. Primary Glomerular Diseases 1. Chronic glomerulonephritis o f various types 2. Goodpasture's syndrome 3. Henoch-Schonlein nephritis 4. Systemic lupus erythematosus 5. Polyarteritis nodosa B. Renal Vascular Disease 1. Bilateral ischemic disease o f the kidney 2. Malignant hypertension 3. Scleroderma 4. Sickle cell disease 5. Renal vein thrombosis C. Inflammatory Disease 1. Chron ic pyelonephritis 2. Tuberculosis D. Metabolic Disease with Renal Involvement 1. Diabetes mellitus 2. Gout 3. Hypercalcemia 4. Mul t ip l e myeloma 5. Amylo idos i s E. Nephrotoxins • 1. Analgesic nephropathy 2. Chronic heavy metal poisoning F. Obstruction Uropathy 1. Ca l cu l i 2. Neoplasm 3. Strictures 4. Retroperitoneal fibrosis 5. Prostatic hypertrophy 6. Congenital anomalies o f the lower urinary tract G. Congenital Anomalies of the Kidneys 1. Hypoplast ic kidneys 2. Polycyst ic kidney disease 3. Medul la ry cystic disease 4. Alpor t ' s syndrome (hereditary nephritis with deafness) 5. Fabry's disease (glycolipid lipoidosis) 6. Cystinosis 7. Oxalosis H. Miscellaneous 1. Chronic radiation nephritis 2. Balkan nephropathy 8 1.7 B a s i c C o n c e p t s of E T s In March 1988, Yanag i sawa et al identified a 21-amino-acid peptide (Figure 1.7.1), named 'endothelin'. They showed that endothel ial cel ls secreted ET and that it was the most potent vasoconstr ictor known (137). Three types of ET s have been identified: endothelin-1 (ET-1), endothelin-2 (ET-2), and endothel in-3 (ET-3) (61). ET-1 m R N A has only a short half-life of approximately 15 minutes (47). ETs are usually not stored in the cytoplasmic vesic les. It is likely that the secretion of ET depends on transcription and translation. Once ET s are secreted, they either bind to specif ic receptors or are rapidly degraded by proteolytic enzymes (1,29). In the kidney, ET-1 seems to be the main isoform responsible for most of the pathophysiology assoc iated with alterations in ET production. ET-2 was found to be produced by the intestine exclusively (105). ET-3 is synthesized in the kidney as well. However, the amount is much less than ET-1 . Human ET-1 m R N A encodes a 212 amino acid prepropeptide that contains a signal sequence at the amino terminal s ide as well as a region encoding an ET-1-l ike peptide of unknown function (47,137). The prepropeptide can be c leaved by one or more dibasic pair-specif ic endopept idases to yield a 38-amino-acid big ET-1 in humans (39 amino acids in other species) (86,137). This Big ET-1 is then converted to mature 21-amino-acid ET-1 by a putative endothelin-converting enzyme (ECE ) (Figure 1.7.2) (135,137). This conversion can occur either inside the cell or after big ET-1 is secreted. ET-1 is not only produced in the cardiovascular system, but also synthesized in the kidney. For instance, endothelial, mesangia l , glomerular 9 epithelial, and medullary collecting duct cells are thought to synthesize ET-1 (54,58-60). 10 Figure 1.7.1 Primary structures of endothelins. Adapted from Sokolovsky et al. 1995. 11 signal sequence V ET-like peptide (212) LYS-ARG ARG-ARG Dibasic pair-specific endopeptidases (s) Big ET (38) TRP-VAL ET converting enzyme(s) Mature ET (21) Neutral endopeptidases and others Inactive metabolites Figure 1.7.2 Maturation of human ET. Big ET-1 is converted to mature ET-1 both inside and outside the cell. (Adapted from Kohan et al. 1993) 12 c Endothelins in the Kidney Four types of cells in the kidney have been identified to produce endothel in. They are endothelial, mesangia l , glomerular epithelial, and tubular epithelial cells. Some factors can induce endothelin production in the renal endothel ial cell, such as vasoact ive agents (46,85,137), cytokines (75,78,141), and thrombin (14,137). Conversely, ET-1 production by endothelial cell is inhibited by oxidized low-density lipoprotein (Ox-LDL), atrial and brain natriuretic peptides, and nitrates (17,45,50,68,70). Mesangia l cells a lso produce ET-1 . In cultured studies, mesangia l cells were shown to synthesize and re lease ET-1 , yet the amount is much less when compared to the cultured endothel ial cel ls (59). Glomerular epithelial cells, like the other two cell types, have the ability to synthesize ET-1 (54). Moreover, many studies showed that renal tubular epithelial cel ls contribute to the production of ET-1 (60). For instance, porcine inner medul la was investigated to have the highest ET-1 concentrat ion in the body (55). Studies on renal epithelial cell lines (72,95), cultured tubular cells (58,63), and microdissected renal tubules (70,121,122) have all pointed out that the collecting duct, particularly the inner medullary collecting duct (IMCD), was the major site for ET-1 production in the kidney. 13 E T a n d C h r o n i c R e n a l Fa i l u re P lasma big-endothelin-1 was found to be significantly increased in hemodia lysed and nonhemodia lysed C R F patients (106). Severa l reports showed that excess ive amount of ET-1 is excreted into the urine of 5/6 nephrectomized rats. In those studies, excess urine ET-1 has been demonstrated to be produced by the remnant renal t issue (14,18). Rena l ET gene express ion has been shown to be increased in the remnant kidney and seems to correlate with d isease progression (98). In situ hybridization study showed that ET-1 m R N A production increased initially in the renal tubules and subsequent ly in glomeruli as renal failure progressed (20). Rena l ET-1 was elevated in the remnant kidneys (79), and the increase of ET-1 production in the C R F kidneys would have pathological implications for the local vasodynamic or diuretic effects. ET-1 causes renal vasoconstr ict ion in both afferent and efferent arterioles and reduces renal blood flow and G F R (10). From its diuretic effects, ET-1 was reported to have diuretic action in the IMCD (119) and to increase sodium and/or water excretion (51,109). Moreover, ET-1 causes an increase in urine excretion in spite of the reduction in G F R by 20% (10). Water c learance is increased because of increased ET-1 production (40,88). Whether ET-1 changes the osmotic permeabil ity of renal collecting ducts and generates its diuretic effects remain to be examined. 1 4 1.10 E T R e c e p t o r s ETs bind to their complementary receptors and form very strong l igand-receptor complexes that are rapidly internalized and inactivated (102). The fact that the sites of ET production are closely linked to the sites of ET receptors suggest ing ETs act mainly as autocrines or paracrines (52). There are two major types of mammal ian ET receptors, named E T A and E T B receptors. E T A receptor was originally c loned from bovine lung t issue (5) and was subsequent ly identified in human t issues (44). Human E T A receptor contains 427 amino acids and binds ET s in the preferential order of ET-1 > ET-2 » ET-3 (5,44). The E T B receptor was originally c loned from rat lung t issue (108) and was subsequent ly c loned from human t issues (107). Human E T B receptor contains 442 amino acids and binds ET-1 , ET-2, and ET-3 with equal affinities (107). A third receptor subtype E T C was isolated and c loned from cultured Xenopus laevis dermal melanophores. It has greater affinity and sensitivity for ET-3 over other ET isoforms (53). Thus far, E T C has not been found in any mammal ian t issues. 1.11 E T R e c e p t o r D is t r i bu t i on in the I M C D Although publ ished data varied in describing the distribution of E T A receptor in the renal IMCD of different spec ies, E T B receptor was consistently found to be abundant in the renal tubules (25,64,116). S ince ET-1 is cons idered to function as an autocrine or paracrine hormone, the amount or type of ET receptors found in the IMCD is crucial. The IMCD is considered to be a major site for 15 renal ET-1 production (62). Bes ides, IMCD contributes to urine concentrat ion by responding to vasopress in, and subsequent ly through the insertion of A Q P 2 water channel into the collecting duct cell membranes. Because E T B receptor is abundant in the IMCD, we proposed that binding of the ET-1 to the putative E T B receptor could have impacts on urine concentration by the IMCD. The role played by ET-1 in affecting the urine concentration in the IMCD is not known. Cou ld this ET-1 modulation relate to changes in A Q P 2 water channel transferring or express ion? If such a relationship exists, what possib le signaling transduction pathway is involved in this modulat ion? Addit ional studies are needed to examine the problem. 