The Renal Damage Caused By Advanced Glycation End Products Relating To Activation Of Na~+/H~+ Exchanger 1

AIM To explore whether renal damages caused by advanced glycation end products relate to activation of Na⁺/H⁺ exchanger 1.METHOD (1) Kidney slice experiment in vitro: The renal slices from normal rats were prepared and incubated in vitro as previous method. The prepared slices were randomly divided into 7 groups as follows: Normal control (only contains DMEM/F12 medium) group; Bovine serum albumin (BSA, 200μg/ml) control group; Advanced glycation end products (AGEs, 200μg/ml); Cariporide (1μmol/ml) control group; AGEs +Cariporide (0.1, 1μmol/ml); AGEs +anti-RAGE (5μg/ml). About seven slices were placed in one incubation flask which was filled with 5 ml DMEM medium and gassed with 95% O₂ and 5% CO₂ at 37℃. After incubation of 120 min, the biochemical parameters in slices and medium were measured including the leakage rate of lactic dehydrogenase (LDH), content malondialdehyde (MDA) and glutathione (GSH), and NHE-1 activity. (2) Animal experiment in vivo: Eight rats were randomly selected to prepare a sham-operation without a left nephrectomy from the normal forty Sprague-Dawley rats. Others had a left nephrectomy and were randomly divided into the following groups: BSA control group; AGEs treated alone group; Cariporide treated group; N-acetylcysteine -treated group. The rats of all groups were received an injection of AGEs (100 mg/kg) by tain vein except BAS-treated and sham-operation groups and meanwhile were administrated Cariporide (1 mg/kg), NAC (200mg/kg) or normal sodium by gavage respectively for 12 weeks. The sham-operated and BSA-treated rats were received an injection of BSA (100 mg/kg) by tail vein and received orally the same volume of normal sodium. Urine was collected by the metabolic cage to determine 24-hour urine creatinine, protein and transforming growth factorβ1 (TGF-β1) three days before the end of the experiment. Blood was collected by arterial catheterization to assay the creatinine and blood urea nitrogen (BUN). The right kidney was removed, weighed and calculated the ratio of kidney weight to body weight (kidney hypertrophy index). One part of the renal cortex was homogenated to examine content of both MDA and GSH; another part was stored at -80℃for detecting the mRNA of both NHE-1 and TGF-β1; The rest was harvested for pathological examination. Glomerular volume, total glomerular surface area and injury score of mesangial matrix were determined with the image analysis system.RESULTS (1) In vitro experiment: The content of MDA and leakage rate of LDH was increased by 2.2 folds and 2.4 folds (P＜0.01) respectively and the GSH content was decreased by 3.5 folds, Moreover, NHE-1 activity in renal cortex also increased (P＜0.01, in comparison to control) after incubation with AGEs alone. On the contrary, high- and low-dose of Cariporide could evidently suppress these changes induced by AGEs (P<0.01). Anti-RAGE could also attenuate the cortex damage induced by AGEs. The incubation of slices with Cariporide or BSA alone had no effect on these biochemical parameters of the renal cortex. 2) In vivo experiment: The injection of AGEs alone resulted in abnormalities of both structure and function in rat kidneys. Compared with the sham-operated rats, the mRNA levels of NHE-l and TGF-β1 in the renal cortex were significantly increased as well as the excretion of TGF-β1 (from 49.25±2.43 to 105.49±5.49, P＜0.01) in urine. The treatment of cariporide plus AGEs significantly improved AGEs-induced disordered structure and dysfunction, down-regulated the expression of both NHE-l mRNA and TGF-β1 mRNA, decreased MDA concertration (from 10.75±1.23 to 7.72±0.83,) and urinary TGF-β1 excretion (from 105.49±5.49 to 69.15±2.51), preserved GSH content (from 9.94±0.81 to 12.40±0.68). There were no changes in body weight, kidney hypertrophy index, renal function, 24 h excretion of both protein and TGF-β1 in urine, content of both MDA and GSH in renal cortex between the sham-operated and operated-rats with BSA groups.CONCLUTION:â‘ The incubation of rat renal slices with AGEs induced lipid peroxidation and led to renal damage in vitro.â‘¡The injection of ectogenic AGEs could induce damages of renal tissue and dysfunction in rats.â‘¢Treatment of cariporide could significantly prevent AGEs induced damages of structures and functions in vivo and vitro.â‘£The mechanisms of AGEs induced renal injuries may be through the reactions with AGE receptor (RAGE) which in turn led to increasing of oxidative stress, and then activated NHE-1 on plasma membrane. Once activated, NHE-1 not only functioned as an exchanger to increase intracellular Ca , but also as a scaffold platform to activate TGFβ-1 signaling pathway. Inhibiting the activation of NHE-1 could prevent AGEs induced renal damage. It was suggested that activation of NHE-1 may play an important role in the renal injuries caused by AGEs and NHE-1 may be expected as a new therapeutic target for diabetic nephropathy.