Effect of hyperglycemia and rosiglitazone on renal and urinary neprilysin indb/dbdiabetic mice

Figures

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  Figure 1. Chronic treatment with rosiglitazone for 10 weeks reduced the increased urinary albumin excretion in 16‐week‐old db/db diabetic mice. Albuminuria in lean control (L), db/db diabetic (db), and db/db diabetic mice treated with rosiglitazone (db+ R) was expressed as albumin/creatinine ratios (a) or as 24‐hr albuminuria (b). Data are expressed as means ± SEM (n = 6, *p < .0001 vs. lean controls, # p < .0001 vs. untreated db/db diabetic mice)

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  Figure 2. (a) Representative light microscopy images of kidney section from lean control (L), db/db diabetic (db), and db/db diabetic mice treated with rosiglitazone (db+ R) stained with periodic acid‐ Schiff (PAS). Magnification: 40×, scale bars:100 μm. (b) Relative mesangial matrix area (%) significantly increased in db/db mice and was attenuated by treatment with rosiglitazone for 10 weeks. Each bar represents mean ± SEM (n = 5–6, *p < .0001 vs. lean controls; # p < .001 vs. untreated db/db diabetic mice)

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  Figure 3. Renal and urinary NEP protein expressions were decreased in 16‐week‐old db/db diabetic mice. To validate the specificity of NEP antibody, western blot analysis of NEP and ACE2 were performed in kidney lysates obtained from NEP KO & NEP WT mice (a). Western blot analysis of (a) renal NEP and β‐actin; (c) urinary NEP and albumin in lean control (L), db/db diabetic (db) and db/db diabetic mice treated with rosiglitazone for 10 weeks (db+ R). One‐way ANOVA using Bonferroni's Multiple Comparison Test showed a significant decrease in the db/db diabetic mice compared to lean controls. Rosiglitazone normalized the decreased renal NEP and increased urinary NEP excretion. Values are represented as mean ± SEM (n = 6–11, *p < .001 vs. lean controls; # p < .0001 vs. untreated db/db diabetic mice)

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  Figure 4. Renal and urinary NEP levels were decreased in db/db diabetic mice. ELISA was used to measure (a) renal NEP, (b) urinary NEP/creatinine, (c) daily urinary NEP excretion, and (d) plasma NEP/ml in lean control (L), untreated db/db diabetic (db), and db/db diabetic mice treated with rosiglitazone (db+ R) for 10 weeks. Kidney lysate samples from NEP KO mice were used as negative control in figure a. Values are represented as mean ± SEM (n = 5–6, *p < .0001 vs. lean controls; # p < .001 vs. untreated db/db diabetic mice)

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  Figure 5. Renal NEP enzymatic activity was decreased in db/db diabetic mice. For validation of NEP activity, the assay was performed at different time of incubation using NEP inhibitor (Thiorphan) and kidney lysate from NEP knockout (NEPKO). (a) Time in minutes versus fluorescence intensity in lean control (Lean), untreated db/db diabetic (dbdb), and db/db diabetic mice treated with rosiglitazone (dbdb+ R). (b) Renal NEP activity in lean control (L), lean + NEP inhibitor (Thiorphan) (L + I), Wild type (WT), and NEPKO. (c) Renal NEP activity in lean control (L), untreated db/db diabetic (db), and db/db diabetic mice treated with rosiglitazone (db+ R) for 10 weeks. Values are represented as mean ± SEM (n = 6–9, *p < .001 vs. lean controls & WT; # p < .001 vs. untreated db/db diabetic mice)

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  Figure 6. Immunofluorescence staining of renal NEP in the cortex and medulla of lean control, db/db diabetic, and db/db diabetic mice treated with rosiglitazone (db/db+ Rosi) for 10 weeks. Staining shows strong immunoreactivity in the brush border of the proximal tubules, distal tubules, and the Bowman capsules. Relative NEP immunostaining was quantified in cortex and medulla using Metamorphe software. Analysis showed significant decrease in NEP immunostaining in db/db diabetic mice compared to controls (*p < .001 vs. lean control, # p < .001 vs. lean control). Rosiglitazone treatment normalized NEP expression in the cortex and not in the medulla. Magnification: 40×, scale bars: 50 μm

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  Figure 7. Immunofluorescence staining for renal ADAM 17 shows strong immunoreactivity in the renal tubules of the cortex (a) and medulla (b) of lean control, db/db diabetic, and db/db diabetic mice treated with rosiglitazone (db/db+ Rosi) for 10 weeks. 40× magnification. ADAM17 immunoreactivity was mainly observed in the db/db diabetic kidney, specifically in the brush border of the proximal and the distal convoluted tubules. (c) Double immunofluorescence staining for ADAM 17 (green) and NEP (red) in cortex of db/db diabetic mice shows significant overlapping reactivity and co‐localization in the proximal renal tubules (magnification: 40×, scale bars: 50 μm)