Homoarginine ameliorates diabetic nephropathy independent of nitric oxide synthase‐3

FIGURE 1. Homoarginine reduces albuminuria in diabetic mice. Urine was collected from wild type and NOS3−/− mice after 24 weeks of diabetes and analyzed for albumin/creatinine ratio. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.

FIGURE 2. Homoarginine reduces plasma BUN in diabetic NOS3−/− mice. Plasma BUN was determined in NOS3−/− mice after 24 weeks of diabetes. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.

FIGURE 3. Expression of NOS1 and NOS2 are not altered in diabetic NOS3−/− mice. RT‐PCR was performed on cDNA extracted from kidney samples of mice and analyzed for (a) NOS1 and (b) NOS2. Data are presented as mean ± SEM.

FIGURE 4. Homoarginine reduces glomerular pathology and fibrosis in diabetic NOS3−/− mice. (a) Paraffin‐embedded mouse kidney sections were subjected to periodic acid‐Schiff (PAS) staining. (b) Kidney sections were fixed in formaldehyde and stained with Masson's Trichrome. (c) Images from b were quantified for percent area fibrosis using ImageJ. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes. Pictures taken at 40× magnification. Black arrow: area of fibrosis.

FIGURE 5. Homoarginine reduces fibrotic markers in diabetic NOS3−/− mice. RT‐PCR was performed on cDNA extracted from kidney samples of mice and analyzed for (a) fibronectin, (b) TGFβ, (c) smooth muscle actin, (d) E‐cadherin. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.

FIGURE 6. Homoarginine reduces glomerular macrophage infiltration in diabetic NOS3−/− mice. Glomerular macrophage recruitment was visualized by immunohistochemical staining using Mac‐2 antibody. The number of glomerular macrophages was counted in 40 glomeruli per section (number of macrophages in glomeruli divided by the number of glomeruli) in blinded fashion under 40× magnification and averaged. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes. Pictures taken at 100× magnification. Black arrow: macrophage.

FIGURE 7. Homoarginine reduces cardiac fibrosis and improves cardiac function in diabetic NOS3−/− mice. (a) Masson's Trichrome staining of heart sections. Pictures taken at 40× magnification. (b) Fibrosis scoring of Masson's Trichrome heart sections from A using ImageJ. (c) Vevo 2100 Imaging Platform was used to image mouse hearts and determine percentage of left ventricular ejection fraction (EF) and fractional shrinking (FS). Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.

FIGURE 8. Homoarginine restores kidney MCU complex and its regulators in diabetic Ins2Akita mice. RT‐PCR was performed on cDNA extracted from kidney samples derived from our previous publication (Wetzel et al., 2019) and were analyzed for (a) MCU complex, (b) MCU positive regulator, MCUR1, and (c) MCU negative regulator, MICU1. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.

FIGURE 9. Homoarginine restores MCU complex and its regulators in diabetic NOS3−/− mice. (a) RT‐PCR analysis of MCU gene expression from mouse kidneys. (b) Protein was isolated from kidneys of NOS3−/− mice. Western blotted for MICU1 with β‐actin as protein‐loading control. (c) Quantification of MICU1 protein expression. Data are presented as mean ± SEM. *p < 0.05 versus normal, # p < 0.05 versus diabetes.