l-Homoarginine supplementation prevents diabetic kidney damage

Abstract

l-homoarginine is an endogenous, non-proteinogenic amino acid that has emerged as a new player in health and disease. Specifically, low l-homoarginine levels are associated with cardiovascular diseases, stroke, and reduced kidney function. However, the role of l-homoarginine in the pathogenesis of diabetic nephropathy (DN) is not known. Experiments were conducted in 6-week-old Ins2^Akita mice supplemented with l-homoarginine via drinking water or mini osmotic pump for 12 weeks. Both plasma and kidney l-homoarginine levels were significantly reduced in diabetic mice compared to nondiabetic controls. Untreated Ins2^Akita mice showed significant increases in urinary albumin excretion, histological changes, glomerular macrophage recruitment, the inflammatory cytokine KC-GRO/CXCL1, and urinary thiobarbituric acid reactive substances (TBARS) excretion as an indicator of oxidative stress, along with a significant reduction in kidney nitrate + nitrite levels compared to control mice at 18 weeks of age. In contrast, l-homoarginine supplementation for 12 weeks in Ins2^Akita mice, via either drinking water or mini osmotic pump, significantly reduced albuminuria, renal histological changes, glomerular macrophage recruitment, KC-GRO/CXCL1 levels, urinary TBARS excretion, and largely restored kidney nitrate + nitrite levels. These data demonstrate that l-homoarginine supplementation attenuates specific features of DN in mice and could be a potential new therapeutic tool for treating diabetic patients.

Keywords: Diabetic nephropathy; l-homoarginine; nitric oxide.

Figure 1

Plasma and kidney l‐homoarginine levels were reduced in type‐1 diabetic mice. Plasma and kidney samples from diabetic and nondiabetic mice at 18 weeks of age were used to measure l‐homoarginine levels using a liquid chromatography–mass spectrometry system. Results are presented as mean ± SEM. *P < 0.05 compared to nondiabetic mice.

Figure 2

l‐homoarginine supplementation reduces urine albuminuria in diabetic mice. l‐homoarginine was supplemented to Ins2^Akita mice for 12 weeks via either drinking water or mini osmotic pumps as indicated. Twenty‐four hour urine was collected for the measurement of UAER after 9 and 12 weeks of supplementation. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01 compared to nondiabetic controls mice; ^# P < 0.05 compared to diabetic mice.

Figure 3

l‐homoarginine supplementation increased kidney homoarginine levels in diabetic mice. Plasma and kidney samples were used after 12 weeks of diabetes using a liquid chromatography–mass spectrometry system or ELISA assay. Results are presented as mean ± SEM. ^# P < 0.0001 compared to diabetic mice.

Figure 4

Effects of l‐homoarginine on kidney pathology. Paraffin‐embedded mouse kidney sections were subjected to periodic acid–Schiff (PAS) staining. Scale bar represents 20 µm.

Figure 5

l‐homoarginine supplementation reduces glomerular macrophage infiltration in diabetic mice. (A) Glomerular macrophage recruitment was visualized by immunohistochemical staining using Mac‐2 antibody 12 weeks after l‐homoarginine supplementation. Macrophage numbers per glomerulus were counted in a blinded manner. Scale bar in images represents 10 µm. (B) The number of glomerular macrophages was counted in 20 glomeruli per section (number of macrophages in glomeruli divided by the number of glomeruli) in blinded fashion under 40× magnification and averaged. Results are presented as mean ± SEM. *P < 0.01 compared to nondiabetic control mice; ^# P < 0.05 compared to diabetic mice. N = 10 for nondiabetic, 9 for diabetes and diabetes + oral HA, 7 for diabetes + pump HA.

Figure 6

l‐homoarginine supplementation reduces kidney KC‐GRO/CXCL1 levels in diabetic mice. A MSD multi‐spot assay was used to measure kidney KC‐GRO levels. Data are presented as mean ± SEM. *P < 0.0005 compared to nondiabetic control mice; ^# P < 0.005 compared to diabetic mice.

Figure 7

l‐homoarginine supplementation reduces urinary TBARS excretion in diabetic mice. Twenty‐four hour urine was collected at 18 weeks of age for the measurement of TBARS. Data are presented as mean ± SEM. *P < 0.0001 compared to nondiabetic controls mice; ^# P < 0.05 compared to diabetic mice.

Figure 8

l‐homoarginine supplementation increases renal nitrate and nitrite concentration in diabetic mice. Mouse kidney lysates were used to measure nitrate and nitrite concentration after 12 weeks of l‐homoarginine supplementation. Data are presented as mean ± SEM. *P < 0.01 compared to nondiabetic control mice; ^# P < 0.05 compared to diabetic mice.

Figure 9

The effect of l‐homoarginine supplementation on l‐arginine levels. Mouse plasma and kidney tissues were prepared to measure l‐arginine levels using a liquid chromatography–mass spectrometry system. Data are mean ± SEM. *P < 0.01 compared to nondiabetic control mice.