Diabetic Nephropathy

Nos3 deficiency markedly accelerates the development and progression of diabetic kidney disease in db/db mice without substantially affecting body weight or postprandial blood glucose levels. The dual-deficient model exhibits earlier and more severe albuminuria, progressive glomerular and tubular injury, increased interstitial inflammation, and pronounced renal fibrosis, demonstrating a robust and clinically relevant model of advanced diabetic nephropathy.

(A) Body weight progression during the study period; (B) postprandial blood glucose levels during the study period; (C) urinary albumin-to-creatinine ratio (UACR); (D) glomerulopathy scores; (E) tubular basophilia scores; (F) interstitial inflammation scores; and (G) representative renal histopathology at 16 weeks assessed by PAS and Masson staining, showing tubular protein casts (green arrow), basophilia (star), glomerulosclerosis (yellow arrow), and interstitial fibrosis (blue arrow). Groups were defined as follows: db/db mice and db/db Nos3-deficient mice, as indicated in the figure. Data are presented as mean ± SD. Statistical significance is indicated in the figure. ****, p < 0.0001 vs. db/db mice by one-way ANOVA with appropriate post-hoc comparisons.

In BKS-Lepr-/-; Nos3-/- mice, Semaglutide markedly improved glycemic control and reduced the progression of diabetic kidney disease, while Lisinopril provided additional renoprotective effects. Combination treatment produced superior reductions in albuminuria and renal histopathological injury compared with either monotherapy, demonstrating synergistic benefits in both metabolic control and kidney protection.

(A) Postprandial blood glucose levels over time following the first dose; (B) HbA1c levels measured at baseline and on Days 28 and 66 post first dosing; (C) longitudinal urinary albumin-to-creatinine ratio (UACR); (D) interstitial inflammation score; (E) glomerulopathy score; (F) tubular basophilia score; (G) tubular protein cast score; (H) interstitial fibrosis score; and (I) representative hematoxylin and eosin (H&E)-stained kidney sections. Green arrows indicate tubular basophilia, and blue arrows indicate interstitial inflammation. Groups included BKS-Lepr(-/-);Nos3(-/-) mice treated with Vehicle, Semaglutide, Lisinopril, or Semaglutide plus Lisinopril, as indicated in the figure. Data are presented as mean ± SD. Statistical significance is indicated in the figure. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 by one-way ANOVA with appropriate post-hoc comparisons. Scale bar = 50 μm.

Figure 1: Therapeutic Effects of Retatrutide, Lisinopril, and Empagliflozin in AAV-mRenin–Induced Diabetic Nephropathy in BKS-Lepr(-/-) Mice

In AAV-mRenin–induced diabetic kidney disease mice, Retatrutide produced robust body weight reduction and strong improvements in glycemic control, while Empagliflozin provided moderate glucose-lowering effects and Lisinopril primarily exerted renoprotective benefits. Among all treatments, Retatrutide and Lisinopril significantly reduced albuminuria, whereas Empagliflozin showed a weaker effect on urinary albumin excretion, highlighting differential efficacy across metabolic and renal endpoints.

(A) Body weight changes during the 49-day treatment period; (B) postprandial blood glucose levels over time; (C) HbA1c levels at study endpoint (Day 50); (D) longitudinal urinary albumin-to-creatinine ratio (UACR); and (E) endpoint UACR levels. Groups included BKS + AAV-Vector + Vehicle, BKS-Lepr(-/-) + AAV-Vector + Vehicle, BKS-Lepr(-/-) + AAV-mRenin + Vehicle, BKS-Lepr(-/-) + AAV-mRenin + 10 nmol/kg Retatrutide, BKS-Lepr(-/-) + AAV-mRenin + ~30 mpk Lisinopril, and BKS-Lepr(-/-) + AAV-mRenin + 20 mpk Empagliflozin, as indicated in the figure. Data are presented as mean ± SD. Statistical significance is indicated in the figure. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 by one-way ANOVA with appropriate post-hoc comparisons.

Figure 2: Histopathological Improvement and Reduction of Renal Fibrosis by Retatrutide, Lisinopril, and Empagliflozin in AAV-mRenin–Induced Diabetic Nephropathy in BKS-Lepr(-/-) Mice

Lisinopril significantly attenuated renal injury in diabetic mice by reducing glomerulopathy, tubular protein cast formation, and interstitial inflammation. Retatrutide also demonstrated marked renoprotective effects, improving both glomerular and tubular pathological changes. In contrast, Empagliflozin showed comparatively weaker effects on these histopathological parameters. Masson staining further revealed that Lisinopril and Retatrutide markedly reduced interstitial collagen deposition and preserved renal parenchymal integrity, supporting robust anti-fibrotic and renoprotective benefits.

(A) Glomerulopathy score; (B) tubular protein cast score; (C) tubular basophilia score; (D) interstitial inflammation score; (E) interstitial fibrosis score; and (F) representative Masson’s trichrome staining images of kidney sections. Yellow arrows indicate areas of interstitial fibrosis. Groups included BKS + AAV-Vector + Vehicle, BKS-Lepr(-/-) + AAV-Vector + Vehicle, BKS-Lepr(-/-) + AAV-mRenin + Vehicle, BKS-Lepr(-/-) + AAV-mRenin + 10 nmol/kg Retatrutide, BKS-Lepr(-/-) + AAV-mRenin + ~30 mpk Lisinopril, and BKS-Lepr(-/-) + AAV-mRenin + 20 mpk Empagliflozin, as indicated in the figure. Data are presented as mean ± SD. Statistical significance is indicated in the figure. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with appropriate post-hoc comparisons.