1.12 Endothelin Signaling Transduction Pathways in the Renal Cells-Role of PKC Despite its short half-life in the circulation, ET-1 can maintain its biological effects for a longer period of time. Prolonged biological effects might be assoc iated with receptor internalization followed by externalization of new or recycled receptors (84,101). In the kidney, multiple signaling transduction pathways have been assoc iated with ET-1 stimulation. First of all, ET-1 activates phosphatidyl inositol-specif ic phosphol ipase C (PI-PLC) in mesangia l cel ls (110), cortical collecting duct cells (71), IMCD cells (22), and renal medullary interstitial cells (131). This results in accumulat ion of inositol tr iphosphate (IP 3), the transient increase of cytoplamic free calc ium concentration [Ca 2 + ] i , and a parallel transient increase of diacylglycerol (DAG) . 16 Transient increase of D A G activates P K C activity. Activation of P I -PLC is thought to be linked to E T A or E T B subtype (94). ET-1 activation of cytosol ic variety of P L A 2 ( cPLA 2 ) is assoc iated with e icosanoid production (mainly P G E 2 ) (11,22,36,66,120) and is thought to be P K C independent. Yet the ET receptor type related to P L A 2 activation remains unknown. ET-1 activation of phosphatidylchol ine-specif ic phosphol ipase D (PC-PLD) also correlates to D A G production (94). However, ET-1 evoked P C - P L D activation (in the renal medullary interstitial cells) is considered to be modulated by the dual regulations of P K C and [Ca 2 +]j (35). ET-1 also stimulates phosphatidylchol ine-specif ic phosphol ipase C (PC -PLC ) . ET-1 evoked signaling pathway might be assoc iated with the prolonged production of D A G (94). Finally, ET-1 can enhance the phosphorylation of protein tyrosine k inases such as mitogen activated protein (MAP) kinase through E T A receptor (129). Taken together, intra-renal ET-1 correlates to at least five different signaling pathways. Among those different pathways, P K C is activated for a transient or prolonged period. Intracellular activated P K C in different renal cell types is involved in the downstream effector activation or inhibition. It may become a modulator for s ome transcriptional factors. Thus, it affects the nuclear gene transcription for long-term regulation. 1.13 R e n a l A n g II a n d S o m e O the r M o d u l a t o r s Renin, which activates Ang II, was thought to be synthesized only in the juxta-glomerular cells. Recently, it was also found to be synthesized in a number of 17 extrarenal and intra-renal cells. Locally synthesized or stored Ang II regulates some distinct physiological functions of the cells in a paracrine or autocrine manner (7,77). Two major types of Ang II receptors have been c loned. They are AT1 and AT2 receptors. Both of them are seven-t ransmembrane domain G-protein coupled receptors. The AT1 receptor is thought to be the major receptor subtype in the adult kidney. It is assoc iated with Gq/11, Gi , or Go proteins (6,7). AT2 receptor mainly exists in fetus and is related to cell differentiation in the fetal kidney (6). In smooth musc le cells, Ang II activates P K C by generating either inositol 1,4,5-triphosphate (IP3) or diacylglycerol (DAG) through AT1 receptor following phosphol ipase C-P i activation (6). Ang II also suppresses c A M P production. This is sequential to the inhibitory effect through G i protein on adenylate cyc lase and various metabol ites of arachidonic acid re leased after stimulation of phosphol ipase C, A2 and D activities (6). In addition, AT1 receptor effects can also be mediated by some growth factors, cytokines, and other peptides, such as ET, transforming growth factor -p i , and platelet-derived growth factor. 1.14 Interactions between ET-1 and the Renin-Angiotensin-Aldosterone System The renin-angiotensin system (RAS) and the E T system are the most potent vasopressor systems. A lot of research has addressed the importance of R A S or ET system in the cardiovascular field. However, relatively little information focused on their interrelationship in the kidney. In in vivo studies, intravenous infusion of Ang II induced threefold increase of ET-1 in the whole kidney, and 18 E T A receptor could be involved in this ET-1 induction. (12,13). Intrarenal renin-angiotensin system was found to be effective in modulating renal hemodynamics and tubular transport function. In the kidney, Ang II can act as a paracrine or autocrine. It was noted that the medullary Ang II content per gram of t issue has been quoted to be four to five t imes higher than the cortical content (89). Reduced renal blood flow and presence of albuminuria caused by intravenous Ang II infusion may be normalized by non-select ive E T A / E T B receptor antagonist bosentan (43). The above evidence suggested that Ang II might exert its effects through the endothelin system. On the other hand, ET-1 has some negative feedback on the RAS . For instance, ET-1 can inhibit renin synthesis (103). ET-1 and Ang II were shown to be upregulated in the remnant kidney of C R F rat (14,18,79,100), suggest ing that these two systems may function independently or cooperatively in the C R F kidney. This phenomenon requires further investigation. 1.15 ET-1 E f fec ts o n c A M P A c c u m u l a t i o n a n d C o l l e c t i n g D u c t P e r m e a b i l i t y ET-1 inhibits vasopress in (AVP)-st imulated, but not basal , c A M P accumulat ion in the rat cortical collecting tubules (CCT) (118). Also, ET-1 inhibits A V P -stimulated osmotic water permeabil ity in the inner medullary collecting duct (96). A n experiment on freshly prepared renal papillary tubules revealed that ET-1, ET-3, and sarafotoxin S 6 b had similar potencies as inhibitors of c A M P 19 accumulat ion (133). This result suggests that E T B receptor might be the key ET receptor that is involved in this mechan ism because E T B receptor has similar affinities to all types of ETs . Bes ides, the result that E T A antagonist had no effect of inhibiting AVP- induced c A M P accumulat ion further supported this idea (33). E T B receptor in porcine kidney tubular epithelium ( LLC -PK i ) was found to be coupled to two different types of signaling transduction cascades . One is to reduce c A M P production, and the other is to stimulate c G M P production via distinct G-proteins (99). Activation of P K G by c G M P was thought to be a factor for preventing c A M P accumulat ion (39). In the IMCD, ET-1 stimulated prostaglandin E 2 ( P G E 2 ) production through E T B receptor (66), an effect that may result in the inhibition of IMCD Na-K-adenosinetr iphosphatase (Na-K-ATPase) activity (142). 1.16 PKC might be an Inhibitor; PKA might be an Activator for V2R Expression In the kidney, P K C activity is triggered by the regional renin-angiotensin or endothel in systems. Studies in our laboratory showed that P K C could be a factor for inhibiting V 2 R gene express ion in renal IMCD. Rat IMCD t issue incubated with P M A (PKC activation) overnight was observed to have significantly less V 2 R m R N A express ion compared to the control with no P M A treatment. In the same study, A Q P 2 m R N A was also downregulated because A Q P 2 express ion was related to the V 2 R express ion. Therefore, one of the possibil it ies for the inability of remnant kidney to concentrate urine could be 20 related to P K C activation, which, probably correlates to the elevated Ang II or ET-1 production in the kidney. This hypothesis needs further confirmation. In contrast, P K A might enhance V 2 R m R N A express ion. Our group also confirmed that 24-hpur incubation of IMCD in dybutyry l-cAMP ( PKA activation) enhanced V 2 R m R N A express ion (132). These data suggested that k inases such as P K C and P K A could be seen as two independent systems that function and ba lance each other in order to maintain renal vasodynamic or tubular transporting homeostas is . More interestingly, in cultured renal L LC -PK1 cell, P K C and P K A signal transduction systems act to potentiate each other (4). It s eems that compl icated but del icate modulat ions by P K C and PKA- induced micro-mechan i sms contribute and maintain the physiological intracellular homeostas is . 