Inhibition of NA(+)/H(+) Exchanger 1 Attenuates Renal Dysfunction Induced by Advanced Glycation End Products in Rats

Abstract

It has been recognized that sodium hydrogen exchanger 1 (NHE1) is involved in the development of diabetic nephropathy. The role of NHE1 in kidney dysfunction induced by advanced glycation end products (AGEs) remains unknown. Renal damage was induced by AGEs via tail vein injections in rats. Function and morphology of kidney were determined. Compared to vehicle- or BSA-treated rats, AGEs caused abnormalities of kidney structures and functions in rats, accompanied with higher MDA level and lower GSH content. Gene expressions of NHE1 gene and TGF-β1 in the renal cortex and urine were also increased in AGEs-injected rats. Importantly, all these detrimental effects induced by AGEs were reversed by inhibition of NHE1 or suppression of oxidative stress. These pieces of data demonstrated that AGEs may activate NHE1 to induce renal damage, which is related to TGF-β1.

Figure 1

AGEs time-/dose-dependently increase LDH leakage in isolated renal cortex from rats. Cortex from isolated rat kidney was sliced into small pieces with the thickness of 0.3–0.5 mm. The slice was incubated with AGEs as indicated times and concentrations. LDH leakage was assayed in slice of renal cortex. (a) Time course of AGEs on LDH leakage. (b) Dose course of AGEs on LDH leakage. All data were expressed as mean ± SD. N is 5 in each group. ^∗ P < 0.05 versus control (0).

Figure 2

Ex vivo inhibition of NHE1 by cariporide reverses AGEs-increased LDH leakage in rat renal cortex. Sliced renal cortex was incubated with BSA, H-car (1 μM cariporide), L-car (0.1 μM cariporide), and Ab-RAGE (antibody of AGEs receptor). After treatment, (a) LDH leakage and NHE1 protein expression, (b) GSH content, (c) MAD level, and (d) NHE1 activity were measured, respectively. All data were expressed as mean ± SD. N is 5 in each group. ^∗ P < 0.05 versus BSA. ^# P < 0.05 versus AGEs alone.

Figure 3

Inhibition of NHE1 improves AGEs-induced abnormality of glomerular structure in rats. The rats received a tail vein injection of AGEs (100 mg/kg) followed by treatment with or without N-acetylcysteine (200 mg/kg/day) or cariporide (1 mg/kg/day) for 12 weeks. At the end of experiments, rats were sacrificed under anesthesia. (a) Morphology of glomerular in kidney by HE staining. (b) Mesangial matrix injury score, (c) cell numbers in glomerular, and (d) glomerular volume were determined. Data are expressed as mean ± S.E.M. N is 10–15 in each group. ^∗ P < 0.05 versus BSA. ^# P < 0.05 versus AGEs alone.

Figure 4

In vivo administration of cariporide prevents renal dysfunction in AGEs-injected rats. The rats received a tail vein injection of AGEs (100 mg/kg) followed by treatment with or without N-acetylcysteine (200 mg/kg/day) or cariporide (1 mg/kg/day) for 12 weeks. At the end of experiments, (a) serum creatinine level, (b) blood urea nitrogen, (c) urinary protein excretion, and (d) clearance of creatinine were measured, respectively. All data were expressed as mean ± SD. N is 5 in each group. ^∗ P < 0.05 versus BSA. ^# P < 0.05 versus AGEs alone.

Figure 5

Cariporide recues AGEs-induced oxidative stress in kidney of rats. The rats received a tail vein injection of AGEs (100 mg/kg) followed by treatment with or without N-acetylcysteine (200 mg/kg/day) or cariporide (1 mg/kg/day) for 12 weeks. At the end of experiments, rats were sacrificed under anesthesia. (a) GSH content and (b) MAD level were determined. All data were expressed as mean ± SD. N is 10–15 in each group. ^∗ P < 0.05 versus BSA. ^# P < 0.05 versus AGEs alone.

Figure 6

Cariporide reduces the gene expressions of NHE1 and TGF-β1 in renal cortex from AGEs-injected rats. The rats received a tail vein injection of AGEs (100 mg/kg) followed by treatment with or without N-acetylcysteine (200 mg/kg/day) or cariporide (1 mg/kg/day) for 12 weeks. At the end of experiments, rats were sacrificed under anesthesia. (a) mRNA levels of NHE1 and TGF-β1 and protein level of NHE-1 were assayed by RT-PCR or Western blot. (b) TGF-β1 excretion of rat urine. All data were expressed as mean ± SD. N is 10–15 in each group. ^∗ P < 0.05 versus BSA. ^# P < 0.05 versus AGEs alone.