Aus der Klinik für Innere Medizin mit Schwerpunkt Nephrologie
der Medizinischen Fakultät der Charité – Universitätsmedizin Berlin

Dissertation

Nitric oxide-cGMP signal transduction in the injury, matrix expansion and progression of anti-thy1-induced renal disease of the rat

Zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)

vorgelegt der Medizinischen Fakultät der Charité -
Universitätsmedizin Berlin

von
Yingrui Wang
aus Guangdong, V.R. China

Gutachter:
1. Prof. Dr. med. Frank Strutz
2. PD. Dr. med. Ulrich Wenzel
3. PD. Dr. med. Harm Peters

Datum der Promotion:14 März 2005

Table of contents

• 1  Introduction
  • 1.1  Pathogenesis and therapy of chronic progressive renal disease
    • 1.1.1 Histological and molecular characteristics
    • 1.1.2 The central role of TGF-beta in renal fibrosis
    • 1.1.3 The sequence of phases leading to progressive renal fibrosis
    • 1.1.4 Therapeutic approaches to chronic progressive renal disease
  • 1.2  The L-arginine-NO pathway in renal disease
    • 1.2.1 Two faces of the L-arginine-NO pathway
    • 1.2.2 NO synthases
    • 1.2.3  Effects of L-arginine supplementation on renal disease
  • 1.3 NO-cGMP signaling in renal disease
    • 1.3.1  Distribution of NO-cGMP signaling in the glomeruli and the tubulointerstitium
    • 1.3.2 The physiology of NO-cGMP signaling
    • 1.3.3 The pathophysiology of NO-cGMP signaling
    • 1.3.4 Pharmacological stimulators of sGC activity
  • 1.4  Aim of the study
• 2  Materials and methods
  • 2.1 Materials
    • 2.1.1 Chemicals, tools and instruments
    • 2.1.2 Computer and software
  • 2.2 Animal experiments
    • 2.2.1 Animals
    • 2.2.2  Production of monoclonal antibody OX-7 and mAb 1-22-3
      • 2.2.2.1 Cell culture
      • 2.2.2.2 Purification of the monoclonal antibody OX-7/mAb 1-22-3
    • 2.2.3  Models of anti-thy1-induced renal disease
      • 2.2.3.1 Acute anti-thy1 glomerulonephritis
      • 2.2.3.2 Chronic anti-thy1-induced glomerulosclerosis
    • 2.2.4 Food and drinking water intakes
    • 2.2.5 Drug administration
  • 2.3 Experimental design
    • 2.3.1 Protocol 1: NO-cGMP signal transduction in the injury phase of acute anti-thy1 glomerulonephritis (day 1 after antibody injection)
    • 2.3.2 Protocol 2: NO-cGMP signal transduction in the matrix expansion phase of acute anti-thy1 glomerulonephritis (day 7 after antibody injection)
    • 2.3.3 Protocol 3: NO-cGMP signal transduction in anti-thy1-induced chronic glomerulosclerosis (progression phase)
  • 2.4 Harvesting of materials
    • 2.4.1 Urine collection
    • 2.4.2 Removal of kidney and blood and further processing
      • 2.4.2.1 Removal of kidney and blood
      • 2.4.2.2  Isolation of glomeruli
      • 2.4.2.3 Preparation of tubulointerstitial tissue
    • 2.4.3 Cell culture
  • 2.5 Measurements
    • 2.5.1 Systolic blood pressure
    • 2.5.2 Renal function
    • 2.5.3  Rat tail bleeding time
    • 2.5.4 Urinary protein
    • 2.5.5 Basal and LPS-stimulated nitrite production
    • 2.5.6  Histology
      • 2.5.6.1 PAS staining
      • 2.5.6.2 ED1 staining
      • 2.5.6.3 Fibrinogen staining
      • 2.5.6.4 Histological evaluation
    • 2.5.7 Enzyme-linked immunosorbent assay (ELISA)
      • 2.5.7.1 TGF-beta1
      • 2.5.7.2 Fibronectin
      • 2.5.7.3 PAI-1
      • 2.5.7.4 cGMP
        • 2.5.7.4.1  Plasma cGMP
        • 2.5.7.4.2  Basal and NO-stimulated cGMP production