1.17 H y p o t h e s i s The inability of the chronic renal failure (CRF) kidney to concentrate urine is due to several factors. These include an increase in single nephron glomerular filtration rate ( SNGFR ) (128), redistribution of the renal blood flow from the cortex to the medul la (21), and altered responses of the kidney to hormone stimulation in order to maintain body fluid homeostas is . C R F rats are found to have elevated renal angiotensin II (Ang II) and endothelin-1 (ET-1) levels (79,100) that can alter protein k inase C (PKC) (94,123) and protein k inase A (PKA) (33) activities in the inner medullary collecting duct (IMCD) and may impact the concentrating ability of the kidney. Recent studies in our laboratory 21 showed P K C and P K A are modulators for V2 receptor (V2R) express ion in the inner medullary collecting duct which in turn controls aquapor in 2 (AQP2) express ion (42). W e hypothesize that Ang II and endothel in modulate the distribution of V 2 R in the IMCD of renal failure kidney. Figure 1.17 illustrates the hypothesis. Thus, the goal of this study is to examine the role of Ang II and endothel in in regulating the express ion of V 2 R in the IMCD of the 5/6 nephrectomized rats. 1.18 Objectives and Rationale The current study was undertaken to examine the mechan i sms for the defective urine concentrating ability of the IMCD during chronic renal failure. Two ser ies of exper iments were performed. 1. Competit ive R T - P C R was performed to evaluate ET-1, E T B receptor, and A Q P 2 m R N A levels in the IMCD. This set of exper iments was to explore inner medullary collecting duct ET-1, ET B , and A Q P 2 express ion in normal and C R F rats. 2. To elucidate the role of Ang II in C R F , changes of V 2 R and A Q P 2 m R N A levels as well as changes of their protein express ion were examined in response to A C E inhibitor enalapri l. 22 It is anticipated that the findings of this study will be significant in interpreting the roles for Ang II and ET-1 play in regulating vasopress in V 2 R and A Q P 2 density in the IMCD of C R F kidneys. Understanding the involvement of Ang II and ET-1 in regulating A Q P 2 levels may help to develop new strategies in preventing polyuria in C R F patients. 23 2.0 M A T E R I A L A N D M E T H O D 2.1 Ma te r i a l s 2.1.1 A n i m a l M o d e l fo r C R F Male Wistar rats weighing between 250-350 g were purchased from the U B C animal center were used in our experiments. Al l animals were housed in controlled temperature, humidity condition and received standard laboratory rat chow and tap water. Renal mass reduction (5/6 nephrectomy) was performed by two consecut ive steps of surgical intervention. In the first surgery, 2/3 of the left kidney was removed. Two weeks after the first operation, we removed the right kidney. All the renal mass reduction procedures were done under intraperitoneal Pentobarbital anesthes ia (0.1 ml/100 g) (Somnotol, 50 mg/kg, M T C Pharmaceut ica ls , Cambr idge, Ontario, Canada) . Studies were performed ten days after 5/6 nephrectomy. Remnant or intact kidneys were removed from the 5/6 nephrectoimized and sham operated rats, respectively for R N A extraction (for the competitive RT -PCR) , cell membrane preparation (for the Westernblots), or t issue fixation (for the in situ hybridization). 2.2 M e t h o d s 2.2.1 24 -Hou r C l e a r a n c e S t u d i e s 24-hour urine collection was done with metabolic cages before animals were sacr i f iced. Blood samples were obtained from the abdominal aorta of each rat. Serum BUN , serum creatinine, and urine creatinine were determined using diagnostic kits purchased from S I G M A (Aldrich, Ontario). Rena l G F R and water excretion were calculated using standard equation. 2.2.2 Enalapril Treatment for the Normal and CRF Rats Male Wis tar rats were divided into two groups. Group 1 cons isted of sham-operated and 5/6 nephrectomized rats. They were not treated with enalapri l (SIGMA, Aldrich, ON). Group 2 also composed of sham-operated and 5/6 nephrectomized rats but were all treated with enalapri l (subcutaneously, 2.5 mg/kg/day) for 11 days. 24-hour c learance study was also performed to monitor the renal function. The IMCD was isolated and extracted to quantify V2R , ET-1 , E T B receptor, A Q P 2 m R N A (using competit ive R T - P C R ) and protein (using Western blot) levels. Some of the kidneys were paraffin sect ioned for the in situ hybridization. 2.3 mRNA Measurement-Competitive Reverse-Transcription Polymerase Chain Reaction (RT-PCR) 2.3.1 Total RNA Extraction Total R N A was extracted from IMCD tissue samples (50-1 OOmg) in 1 ml of TriZol Reagent (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) . Total R N A was extracted and precipitated using chloroform (SIGMA, Aldr ich, Canada) and isopropanol (SIGMA, Aldrich, Canada), respectively. R N A pellet was washed and dissolved with 75% ethanol and DNase & RNase- f ree water (SIGMA, Aldrich, Canada), respectively. 26 2.3.2 Total RNA Level Determination Total R N A concentration was determined by spectrophotometr ic analys is ( PERK IN E L M E R , Miss issauga, ON). OD 2 6o /OD 2 8 o ratio was calculated to monitor the R N A dissolved efficiency (partially dissolved R N A samples would have an A26o/28o ratio < 1.6). 2.3.3 cDNA Synthesis Synthes is of c D N A from total R N A was carried out by mixing Ol igo (dT) i 2 - is random primer (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , total R N A sample, and DNase & RNase- f ree water. The mixture was heated to 70°C for 10 minutes and quickly chilled on ice. The contents of the tubes were centrifuged and 5x First Strand Buffer (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , 0.1 M DTT (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , and d N T P Mix (10mM each dATP , dGTP , dCTP , and dTTP at neutral pH) (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) were added. Contents of the tubes were gently mixed and incubated at 42°C for 2 minutes. S U P E R S C R I P T ™ II (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) was then added and the contents incubated at 42 °C for 50 minutes. The reaction is inactivated by heating at 70 °C for 15 minutes. 2.3.4 Generation of Standard Curves for the Competitive RT-PCR Target and competitor D N A products were synthesized with appropriate primers. The D N A products were separated in 1.5% agarose gels. A gel 27 extraction kit (Q IAGEN Inc, Miss issauga, ON) was used to gene c lean target and competitor DNA. In order to make a linear titration curve between the logarithm of the band ratio (Target/Competitor) and the logarithm of the initial amount of target band, serial dilutions of the target DNA product were mixed with s ame amount of known concentration of competitor D N A to compete in the P C R amplif ication. A representative gel of competitive R T - P C R standard curve is shown in Figure 2.3.4. 28 LO C\l CO CO o + X CT> CO o CO CD CD - r CM oo CD \ r ^ cp o o o 0 S (louie) i Bo| LO • o o Ui o LO d LO or o a. H a: > & s E> a. ^ E H o ° * ^ •S £ o Q) U ^ 8 "o o ® £ ° _ Co | IX o CO a> i_ 3 14. CD CM 2.3.5 C o m p e t i t i v e R T - P C R P C R was performed by adding 10X buffer (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , d N T P Mix (10mM each dATP , dGTP , dCT P , and dT TP at neutral pH) (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , MgCI 2 (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , primers for targets, c D N A products from tissue samples, Taq (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) , and RNa se & DNase-free water (SIGMA, Aldr ich, Canada) . The amplif ication profile for A Q P 2 and its competitor was as follows: 95°C for 30 sec, 55°C for 45 sec, 72°C for 1 min for 30 cycles, fol lowed by strand extension at 72°C for 7 min. The amplification profile for E T B and its competitor was as follows: 95°C for 30 sec, 60°C for 45 sec, 72°C for 1 min for 30 cycles, fol lowed by strand extension at 72°C for 7 min. The amplif ication profile for ET-1 and its competitor was as follows: 95°C for 30 sec, 65°C for 45 sec, 72°C for 1 min for 30 cycles, followed by strand extension at 72°C for 7 min. The amplif ication profile for V 2 R and its competitor was as follows: 95°C for 30 sec, 65°C for 1 min, 72°C for 1 min for 25 cycles, followed by strand extension at 72°C for 7 min. DNA primers used in competitive R T - P C R are listed in Table 2.3.5. 