Volume 72, Issue 11, Pages 1345-1357 (December 2007) Blockade of cannabinoid CB1 receptors improves renal function, metabolic profile, and increased survival of obese Zucker rats  P. Janiak, B. Poirier, J.-P. Bidouard, C. Cadrouvele, F. Pierre, L. Gouraud, I. Barbosa, J. Dedio, J.-P. Maffrand, G. Le Fur, S. O'Connor, J.-M. Herbert  Kidney International  Volume 72, Issue 11, Pages 1345-1357 (December 2007) DOI: 10.1038/sj.ki.5002540 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding on the (a) body weight (b) and food intake of obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats over a 12-month treatment period. Values are expressed as means±s.e.m. Statistical analysis was performed using two-way ANOVA followed by Newman–Keuls post hoc test. Body weight gain was significantly (P<0.05) reduced by rimonabant 10 mg/kg/day and pair-feeding from 2 weeks to 9 months and 8 months of treatment, respectively, in comparison with vehicle-treated group. Hyperphagia was suppressed transiently and dose-dependently by rimonabant. This reduction in food intake remained significant (P<0.05) up to 2 and 6 weeks after initiating the treatment with rimonabant at 3 and 10 mg/kg/day, respectively. *P<0.05 rimonabant 10 mg/kg vs obese fa/fa-vehicle; #P<0.05 rimonabant 3 mg/kg vs obese fa/fa-vehicle; §P<0.05 pair-feeding vs obese fa/fa-vehicle. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding on the survival rate (%) of obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats over a 12-month treatment period. Statistical analysis was performed with a log-rank test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on (a) plasma triglycerides, (b) cholesterol, (c) LDL-cholesterol, (d) LDL-cholesterol/HDL-cholesterol ratio, (e) non-esterified fatty acids in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 9 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using Kruskal–Wallis test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats; no. P<0.05 rimonabant 10 mg/kg vs pair-feeding. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on plasma adiponectin in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 6 and 9 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using one-way ANOVA followed by a Newman–Keuls post hoc test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats; #P<0.05 rimonabant 10 mg/kg vs pair-feeding. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Representative examples of pancreatic β-islet structure revealed by insulin immunostaining. (a) Normal β-islet in lean fa/+ Zucker rats, (b) fragmentation of β-islet structure in obese fa/fa Zucker rats, and (c) preservation of β-islet integrity in rimonabant-treated (10 mg/kg/day) obese fa/fa Zucker rats. Original magnification × 200. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 6 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on plasma norepinephrine in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 9 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using Kruskal–Wallis test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 7 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on pressor response to angiotensin II injected intravenously at 30 and 100 ng/kg in anesthetized obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using two-way ANOVA followed by a Newman–Keuls post hoc test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats; #P<0.05 rimonabant 10 mg/kg vs pair-feeding. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 8 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on the (a) proteinuria/creatininuria ratio (b) N-acetyl-glucosaminidase excretion rate in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 1, 3, 6, 9, and 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using Kruskal–Wallis test. The reduction in proteinuria/creatininuria ratio produced by rimonabant was significant (P<0.05) up to 9 and 12 months of treatment at 3 and 10 mg/kg/day, respectively. Prevention in the elevation of N-acetyl-glucosaminidase excretion by rimonabant remained significant up to 9 months for both doses. Pair-feeding was effective up to 9 months on proteinuria/creatininuria ratio and on N-acetyl-glucosaminidase excretion rate. *P<0.05 rimonabant 10 mg/kg vs obese fa/fa-vehicle; #P<0.05 rimonabant 3 mg/kg vs obese fa/fa-vehicle; §P<0.05 pair-feeding vs obese fa/fa-vehicle; $P<0.05 rimonabant 10 mg/kg vs pair-feeding. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 9 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on the (a) plasma creatinine, (b) plasma urea nitrogen, (c) creatinine clearance in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using Kruskal–Wallis test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 10 Representative examples of CB1 receptor immunostaining in obese fa/fa Zucker rats. (a) Glomerulus showing a positive focal immunostaining for CB1 receptors. (b) Tubules of the renal medulla did not display any CB1 receptor immunoreactivity. (c) Positive control for CB1 receptor immunostaining found in the brain. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 11 Representative examples of glomerular structure. (a) Normal glomeruli in lean fa/+ Zucker rats, (b) severe diffuse glomerulosclerosis in obese fa/fa Zucker rats, and (c) less severe glomerulosclerosis with focal pattern in rimonabant-treated (10 mg/kg/d) obese fa/fa Zucker rats. Masson's trichrome. Original magnification × 400. (d) The effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on glomerular lesions in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using one-way ANOVA followed by Newman–Keuls post hoc test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats; #P<0.05 rimonabant 10 mg/kg vs pair-feeding. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 12 Representative examples of tubulointerstitial structure. (a) Normal tubules and absence of fibrosis and inflammation in the interstitium in lean fa/+ Zucker rats, (b) tubular lesions characterized by tubular dilatation and casts (arrows) associated with interstitial fibrosis and inflammation in obese fa/fa Zucker rats, and (c) less intense tubulointerstitial lesions in rimonabant-treated (10 mg/kg/day) obese fa/fa Zucker rats. Masson's trichrome. Original magnification × 400. (d) The effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on tubulointerstitial lesions in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis was performed using Kruskal–Wallis test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 13 Effects of rimonabant, 3 and 10 mg/kg/day p.o., and pair-feeding, on the (a) glomerular fibrosis (%), (b) plasma PAI-I levels, (c) kidney weight in obese fa/fa Zucker rats in comparison with control lean fa/+ Zucker rats after 12 months of chronic treatment. Values are expressed as means±s.e.m. Statistical analysis for glomerular fibrosis and plasma PAI-1 levels was performed by Kruskal–Wallis test. Kidney weight was analyzed by one-way ANOVA followed by a Newman–Keuls post hoc test. *P<0.05 vs vehicle-treated obese fa/fa Zucker rats. Kidney International 2007 72, 1345-1357DOI: (10.1038/sj.ki.5002540) Copyright © 2007 International Society of Nephrology Terms and Conditions