    • 2.5.8 mRNA analysis
      • 2.5.8.1 RNA isolation by Trizol
      • 2.5.8.2 Reverse transcription-polymerase chain reaction (RT-PCR)
      • 2.5.8.3 Polymerase chain reaction
  • 2.6  Statistical analysis
• 3  Results
  • 3.1 Blood pressure, bleeding time and plasma cGMP levels in acute anti-thy1 glomerulonephritis (injury phase and matrix expansion phase)
    • 3.1.1 Systolic blood pressure
    • 3.1.2 Bleeding time
    • 3.1.3 Plasma cGMP levels
  • 3.2 Protocol 1: NO-cGMP signaling in the injury phase 1 day after induction of acute anti-thy1 glomerulonephritis
    • 3.2.1 Body weight
    • 3.2.2 Proteinuria
    • 3.2.3 Mesangial cell lysis
    • 3.2.4 Glomerular iNOS-NO pathway
    • 3.2.5  Glomerular eNOS-NO-cGMP signaling cascade
  • 3.3  Protocol 2: NO-cGMP signaling in the matrix expansion phase 7 days after induction of acute anti-thy1 glomerulonephritis
    • 3.3.1 Body weight
    • 3.3.2 Proteinuria
    • 3.3.3  Markers of glomerular matrix expansion
    • 3.3.4 Glomerular eNOS-NO-cGMP signaling cascade
    • 3.3.5 Mechanisms of Bay 41-2272’s renoprotective effects
      • 3.3.5.1 Blood pressure
      • 3.3.5.2 TGF-beta1 production in vitro
      • 3.3.5.3 Glomerular platelet deposition
      • 3.3.5.4  Glomerular macrophage infiltration
  • 3.4  Protocol 3: NO-cGMP signaling 16 weeks after induction of anti-thy1-induced chronic glomerulosclerosis (progression phase)
    • 3.4.1 Body weight
    • 3.4.2 Systolic blood pressure
    • 3.4.3 Proteinuria
    • 3.4.4 Markers of tubulointerstitial matrix accumulation
      • 3.4.4.1 Matrix score
      • 3.4.4.2  Protein and mRNA expression of TGF-beta1, fibronectin and PAI-1
    • 3.4.5 Markers of glomerular matrix accumulation
    • 3.4.6 Markers of renal function
    • 3.4.7 Tubulointerstitial eNOS-NO-cGMP signaling cascade
    • 3.4.8 Glomerular sGC activity
    • 3.4.9 Renal macrophage infiltration
• 4  Discussion
  • 4.1 Critical evaluation of the methodology used
    • 4.1.1 Animal model of anti-thy1-induced acute and chronic renal disease
    • 4.1.2 Analysis of markers of renal fibrosis
    • 4.1.3 TGF-beta1 as a key marker of matrix expansion
    • 4.1.4 Stimulation of sGC by Bay 41-2272
  • 4.2 The NO-cGMP pathway in the injury phase of acute anti-thy1 glomerulonephritis
    • 4.2.1 Regulation of NO-cGMP signal transduction
    • 4.2.2 Effects of Bay 41-2272 on mesangial cell lysis
  • 4.3 The NO-cGMP pathway in the matrix expansion phase of acute anti-thy1 glomerulonephritis
    • 4.3.1 Regulation of NO-cGMP signal transduction
    • 4.3.2 Antifibrotic effects of Bay 41-2272
  • 4.4 The NO-cGMP pathway in anti-thy1-induced chronic glomerulosclerosis
    • 4.4.1 Regulation of NO-cGMP signal transduction
    • 4.4.2 Antifibrotic effects of Bay 41-2272
  • 4.5 Transcriptional regulation of NO-cGMP signal transduction from acute to chronic renal disease
  • 4.6 Mechanisms of Bay 41-2272’s antifibrotic effects
    • 4.6.1  Reduction in blood pressure
    • 4.6.2 Blood pressure-independent effects of Bay 41-2272
      • 4.6.2.1 Direct reduction in TGF-beta1 expression
      • 4.6.2.2 Inhibition of renal platelet deposition
      • 4.6.2.3 Inhibition of renal macrophage infiltration
  • 4.7 To differentiate the beneficial and detrimental actions of the L-arginine-NO pathway
• 5  Summary
• 6  Zusammenfassung
• Reference list
• Abbreviations
• Acknowledgements
• Erklärung an Eides Statt