30 Table 2.3.5 Primers for Competitive RT-PCR Sense primer Anti-sense primer AQP2 5' - T C C T T C CTT C G A G C T G C C TT- 3' 5' - A C G T T C C T C C C A G T C G G T GT- 3' AQP2 competitor 5' - T C C T T C C T T C G A G C T G C C T T - 3 ' 5' - A C G T T C C T C C C A G T C G G T G T C A G G G G T C C G A T C C A G A A G A- 3' ET-1 5' - C G T T G C T C C T G C T C C T C C T T G A T G G- 3' 5' - A A G T C C C A G C C A G C A T G G A G A G C G - 3' ET-1 competitor 5' - C G T T G C T C C T G C T C C T C C T T G A T G G- 3' 5' - A A G T C C C A G C C A G C A T G G A G A G C G T G C T G T T G C T G A T G G C C T C C - 3' E T B 5' - T T A C A A G A C A G C C A A A G A C T - 3 ' 5' - C A C G A T G A G G A C A A T G A G A T - 3 ' 31 E T B c o m p e t i t o r 5' - T T A C A A G A C A G C C A A A G A C T - 3 ' 5' - C A C G A T G A G G A C A A T G A G A T A G C A G C A C A A A C A C G A C T T A- 3' V2R 5' - A G C A A C A G C A G C C A G G A G G A A C- 3' 5' - G G C C C A G C A A T C A A A C A C C C - 3 ' V2R c o m p e t i t o r 5' - A G C A A C A G C A G C C A G G A G G A A C- 3' 5' - G G C C C A G C A A T C A A A C A C C C G C C A G G A T C A T G T A G G A G G A G G - 3' 32 2.3.6 D N A S i g n a l Quan t i f i ca t i on D N A competit ive R T - P C R products were separated in 1.5% agarose gels and stained with ethidium bromide. Densit ies were analyzed by computer densitometry (Alphalmager 1200 A lpha Innotech Corporat ion, San Leandro, CA) . A representative gel of competitive R T - P C R standard curve of rat ETB receptor is presented in Figure 2.3.6. 33 LANE 1 1KB DNA LADDER LANE 2 target 27.27769 amol + competitor 3.60295 amol LANE 3 target 13.63884 amol + competitor 3.60295 amol LANE 4 target 6.819422 amol + competitor 3.60295 amol LANE 5 target 3.409711 amol + competitor 3.60295 amol LANE 6 target 1.704855 amol + competitor 3.60295 amol LANE 7 target 0.852428 amol + competitor 3.60295 amol LANE 8 target 0.426214 amol + competitor 3.60295 amol LANE 9 target 0.213107 amol + competitor 3.60295 amol LANE 10 target 0.106553 amol + competitor 3.60295 amol LANE 11 rat sample control LANE 12 rat sample chronic renal failure mm mat mm mm mm img. • - - — - ~ | Nov 13.1998 RAT ETBR Stardard cuve_ Target=564bp competitor=427bp Figure. 2.3.6 REPRESENTATIVE GEL OF COMPETITIVE RT-PCR STANDARD CURVE OF RAT ETBR 34 In Situ Hyb r i d i za t i on The animals were anaesthet ised with sodium pentobarbital (Somnotol; M T C Pharmaceut ica ls , Cambr idge, ON), and transcardially perfused with phosphate-buffered sal ine (PBS), pH 7.4, followed by a fixative containing 4% paraformaldehyde in P B S on ice. The kidneys were removed and placed overnight in the same fixative at 4°C. After 24 hours, the perfusion-fixed kidneys were rinsed in cold PBS , dehydrated in ascending concentrat ions of ethanol, and embedded in paraffin. K idney sect ions were cut at 7urn, p laced on electrostatically-treated sl ides (Superfrost*Plus; Fisher, Nepean, ON), and dried overnight at 37°C. The sect ions were heat fixed for 30 minutes at 55°C and deparaff inized through toluene and a graded ethanol series. Sect ions were rehydrated in P B S containing 0.1% active diethyl pyrocarbonate (DEPC) , deproteinated in 0.1 M HCI for 10 minutes, and rinsed briefly in P B S . Fol lowing digestion with proteinase K (Dako Corporation, Carpinteria, CA), s l ides were postfixed with 4% paraformaldehyde, rinsed with PBS , dehydrated through ethanol, and air dried. The sect ions were prehybridized for 2 hours at 37°C in a hybridization solution containing 50% formamide (OMNIPUR, E M Sc ience, Gibbstown, NJ). Hybridization was performed with a heat-denatured digoxigenin-labeled D N A probe ( P C R DIG Label ing Mix, Boehringer Mannheim) diluted in the same hybridization solution at 37°C for 16 hours in a humid chamber. The sl ides were washed sequential ly with 2x S S C and 1x S S C at 55°C, fol lowed by 0.5x S S C and 0.2x S S C at room temperature. S l ides were washed with buffer 1 (0.1 M Tris-HCI, 0.15 M NaC l , pH 7.5) and incubated in buffer 1 containing 1% blocking reagent (Boehringer Mannheim) for 30 minutes at room temperature. Immunological detection of the DIG-labeled hybrids with alkal ine phosphatase-conjugated sheep anti-digoxigenin antibody was performed as recommended by the manufacturer (DIG Nucleic Ac id Detection Kit, Boehringer Mannheim). The sl ides were washed twice with buffer 1 fol lowed by immersion in buffer 3 (0.1 M Tris-HCI, 0.1 M NaC l , 0.05 M MgCI 2 , pH 9.5). The site of alkaline phosphatase was developed by reaction with nitroblue tetrazol ium and 5-bromo-4-chloro-3-indolyl-phosphate. The color reaction was stopped with 10 mM Tris-HCI, 1 mM EDTA, pH 8.0. S l ides were rinsed with distilled water and covered with an aqueous mounting medium. Visual izat ion of the in situ hybridization signal was performed using a Z E I S S Ax ioskop 2 light microscope. Digital images of the kidney medul la ( IMCD cells) were captured using a monochrome C C D camera (Pentamax, Pr inceton Instruments, Inc, Trenton, NJ). 2.4.1 C o n t r o l s The specificity of the probes for in situ hybridization in kidney t issue were confirmed by the following: (1) competitive hybridization in which the sect ions were incubated with the labeled probe in the presence of a 100-fold excess of 36 the s ame non-labeled probe; (2) pre-treatment of the t issue sect ions with R ibonuc lease A (0.1 mg/ml; S igma) for 60 minutes at 37°C; (3) omiss ion of the probe in the hybridization mixture. All controls resulted negative. 2.5 Western blotting 2.5.1 Cell Membrane Preparation Kidneys removed from the C R F or sham operated rats were placed in Petri d ishes containing 4°C P B S (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) . IMCD were dissected from these kidneys and placed in Petri d ishes containing a small amount of homogenizat ion buffer (contains protease inhibitors P M S F 250 nM, Leupeptin 2 mg/ml, Pepstat in A 1.4 mg/ml, and Aprotinin 5 mg/ml) (SIGMA, Aldrich, Canada) . The IMCD were then frozen at - 70°C for 10 min then thawed at room temperature. After thawing, the IMCD were minced into smal l p ieces and transferred into centrifugation test tubes containing 2 ml of homoginization buffer solution (contains protease inhibitors), then homogenised at 4°C. After homogenizat ion, the t issue-homogenizat ion fluid mixture was centrifuged at 4°C at 1,000 rpm for 15 min. The supernatants were transferred into centrifuge tubes and centrifuged at 4°C at 15,000 rpm for 20 min. The supernatants were discarded and the pellets resuspended with equal amounts of TRIS-glyc ine S D S solution (contains protease inhibitors as the above). In the end, a smal l amount of beta-mercaptoethanol (SIGMA, Aldrich, Canada) was added to each sample. 37 2.5.2 S a m p l e To ta l P ro te i n L e v e l De te rm ina t i on Protein concentration was determined by the B io-Rad Protein M ic roassay (5x concentrated from Bio-Rad, Miss issauga, ON). Using this protein assay kit, O.D. was measured at 595 nm (PERK IN E L M E R , Miss i ssauga, ON). An example curve for determining protein concentration by the kit is presented in Figure 2.5.2. 2.5.3 P ro te i n M o l e c u l a r W e i g h t S t a n d a r d A high-ranged prestained protein molecular weight standard (LIFE T E C H N O L O G I E S , Burlington, Ontario, Canada) was used in the Westernblott ing. 2.5.4 E l e c t r o p h o r e s i s a n d P r o t e i n T r a n s f e r Samp le proteins were separated in a 10% S D S - P A G E . Components for the stacking and resolving gels are showed in Table 2.5.4. Separated proteins were then transferred to a nitrocellulose membrane (0.45 pm Trans-Blot Transfer Med ium BIO-RAD, Miss i ssauga, ON) using Trans-Blot S D Semi-Dry Electrophoretic Transfer Cel l (BIO-RAD, Miss issauga, ON). 38 Table. 2.5.4 Components for Westernblotting mini-gel Resolving gel 10% H 2 0 2.1 ml 30% acrylamide mix 2 ml 1.5 M Tris (pH 8.8) 1.3 ml 10% SDS 0.05 ml 10% ammonium persulfate 0.05 ml TEMED 0.002 ml Total volume 5.502 ml Stacking gel 5% H20 0.884 ml 30% acrylamide mix 0.22 ml 1.0 M Tris (pH6.8) 0.17 ml 10% SDS 0.013 ml 10% ammonium persulfate 0.013 ml TEMED 0.002 ml Total volume 1.302 ml 10% ammonium persulfate was renewed every 7 days 40 2.5.5 Blocking Procedure for the Western Blotting Blocking procedure was performed by incubating nitrocellulose membranes in a 5% skim milk powder solution containing 0.02% sodium az ide at room temperature overnight. 2.5.6 Antibody Incubation 2.5.6.1 Primary Antibody After blocking procedure, nitrocellulose membrane was incubated in the primary antibody. Anti-rat A Q P 2 antibody was purchased from C H E M I C O N International, Inc, Temecu la , CA . Anti-rat V 2 R antibody was purchased from A lpha Diagnostic Intl, Inc, San Antonio, TX. Primary antibody incubation was performed for an hour at room temperature with agitation. After incubation, membrane was soaked and rinsed with 0.1% P B S T solution to rid of the excess primary antibody. 2.5.6.2 Secondary Antibody Secondary antibody was a donkey anti-rabbit horseradish peroxydase linked antibody (Amersham LIFE S C I E N C E , Little Chalfont Buckinghamshire, England). Incubation time for the secondary antibody was an hour at room temperature with agitation. After incubation, blot was also soaked and rinsed in 0.1 % P B S T solution to abol ish excess secondary antibody. 41 2.5.7 Signal Detection An enhanced chemi luminacence detection solution ( EC L detection kit, Ame r sham Pharmac ia Biotech, Buckinghamshire, England) was mounted to the protein s ide of the whole nitrocellulose membrane for one minute at room temperature. Principals for the E C L detection are shown in Figure 2.5.7. The membrane was then wrapped in an appropriate s ized c lean siren wrap and placed in a film development cassette to expose to a K O D A K X - O M A T film ( E A S T M A N K O D A K C O M P A N Y , Rochester, NY). Fi lms were deve loped by K O D A K M35A X - O M A T Processor ( K O D A K Diagnostic Imaging). 2.5.8 Semi-guantification on the Immunoblotting Signals Fi lms ( K O D A K X -OMAT) for Westernblott ing were developed and then scanned by a scanner (Hewlett Packard, NY). Fi les were unfolded in an image analytical program (Scion Image. Wayne Rasband, National Institutes of Health, USA , modified by Sc ion Corporation, frederick, Maryland) for the immunoblot protein density analysis. Blot density was picked up and est imated by this analytical program. 42 Experimental Protocol for Competitive RT-PCR Wistar rats weighing 250~350 g Experimental group (n=19) Control group (n=19) First operation Left kidney 2/3 renal mass reduction Sham-operated Wait for two weeks Second operation Right kidney total nephrectomy Optimal humidity, temperature Chow food, tap water accessible for 10 days Metabolic cage set-up 24-hour urine collection before animal sacrifice IMCD mRNA extraction Competitive RT -PCR ET-1, ET B , V2R, A Q P 2 44 Experimental Protocol for In Situ Hybridization Wistar rats weighing 250-350 g Experimental group Control group First operation Left kidney 2/3 renal mass reduction , rWait for 2 weeks Second operation Right kidney total nephrectomy Optimal humidity, temperature Chow food, tap water accessible for 1 0 days Metabolic cage set-up 24-hour urine collection before animal sacrifice i Animal kidney perfusion and P F A fixation In situ hybridization for IMCD ET-1, E T B , V2R , A Q P 2 Sham-operated 45 Experimental Protocol for Westernblot Wistar rats weighing 250-350 g Experimental group First operation Left kidney 2/3 renal mass reduction , rWait for two weeks Second operation Right kidney total nephrectomy Optimal moisture, temperature Chow food, tap water accessible for 10 days Metabolic cage set-up 24-hour urine collection before animal sacrifice IMCD cell membrane preparation Westernblot AQP2 , V2R Control group r Sham-operated 46 Experimental Protocol for Enalapril-treated Rats in Competitive RT-PCR, In S i t u Hybridization, and Westernblot Wistar rats weighing 250~350 g 1 r 1 Experimental group Control group First operation Left kidney 2/3 renal mass reduction Sham-ope rated r Wa i t for 3 weeks Second operation Right kidney total nephrectomy r r Optimal humidity, temperature Chow food, tap water accessib le, Enalapri l subcutaneous injection for consecutive 11 davs Y Metabolic cage set-up 24-hour urine collection before animal sacrifice r Competit ive R T - P C R ET-1 , E T B , V2R , A Q P 2 In situ hybridization ET-1 , E T B , V 2 R , A Q P 2 Westernblot V 2 R , A Q P 2 47 Data Analysis Results were expressed as mean ± standard error of the mean (SEM) . Statistical analysis was performed using unpaired Student's t-test or analys is of var iance when appropriate, to evaluate the signif icance of the difference between means, and a p level below 0.05 was cons idered to be significant. 48 3.0 R E S U L T S 3.1 Clearance Studies 3.1.1 Series One: Rats without Enalapril Treatment Data in detail are presented in Table 3.1.1. In summary, serum B U N increased significantly by 53.2% (p < 0.01), G F R decreased significantly by 61.5% (p < 0.01), and FEH2o increased significantly by 81.0% (p < 0.01) in the C R F rats (n=19) compared to the control rats (n=19). Figure 3.1.1 demonstrates an inverse relationship between G F R and FEH2o-3.1.2 Series Two: Rats with or without Enalapril Treatment Clearance data are shown in Table 3.1.2. Briefly, serum B U N significantly increased by 50.9% (p < 0.01), G F R significantly decreased by 53.6% (p < 0.01), and FEH20 significantly increased by 79.3% (p < 0.01) in the untreated C R F rats (n=5) compared to the untreated control rats (n=5). Serum B U N significantly increased by 55.6% (p < 0.01), G F R significantly dec reased by 58.0% (p < 0.01), and FEH2o significantly increased by 74.3% (p < 0.01) in the treated C R F rats (n=5) compared to the treated control rats (n=6). Figure 3.1.2 demonstrated the relationship between mean G F R and FEH2o in this series. Between these two groups (treated and untreated rats), serum B U N significantly decreased by 23.2% (p < 0.01) in the treated control rats compared to the untreated control rats. There were no statistical significant difference in BUN , mean GFR , and FEH2o between the two groups. It appears that enalapri l 49 treatment has no effect on urine water excretion in the C R F rats. Figure 3.1.2 shows the relationship between G F R and F E H 2 0 . 50 Table 3.1.1 Clearance studies for the experiment series 1 animals Control Rats C R F rats No. of rat 19 19 Se rum B U N (mg%) 23.27 ± 1.08 49.75 ±3.13* G F R (ml/min) 2.31 ±0.09 0.89 ±0.04^ F E H 2 o (%) 0.68 ±0 .05 3.57 ±0.27^ * P < 0.01 versus the control rats v P < 0.01 versus the control rats ^ P < 0.01 versus the control rats Va lues are expressed as mean ± S E M Abbreviat ions: B U N -Blood Urea Nitrogen G F R -Glomerular Filtration Rate FEH2o -Fractional Excretion of Water 51 (%) jajey\A jo uojjejoxg leuojjoejj Table 3.1.2 Clearance studies for the experiment series 2 animals Control rats without enalapril treatment C R F rats without enalapri l treatment Control rats with enalapri l treatment C R F rats with enalapri l treatment No. of rats 5 5 5 6 Serum B U N (mg%) 30.02 ± 0 .35 v 61.15 ±3.00* 23.06 ±0.68 51.95 ±4.05* 3 G F R (ml/min) 1.92 + 0.17 0.89 + 0.03* 1.81 ±0 .23 0.76 ± 0.11* 3 FEH20(%) 0.85 ±0.07 4 .10±0 .15* 0.98 ± 0.14 3.81 ±0.79* 3 * - p < 0.01 versus comparable control rats \\i - p < 0.01 versus the treated control rats a - statistically insignificant versus untreated C R F rats Va lues are expressed as mean ± S E M Except for serum B U N in control rats of the two groups, the increased or decreased values of serum BUN , GFR , and FEH2o in the treated group were evaluated statistically insignificant when they were compared to the va lues of the untreated group. Abbreviat ions: B U N -Blood Urea Nitrogen G F R -Glomerular Filtration Rate FEH20 - Fractional Excretion of Water 53 (%) jejeM jo uo|}8J0X3 |euo;pej j 3.2 ET-1, E T R . and AQP2 mRNA Level in the Series 1 Animals: ET-1 m R N A increased, ET B , A Q P 2 m R N A decreased in the C R F rats compared to the control rats. In this study, ET-1 m R N A increased by 56.3% (p < 0.01), E T B m R N A decreased by 55.7% (p < 0.01), and A Q P 2 m R N A decreased by 31.9% (p <0.01) in the C R F (n=19) compared to the control rats (n=19). The data are tabulated in Table 3.2. ET-1 production in the IMCD increased with serum B U N (Figure 3.2.1). The reduction in G F R is assoc iated with an increase in ET-1 production in the IMCD (Figure 3.2.2). Higher water excretion in urine might be related to higher ET-1 content in the IMCD. Figure 3.2.3 shows a linear relationship between FEH2o and ET-1 production. FEH2o was related to V 2 R m R N A level. In this study, A Q P 2 m R N A express ion was found inversely related to FEH2o (Figure 3.2.4). ET-1 production is also inversely related to the reduction of A Q P 2 express ion (Figure 3.2.5). 