Tables

• Table 1: Tools and instruments used during the experiments.
• Table 2: Contents of the food (average content in 1 kg diet).
• Table 3: Mixture of reverse transcription components.
• Table 4: Procedure for reverse transcription.
• Table 5: Mixture of PCR components.
• Table 6: Templates of PCR.
• Table 7: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on rats’ body weights at the beginning and the end of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. (*p<0.05 vs. Control)

Images

• Figure 1: Distribution of NO-cGMP signaling in the glomeruli. L-Arginine (L-Arg), NO (Nitric oxide), Endothelial NO synthase (eNOS), Neuronal NOS (nNOS), cGMP (Cyclic guanosine monophosphate), GTP (Guanosine 5`-triphosphate), sGC (Soluble guanylate cyclase).
• Figure 2: Distribution of NO-cGMP signaling in the tubulointerstitium. L-Arginine (L-Arg), NO (Nitric oxide), Endothelial NO synthase (eNOS), Neuronal NOS (nNOS), cGMP (Cyclic guanosine monophosphate), GTP (Guanosine 5`-triphosphate), sGC (Soluble guanylate cyclase).
• Figure 3: Effects of Bay 41-2272 (+Bay 41-2272) on systolic blood pressure in acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Blood pressure was measured in conscious rats using tail cuff. (**p<0.01 vs. aGN and Control)
• Figure 4: Effects of Bay 41-2272 (+Bay 41-2272) on bleeding time in acute anti-thy1 glomerulonephritis (aGN). Bleeding was induced by a standard tail incision (10 mm long and 1 mm deep) in anesthetized animals. (**p<0.01 vs. aGN and Control)
• Figure 5: Effects of Bay 41-2272 (+Bay 41-2272) on plasma cGMP levels in acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. (**p<0.01 and *p<0.05 vs. Control, #p<0.05 vs. aGN)
• Figure 6: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular nuclei count indicating mesangial cell lysis 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control rats (Control) were injected with PBS. (***p<0.001 vs. Control)
• Figure 7: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular iNOS mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (**p<0.01 vs. Control)
• Figure 8: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular basal and LPS-stimulated NO production 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Nitrite as indicator of NO production was determined by the Griess reaction. Glomeruli were harvested from individual animals and cultured at a density of 2000 per ml for 48 hours. (***p<0.001 and **p<0.01 vs. Control)
• Figure 9: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular eNOS mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group.
• Figure 10: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular alpha1 sGC and beta1 sGC mRNA expression 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group.(***p<0.001 vs. Control)
• Figure 11: Effects of Bay 41-2272 (+Bay 41-2272) on basal and DEA/NO stimulated glomerular cGMP production 1 day after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 vs. Control)
• Figure 12: Effects of Bay 41-2272 (+Bay 41-2272) on proteinuria 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Urine was collected for 24 hours using metabolic cages. (*p<0.05 vs. aGN)
• Figure 13: Effects of Bay 41-2272 (+Bay 41-2272) on markers of glomerular matrix expansion, including matrix score, TGF-beta1, fibronectin and PAI-1 production, 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Matrix expansion was scored on PAS-stained slides. Glomeruli were harvested from individual animals and cultured at a density of 2000 per ml for 48 hours. (***p<0.001, **p<0.01, *p<0.05 vs. aGN)
• Figure 14: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular eNOS mRNA expression 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (*p<0.05 vs. aGN)
• Figure 15: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular sGC mRNA expression and sGC activity to produce cGMP 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (***p<0.001 and *p<0.05 vs. Control, #p<0.05 vs. aGN)
• Figure 16: In vitro effects of Bay 41-2272 on TGF-beta1 production in glomeruli isolated from control and nephritic rats 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomeruli were cultured at a density of 2000 per ml for 48 hours in the presence of Bay 41-2272 in a concentration sequence of 0.1 µM, 1 µM, 5 µM and 10 µM. (*p<0.05 vs. glomeruli of Control and aGN without Bay 41-2272)
• Figure 17: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular fibrinogen deposition 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomerular fibrinogen deposition is expressed as the percentage of fibrinogen-positive area per glomerular cross-section. (*p<0.05 vs. aGN)