55 Table 3.2 ET-1, ET B, & AQP2 mRNA level in the experiment series 1 rats Control rats C R F rats No. of rats 19 19 ET-1 m R N A (amol/ug) 0.31 ±0.04 0.71 ±0.10* E T B m R N A (amol/ug) 14.00 ±0.70 6.20 ± 0.43* A Q P 2 m R N A (amol/ug) 13.90 ±0.73 9.74 ± 0.83* *P < 0.01 versus control rats Va lues are expressed as mean ± S E M CD CO h -CO CM d + X ^1-CJ) CO CO C M L O C O CN CO o * o O • < r - -1— CD - r -L O - T -CO " T " C M C M 00 O CD O (0 c o < t> Z C o o Ui 3 o E E "3" uj d C M d LU C CD CD < .s-CO c o CD CO CD c CO CM CO CD i _ 3 Ui 0 0 (0 +-< c 0 E 0 Q. X 0 0 CO c o +•» 0 v. o X LU (%) uaie/w J ° uojiejoxg IBUOSJOBJJ I— CN CO CO CM O CD OO 00 LO o II T _ CM ll <z X CD CN 03 i _ "o c o o 01 o —i 1 1 1 1 1 1 r-O ^ N L O C O T - Q N L O (VNU lejoj jo Bn/|0UJB) VNUw ZdOV h CD h LO CD C o </> c .92 ^ _Q CD £ Q. E 0 CO CO c o 0 Q. X **z JS .£ o CM !" a. O < CD CO c o CD I . CO o X LU 15 C o CO CD c 1 3 U CU CO • M H w r CN O CO > CD C o • M H LU CN CO CD ro c o 3 O CO CO CO o CD (VNU IBWjo 6n/|owe) VNUW zdOV 3.3 ET-1, E T R , V2R, and AQP2 mRNA Level in Series 2 Animals: 3.3.1 Rats with or without Enalapril Treatment: There were two groups of rats in this series. One group was not treated with enalapri l, the other was enalapril treated. In the untreated groups, ET B , V2R , and A Q P 2 m R N A were downregulated whereas ET-1 m R N A was upregulated in the C R F rats compared to the control rats. In the treated group, ET-1 , E T B , V2R, and A Q P 2 m R N A were partially normalized in the C R F rats (n=6) compared to the treated control rats (n=5). The restoration of E T B m R N A in the treated C R F rats compared to the untreated C R F rats was statistically significant (p < 0.01). Absolute va lues in each category are shown in Tab le 3.3.1. In this series, both V 2 R and A Q P 2 m R N A express ion were elevated when ET-1 m R N A decreased or G F R increased (Figure 3.3.1.1, Figure 3.3.1.2, and Figure 3.3.1.3). ET-1 increased with elevated FEH2o (Figure 3.3.1.4) whereas, V 2 R and A Q P 2 were downregulated when FEH2o was increased (Figure 3.3.1.5, Figure 3.3.1.6). V 2 R express ion is related to A Q P 2 express ion (Figure 3.3.1.7). When ET-1 was upregulated, V 2 R and A Q P 2 were downregulated (Figure 3.3.1.8, Figure 3.3.1.9). 62 T a b l e 3.3.1 E T - 1 , E T B , V 2 R , a n d A Q P 2 m R N A leve l in s e r i e s 2 rats Control rats without enalapri l treatment C R F rats without enalapri l treatment Control rats with enalapri l treatment C R F rats with enalapri l treatment No. of rats 5 5 5 6 ET-1 m R N A (amol/|ag) 0.31 ± 0.03 1.03 ± 0.26* 0.33 ± 0.07 0.56 ± 0.05* 3 E T B m R N A (amol/(j.g) 15.50 ± 0.38 "5.30 ± 0.36* 16.18 + 1.09 9.07 + 0.53* V 2 R m R N A (amol/pg) 1493.48 ± 137.72 546.63 ± 79.40* 1488.79 ± 97.72 728.00 + 160.69* 3 A Q P 2 m R N A (amol/^g) 13.45 ± 1.34 9.46 ± 0.60* 14.47 ± 0.51 11.25 + 1.41^ co - p < 0.01 versus the treated C R F rats * - p < 0.02 versus the comparable control rats # - p < 0.01 versus the comparable control rats T - p < 0.05 versus the comparable control rats 3 - statistically insignificant versus the untreated C R F rats Va lues are expressed as mean ± S E M 63 (VNU leiouo Bn/|OLUB) VN^w 1,-13 (VlSia | B * O H O Bn/|ouiB) V N U W ZdOV r CO (VNa IBWjo 6n/|oiue) VNUW k!3 (VNfcl lejouo Bn/|OLUB) VNUW 3dOV (VNH mo} jo Bn/ioiue) VNUW ZdOV ( V N H |B»ouo 6n/|ouiB) V N H W U Z A (VNU ie*<n jo 6n/|ouie) VNfciw ZdOV 3.4 In Situ Hybridization mRNA Staining for ET-1, E T R , V2R, and AQP2 3.4.1 ET-1 mRNA: Per inuclear m R N A signal was higher in the untreated C R F IMCD than in the control (normal untreated rats) section. m R N A was slightly lower for the treated C R F rats compared to the untreated C R F ones (ET-1 m R N A restoration). The result is consistent to the m R N A measurements by the competit ive RT - PCR . RNase -A treated negative control section showed no specif ic m R N A signals (Figure. 3.4.1). 3.4.2 E T R mRNA: Per inuclear m R N A signal was lower in the untreated C R F rat IMCD than in the normal untreated one. m R N A signal was slightly higher in the treated C R F rat IMCD than in the untreated C R F rats (mRNA change restoration). The image findings confirmed the m R N A measurements by competit ive R T - P C R . m R N A signal was void in the RNase -A treated negative control sect ion (Figure 3.4.2). 3.4.3 V2R mRNA: Per inuclear m R N A signal was much lower in the untreated C R F rat IMCD than in the untreated controls. m R N A signal was moderately higher in the treated C R F rat IMCD than in the untreated C R F IMCD (mRNA restoration). The result is a lso consistent to the results of competitive RT - PCR . No specif ic m R N A signal was visual ized in the RNase -A treated negative control sect ion (Figure 3.4.3). 3.4.4 AQP2 m R N A : Per inuclear m R N A signal stained lower in the untreated C R F IMCD than in the untreated control rat IMCD. m R N A signal was higher in the treated C R F IMCD than in the untreated C R F ones (mRNA restoration). The above image findings verified the quantitative results in our competitive RT - PCR . There was no specif ic m R N A signals in the RNase -A treated negative control sect ion as well (Figure 3.4.4). 74 CD CD CD , Figure 3.4.1 In situ hybridization with ET-1 cDNA probe demonstrates the mRNA levels in different conditions in the rat IMCD. (A) ET-1 mRNA in normal rat IMCD. (B) ET-1 mRNA in CRF rat IMCD. (C) ET-1 mRNA in IMCD of CRF rat treated with enalapri l . (D) ET-1 mRNA probe hybridized with RNase A-treated section. Arrows denote mRNA signal. Magnification: 200x 75 C D Figure 3.4.2 In situ hybridization with ETB cDNA probe demonstrates the mRNA levels in different conditions in the rat IMCD. (A) ETB mRNA in control rat IMCD. (B) ETB mRNA in CRF rat IMCD. (C) ETB mRNA in IMCD of CRF rat treated with enalapri l . (D) ETB mRNA probe hybridized with RNase A-treated section. Arrows denote mRNA signal. Magnification 200x. 7 6 F igu re 3 . 4 . 3 In situ h y b r i d i z a t i o n wi th V 2 c D N A p r o b e demons t ra tes the m R N A leve ls in d i f f e ren t cond i t ions in the rat I M C D . (A) V 2 m R N A in n o r m a l ra t I M C D . (B) V 2 m R N A in C R F ra t I M C D (C) V 2 m R N A in I M C D of C R F rat t r e a t e d with e n a l a p r i l . (D) V 2 m R N A p r o b e h y b r i d i z e d wi th R N a s e A - t r e a t e d sec t ion . A r r o w s in e a c h i m a g e d e n o t e m R N A s i g n a l . M a g n i f i c a t i o n : 2 0 0 x Figure 3.4.4 In situ hybridization with A Q P 2 cDNA probe demonstrates the mRNA levels in different conditions in the rat IMCD. (A) A Q P 2 mRNA in normal rat IMCD. (B) A Q P 2 mRNA in CRF rat IMCD. (C) A Q P 2 mRNA in IMCD of CRF rat treated with enalapril. (D) A Q P 2 probe hybridized with RNaseA-treated section. Arrows in each image denote mRNA signal. Magnification: 200x 78 3.5 Westernblotting Protein Semi-Quantification 3.5.1 V2R Mean V2R /p-Act in value in the untreated normal rats was 52.4% higher (n=4, p <0.01) than in the untreated C R F rats (n=3). With enalapri l treatment, mean V2R /p-Act in value was 42.9% greater in the normal rats (n=3) compared to the C R F rats (n=3, p < 0.05). In control rats, there was no significant difference between enalapri l treated and untreated rats. For C R F rats, the difference of V 2 R express ion between before (0.39 ± 0.02) and after (0.56 ± 0.06) treatment was significant (p = 0.049). The data are listed in Table 3.4. 3.5.2 AQP2 Mean AQP2 /p -Act in was 29% higher in the untreated control rats (n=4) when compared to untreated C R F rats (n=3, p < 0.01). After treatment, A Q P 2 was only 1 1 . 8 % (p < 0.03) greater in control rats (n=3) than in the C R F rats (n=3). There was no significant difference between treated and untreated control rats. The elevation of A Q P 2 level in the treated C R F was significant higher than in the untreated C R F rats (p < 0.01). The data are tabulated in Tab le 3.4. 79 T a b l e 3.4 V 2 R & A Q P 2 in W e s t e r n b l o t t i n g Untreated Untreated Treated Treated C R F control rats C R F rats control rats rats No. of rats 4 3 3 3 V2R /p -Act in 0.82 ± 0.39 ± 0.98 ± 0.56 ± 0.09 0.02* 0.15 0.06 V 8 AQP2 /p -Ac t in 0.31 ± 0.22 + 0.34 ± 0.30 ± 0.0058 0.0067* 0.01 0 . 0067 # 3 * - p < 0.01 versus the untreated control rats - p < 0.05 versus the treated control rats # - p < 0.03 versus the treated control rats 5 - p < 0.05 versus the untreated C R F rats 3 - p < 0.01 versus the untreated C R F rats 80 Lanel Lane 2 Lane 3 Lane 4 v 2 R H t f • § M l 3 7 k D beta- ^ ^ - - ^ 4 n k n Figure 3.5.1.1 Vasopress in receptor type 2 (V2R) protein level was shown to be downregulated in 5/6 nephrectomized rats (Lane 3 and Lane 4) compared to the sham-operated rats (Lane 1 and Lane 2). 8! Lane 1 Lane 2 Lane 3 Lane 4 V 2 R beta-actin Figure 3.5.1.2 Vasopress in receptor type 2 (V2R) protein level was shown to be partially restored by enalapril in Lane 4 (5/6 nephrectomized rat with treatment) compared to Lane 2 and Lane 3 (untreated 5/6 nephrectomized rats) Lane 1 represents untreated sham-operated rat. 82 Lane 1 Lane 2 Lane 3 Lane 4 A Q P 2 i ^ p . - ~ — -rnmm 29 kD beta- — ^ g| ^ ^ ^ ^ ^ ^ 40 kD Figure 3.5.2.1 Downregulation of A Q P 2 protein level was shown in Lane 3 and Lane 4 (5/6 nephrectomized rats) compared to the sham-operated rats (Lane 1 and Lane 2). 83 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 A Q P 2 beta-actin Figure 3.5.2.2 A Q P 2 protein level was partially restored by enalapril in Lane 4 and Lane 5 (5/6 nephrectomized rats with treatment) compared to Lane 2 and Lane 3 (untreated 5/6 nephrectomized rats). Lane 1 represents untreated sham-operated rat. 84 DISCUSSION One of the characteristic features of chronic renal failure is polyuria. The inability of the kidney to concentrate urine can be due to morphological, hemodynamic or hormonal changes. Morphological changes resulting from partial t issue loss cause the increase of single nephron glomerular infiltration rate which in turn leads to more water excretion into the urine (128). A lso, hemodynamic redistribution of renal blood flow to the medul lary region diss ipates the concentration gradient (21), which reduces water diffusion from the collecting duct lumen to the medullary interstitium. Furthermore, several hormones that control renal water reabsorption are known to be elevated or reduced in chronic renal failure: An increased secret ion of atrial natriuretic peptide will cause water loss into the urine (24,126,133). Impaired vasopress in secret ion (8,9) and the decrease in the number of A Q P 2 channe ls in the collecting duct cause an increase in water excretion (30,83). A Q P 2 is the most dominant subtype of water channels in this segment of the nephron (136). Interestingly, recent studies showed that A Q P 2 express ion is also significantly reduced in C R F (76), suggest ing a possibility for A Q P 2 in compromis ing urine concentration under this condition. In normal individuals A Q P 2 is recruited by vasopress in. Vasopress in increases A Q P 2 density through V 2 R activation via the c A M P pathway. This in turn activates P K A and facilitates the insertion of A Q P 2 protein into the IMCD 85 membrane (19,41,57,87,90,91,127,139). Another way to control the express ion of A Q P 2 in the collecting duct is by regulating the express ion of V 2 R (42). This was supported by the observation of Teite lbaum et al suggest ing a downregulat ion of the V 2 R express ion in the kidney of 5/6 nephrectomized rats (115). S ince vasopress in levels in C R F remain constant or even higher (49), further factors regulating A Q P 2 express ion have to be cons idered. Poss ib le candidates are Ang II (100) and ET-1 (14,20,79,98), which have been shown to be increased in C R F . Increase in ET-1 in C R F can be due to the action of Ang II. Ang II has been shown to induce ET-1 production (12,26,34,46,67,103,113,134). Both hormones are known to be potent vasoconstr ictors that act on the kidney to reduce renal blood flow and glomerular filtration rate (7,15,61,62,130,137) and cause water retention. C learance studies have shown that ET-1 has a diuretic effect (40,109). This effect can be due to reduction in c A M P levels in the IMCD induced by ET-1 (33). One could speculate that a decl ine in c A M P would reduce the number of A Q P 2 units inserted into the apical membrane of the IMCD. Therefore, the aim of the study was to examine the effects of elevated Ang II and ET-1 levels in C R F on V 2 R and A Q P 2 express ion in the IMCD. 86 4.1 5/6 N e p h r e c t o m y a s an A n i m a l M o d e l fo r C R F Severa l animal mode ls have been used to study chronic renal failure (23,28,97,140). W e used the 5/6 nephrectomy rat model, which is the most commonly accepted model (56,112). 5/6 nephrectomy is usual ly obtained through the extensive reduction of the renal t issue mass by either ligating of both poles of the kidneys or the branches of renal artery on one s ide of the kidney fol lowed by contralateral nephrectomy (23). Under these conditions, the nephron in experimental C R F resembles human nephron with C R F as proposed by the intact nephron hypothesis(56). In order to confirm that the examined rats were indeed in chronic renal failure state we performed renal c learance studies on each spec imen to monitor renal function. Our twenty-four-hour c learances showed that rats subjected to 5/6 nephrectomy had a significant reduction in overall glomerular filtration rate. In addition, there was significant elevation in serum creatinine and blood urea nitrogen levels. These data confirmed that the chronic renal failure animals used in our present report do indeed have impairment in renal function. 4.2 V 2 R E x p r e s s i o n is D o w n r e g u l a t e d in C R F The relationship between V 2 R and A Q P 2 has been examined. Recent reports suggested that A Q P 2 channels are regulated by V 2 R express ion (42). Studies were done to determine whether V 2 R numbers in the IMCD were altered in C R F as a possib le cause for increase in water excret ion seen in 5/6 nephrectomized animals. In our study, fraction of filtered water excreted was 87 increased in C R F animals compared to the sham operated animals. Thus, it will be of interest to determine if V 2 R express ion was changed in C R F . Employ ing competitive R T - P C R and Western blot analysis we showed that both V 2 R m R N A and protein were significantly reduced in the C R F rats. These results are in agreement with Teite lbaum et al (115). These data confirmed that V 2 R played an important role in enhanced water excretion in chronic renal failure. In support of this idea was other pathological condition where concentrating impairment is assoc iated with a defective V2R , such as congenital nephrogenic diabetes insipidus (NDI), a defective nonfunctioning V 2 R causes a poor response to vasopress in, that results in urine concentrat ion impairment (3,16,104). The acquired form of NDI is also assoc ia ted with a reduction in V 2 R density (16). These data suggest that downregulat ion of V 2 R can lead to polyuria. A Q P 2 is D o w n r e g u l a t e d in the C R F R a t s Downregulat ion of A Q P 2 in the 5/6 nephrectomized rats has been reported. (76). This report suggested that increase in water excretion in renal failure is due to reduction in water channel protein to reabsorb water from the renal tubule in renal failure. Studies were done in the present report to determine if A Q P 2 was altered in the collecting duct of 5/6 nephrectomized rats. Using quantitative R T - P C R and Western blot analysis on t issue obtained from the IMCD of 5/6 nephrectomized rats, we found that both A Q P 2 m R N A and protein levels were lowered in C R F rats. These results are in agreement with that of Kwon et al (76). S ince V 2 R has been shown to regulate the express ion of A Q P 2 in the collecting duct (42), it is possib le that the downregulat ion of A Q P 2 in C R F could be due to a low p lasma vasopress in concentration or a decrease of membrane V 2 R units. Vasopress in level was