• Figure 18: Effects of Bay 41-2272 (+Bay 41-2272) on glomerular ED1-positive cell infiltration and P-selectin mRNA expression 7 days after induction of acute anti-thy1 glomerulonephritis (aGN). Normal control animals (Control) were injected with PBS. Glomerular macrophage infiltration is expressed as number of ED1-positive cells per glomerular cross-section. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the normal control group. (*p<0.05 vs. aGN)
• Figure 19: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on systolic blood pressure 8 weeks and 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Blood pressure was measured in conscious animals using a tail cuff method. (**p<0.01 vs. Control, ***p<0.001 vs. cGS)
• Figure 20: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on time course of proteinuria after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Urine was collected for 24 hours using metabolic cages.
• Figure 21: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial matrix protein accumulation 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Matrix expansion was scored on PAS-stained slides. (**p<0.01 vs. cGS, #p<0.05 vs. cGS+Hydralazine)
• Figure 22: Effects of Bay 41-2272 and hydralazine on the histological picture 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatment was started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Shown are characteristic PAS-stained renal sections from A) a non-diseased animal without (2-K Control) and B) with uni-nephrectomy (1-K Control), and C) an animal with anti-thy1-induced chronic glomerulosclerosis without treatment and with Bay 41-2272 (D) or with hydralazine treatment (E) (Magnification x200).
• Figure 23: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial TGF-beta1, fibronectin and PAI-1 protein and mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Matrix protein production was determined in extensively minced individual cortical tissues cultured at a density of 10 mg/ml for 48 hours. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (***p<0.001, **p<0.01 and *p<0.05 vs. cGS, ###p<0.001 and #p<0.05 vs. cGS+Hydralazine)
• Figure 24: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on glomerular matrix protein accumulation and TGF-beta1, fibronectin and PAI-1 protein expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Matrix expansion was scored on PAS-stained slides. Glomeruli were harvested from individual animals and cultured at a density of 2000 per ml for 48 hours. (***p<0.001, **p<0.01 and *p<0.05 vs. cGS, ###p<0.001 vs. cGS+Hydralazine)
• Figure 25: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on markers of renal function 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatment was started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. (**p<0.01 and *p<0.05 vs. cGS, #p<0.05 vs cGS+Hydralazine)
• Figure 26: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial eNOS mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (*p<0.05 vs. Control)
• Figure 27: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial sGC mRNA expression and activity to produce cGMP 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. cGMP generation was measured by ELISA in cortical tissue from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 vs. cGS+Hydralazine, *p<0.05 vs. Control, #p<0.05 vs. cGS)
• Figure 28: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on glomerular sGC activity to produce cGMP 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. cGMP generation was measured by ELISA in glomeruli harvested from individual animals in the presence or absence of the NO donor DEA/NO. (**p<0.01 and ##p<0.01 vs. cGS and cGS+Hydralazine)
• Figure 29: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on cortical and glomerular ED1-positive cells infiltration 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. Data are expressed as ED1-positive cells per cortical section observed at x200 magnification and per glomerular cross-section. (**p<0.01 vs. cGS, ##p<0.01 and #p<0.05 vs. cGS+Hydralazine)
• Figure 30: Effects of Bay 41-2272 (+Bay 41-2272) and hydralazine (+Hydralazine) on tubulointerstitial P-selectin mRNA expression 16 weeks after induction of chronic-progressive anti-thy1 glomerulosclerosis (cGS). Treatments were started 7 days after injection of anti-thy1 antibody into uni-nephrectomized rats. Non-diseased animals without (2-K Control) or with uni-nephrectomy (1-K Control) received a PBS injection. mRNA was analyzed by a real-time PCR method using GAPDH as housekeeping gene. mRNA is shown as a percentage of the untreated chronic anti-thy1 animals. (*p<0.05 vs. cGS)