shown to be normal or even higher in C R F patients (49), which would make vasopress in unlikely as a mediator for the downregulation of A Q P 2 . The downregulation of A Q P 2 in C R F can be a consequence of the V 2 R downregulation. Cau se s for V 2 R to downregulate in C R F are not known. Recent studies in our laboratory suggested that P K C might induce downregulation of V 2 R in the IMCD of normal rats. It follows that hormones whose signaling pathway is mediated through P K C can play a role in downregulating V2R . ET-1 is I nc reased in the C h r o n i c R e n a l Fa i l u re IMCD ET-1 acts as an autocrine or paracrine hormone in the kidney (48,65). In this study, ET-1 m R N A express ion was found to be higher in the IMCD of C R F rats and these results are in agreement with early reports (20,79). At least three mechan i sms are probable for the upregulation of ET-1 in C R F . First, it could be due to Ang II induction (12,26,46,67,103) because Ang II was found to be elevated in C R F state (100). Second, it could be due to E T B receptor. S ince E T B is known as a clearing receptor for ET-1, (27,37) a decrease of E T B could cause an increase in ET-1. Our experiments documented that E T B receptor express ion in the IMCD was diminished and can be a cause for ET-1 increase. Third, other factors such as IL-1 and tumor necrosis factor might be upregulated 89 under C R F conditions. They have been shown to raise ET-1 levels (2,59,114,117,141). 4.5 P o s s i b l e M e c h a n i s m s for C o n t r o l l i n g V 2 R in C R F It is known that Ang II and ET-1 levels are elevated in renal failure. In addition, when Ang II and ET-1 bind to their respective receptors AT1 and ET A , P K C formation is induced (7,103,123) which has been shown to reduce the express ion of V 2 R in the collecting duct according to our unpubl ished IMCD cultured studies. In addition, AT1 receptor activation induces further ET-1 production (34,67,113). In the collecting duct, ET-1 can be a modulator for V 2 R gene express ion through the P K A and P K C pathways. Prel iminary studies done in our laboratory showed that ET-1 reduces V 2 R gene express ion either through P K C or an inhibition of P K A pathway (132). Furthermore, P K A activity is compromised by the activation of the E T B receptor that inhibits adenylate cyc lase (33). A reduction in P K A may affect V 2 R express ion s ince P K A was shown to induce upregulation of V2R . 4.6 V 2 R E x p r e s s i o n R e s c u e E x p e r i m e n t s w i th Ena lap r i l in C R F R a t s The beneficial effect of A C E inhibitors in chronic renal failure and diabetic nephropathy is its ability to delay the progression of the d i sease (73,81,81,124). Thus, these studies were done to determine whether enalapri l an A C E inhibitor would improve renal function in an animal model of renal failure. In our studies, the A C E inhibitor, enalapril was administered in normal rats as well as in C R F 90 rats. G F R and serum B U N were determined and compared to the respective control groups. Enalapri l did not significantly improve the impaired renal function of the C R F rats. The reason for this finding may be due to the fact that the length of the treatment period was not sufficient. Another poss ib le explanation is the nature of this animal model. Rena l function was reduced by surgical ablation and intervention with A C E inhibitors may not be able to restore the defect in overall renal function but able to induce b iochemical changes which was noted in our results. A l though no improvements were seen in overall c learances following enalapri l treatment, we were able to show some normalization of ET-1 , A Q P 2 and V2R . Our R T - P C R and Western blot result showed that V 2 R and A Q P 2 gene express ions and protein synthesis in the C R F rats were partially restored after enalapri l treatment. The changes in these parameters after enalapri l was almost normal ized but did not achieve complete improvement. This will suggest that Ang II may not be the only factor that is responsible for inducing changes in these measured parameters. If V 2 R and A Q P 2 express ion levels can be partially normal ized by enalapri l treatment, one would expect that this would have an impact on the fractional excretion of water (FEH2o)- Our c learance data showed that, the F E H 2 O difference between treated and untreated C R F rats was the same. Other possib le mechan isms have to be evoked to explain our observat ions. This 91 result can be explained by two possible mechanisms: First, changes in the intra-renal blood flow pattern in chronic renal failure will induce a loss of medullary interstitial hypertonicity in the renal medul la that plays an important role in concentrating and diluting processes within the kidney (21). Second , A C E inhibition prevents intra-renal bradykinin degradation and contributes to the natriuresis in the proximal renal tubule (111). W e as sume that the increase in the number of V 2 R and A Q P 2 after enalapri l will increase water reabsorption by the remnant kidney. This was not seen in our c learance data. These changes in V 2 R and A Q P 2 were not able to overcome other factors that are at play in chronic renal failure to increase water excretion. It can be said that downregulat ion V 2 R and A Q P 2 is one of the many factors that are responsible for polyuria seen in this syndrome. Our data showed that ET-1 as well as the E T B receptor density was only partially normal ized by enalapril in C R F . This is in agreement with the data of Lariviere et al. (80). Many humoral factors other than angiotensin II are activated during the progression of the d isease (128). Subs tances such as IL-1 (2,114,117) or T N F - a (2,114) can induce ET-1 production. W e can block the effect of angiotensin II induced ET-1 production with enalapri l but not able to prevent other humoral agents that are active during renal failure to induce further ET-1 formation. Therefore, with enalapri l treatment, ET-1 level would be partially normal ized and did not fall to a level seen in animals with intact renal function. Another reason that might cause the inability of enalapri l to normal ize 92 ET-1 level is the relatively short period of treatment (11 days) as opposed to the long-term treatment given to C R F patients. In the inner medullary collecting duct of the kidney, the predominant form of angiotensin receptor is the AT-1 subtype (7). The binding of angiotensin II to this site will induce the formation of P K C (123). Studies in our laboratory suggested that P K C induce downregulation of the V 2 R receptor. Therefore during enalapri l treatment, the lack of P K C formation will up regulate V 2 R receptors. However, our results showed that V 2 R are not completely restored to normal after enalapri l. This will suggest other factors are involved in inducing downregulat ion. These factors are not known. It could be speculated to include medullary interstitial hypertonicity. Studies have shown that osmolal ity can induce upregulation of ET-1 (138). The effect of osmolal ity on V 2 R is not known and remains to be examined. 93 S U M M A R Y A N D C O N C L U S I O N Polyuria is one of the symptomatic features of chronic renal failure (CRF) . More recently, aquaporin-2 (AQP2), a collecting duct cell membrane water channel was shown to be downregulated in C R F . Studies were done to examine the role of the vasopress in type II receptor (V2R) in regulating the express ion of A Q P 2 in the inner medullary collecting duct (IMCD) of C R F rats. Us ing quantitative competit ive R T - P C R and Western Blot analys is we showed that V 2 R and A Q P 2 gene as well as protein express ion is reduced. Studies were also done to determine the role of angiotensin II in downregulating V 2 R and A Q P 2 by treating 5/6 nephrectomized and sham operated rats with A C E inhibitors. Us ing enalapri l we were not able to restore V 2 R and A Q P 2 express ion in the inner medullary collecting duct of the kidney. Endothel in-1 (ET-1) production was also incompletely normal ized. W e suggested that beside angiotensin II other factors are involved in promoting changes in the express ion of V2R , A Q P 2 and ET-1 in chronic renal failure. 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