| Diabetes has become a global disease, which is the fifth leading cause of death. The prevalence rate of diabetes in China has increased four times during the past ten years. Among the various of secondary problems associated with diabetes, diabetic nephropathy(DN) significantly contributes to the end-stage renal diseases. The mechanism of DN is very complex and a great various factors contribute to the progression of DN. Accumulated evidences indicate that progressive tubular epithelial cells injury and transformation of tubular epithelial cells to a fibroblast-like phenotype further interstitial fibrosis, have been proved to correlate with the magnitude of proteinuria.The aging-suppressor gene that was identified by Kuro-o et al in 1997 was named Klotho. The Klotho(KL) gene encodes the Klotho protein. Three types of Klotho proteins have been detected: the full-length transmembrane Klotho, soluble Klotho, and secreted Klotho. All three proteins have been detected in humans and mice, but only the transmembrane and soluble Klotho proteins have been detected in rats. The Klotho protein, a multifunctional protein, inhibits oxidative stress, modulates iron channels and regulates the metabolism of phosphate, calcium, and vitamin D. Klotho also may function as a hormone, although the Klotho receptor(s) has not been found. The Klotho protein in humans is expressed primarily in the tubular epithelial cells of the kidney and brain choroid plexus, which suggests that Klotho may be essential to the maintenance of normal renal function.Endoplasmic reticulum(ER) stress is one of the most important mechanisms against stress in cells. Synthesis of transmembrane and secretory proteins occurs within the ER. The dysregulation of protein synthesis/processing within the ER causes the accumulation of unfolded proteins, thereby leading to ER stress and the activation of the unfolded protein response(UPR). To some extent, ER stress can activate various intrinsic protective response by clearing the unfolded protein or degrading old proteins. However, if the UPR is oversaturated and misfolded proteins accumulate, the ER can shift into a cytotoxic response, a physiological phenomenon known as ER stress. Emerging evidence from experimental and clinical research indicates that endoplasmic reticulum(ER) stress is involved in renal diseases including DN. It was shown that ER stress-mediated apoptosis could be a crucial pathway contributing to the pathogenesis of DN. ER stress-induced apoptosis was mediated by caspase-12 activation.This study is to observe the expressions of Klotho and Caspase-12 in renal tubular epithelial cells in diabetic rats and to explore the pathogenesis and the possible treatment target of DN by establishing the type 2 diabetes mellitus rat model.Objective: To explore the effect and possible mechanism of the expressions of Klotho and Caspase-12 in DNMethods: 40 healthy male Wistar rats weighing 180 to 220 gram were employed. The rats were randomly divided into 2 groups: normal control group(Control group) and diabetic nephropathy group(DN group). Each group included 20 rats. The control group was fed with a standard rat diet for 16 weeks and in the fifth weeks, 3 ml of 0.1mmol/L citrate buffer solution was administrated by peritoneal injection. The DN group received a high-fat, high-energy diet for 16 weeks( to induce insulin resistance) and a 30 mg/kg streptozotocin peritoneal injection in the fifth week to induce diabetes. As the diagnostic criteria of diabetes in Wistars rats is that the fast blood glucose level is higher than 16.7mmol/L, the rats with fast blood glucose continually higher than 16.7mmol/L were identified diabetes. The other rats were excluded. The following indexes were evaluated during the experiment:(1) The rats were weighed at the start and every four weeks afterwards until the animal were sacrificed at week 16.(2) The fast blood glucose(FBG) was determined from tail bleeding at the start and every four weeks.(3) The serum content of Triglyceride(TG), Total cholesterol(T-CHO) were determined every four weeks. The fast blood insulin was detected every eight weeks. And the insulin sensitive index(ISI) was calculated.(4) The kidneys of rats were weighed. Then renal tissues were collected. And the pathological changes of kidney were observed by light microscope.(5)The expressions of Klotho and caspase-12 in tubules were detected by immunohistochemistry staining.Results:1 General status of two groups:Compared with the control group, the body weight of rats declined remarkably in DN group at the end of experiment(P<0.01). The FBG in DN group was significantly higher than that of control(P<0.01). The serum TG and T-CHO content were remarkably higher in DN group than those of control group(P<0.05). The fast insulin level in rats of DN group was markedly lower than that of control group(P<0.01). And the insulin sensitivity index(ISI) in DN group(-6.41±0.38) was significantly lower than that of control group(-5.81±0.38)( P<0.01). The insulin resistance index(HOMO-IRI) in DN group was 28.86±12.05, which was much higher than the HOMO-IRI of control group(15.76±5.84)(P<0.05).2 The pathological changes of renal tissue:The pathological changes of renal tissue were examined by light microscope. The pathological changes of renal tissue in rats of DN group are followings: the glomeruli were enlarged and proximal tubular epithelial cells were swelling in the kidneys in DN group at weeks 8. Glomerular sclerotic lesion, dilation and atrophy of the tubules, interstitial fibrosis and inflammatory changes were found at week 12. The extent of glomerular damage and interstitial fibrosis was increased at week 16.By PAS staining, it is shown that compared with control group, volume of glomeruli, mesangial matrix, thickness of glomerular and tubular basement membrane increased with the progression of diabetic in DN group.3 The expressions of Klotho and caspase-12 in renal tubular epithelial cellsKlotho protein is primarily expressed in renal proximal tubules. By immunohistochemistry staining, the positive reaction of Klotho was identified in cytoplasm, characterized by brown-yellowish granules in cytoplasm. There was positive basal expression in the renal proximal tubular epithelial cells in the control group. The expression of Klotho in DN group were significantly lower than that of control group(P<0.05).Caspase-12 protein is primarily expressed in renal glomeruli and tubules. By immunohistochemistry, the positive reaction of caspase-12 protein was identified in cell nucleus and /or cytoplasm, represented by brown-yellowish granules in the cytoplasm and/or nuclear. There was light positive basal expression in the control group. In the DN group, the numbers of positive-expressed cells and the expression intensity of Caspase-12 in renal tissue were significantly higher than that of control group(P<0.01, P<0.05).According to the correlation analysis, there is a negative correlation between the expression of Klotho and caspase-12(r=-0.643, P<0.05) in rats with DN.4 The apoptosis of tubular epithelial cells by TUNEL stainingBy TUNEL staining, the apoptotic cells were identified by brown-yellowish granules in cell nucleus. The apoptotic cells were counted to calculate the apoptotic index. The apoptotic index of tubular epithelial cells in DN group(34.6±5.2) was significantly higher than that of control group(1.6±1.2)(P<0.01).Conclusion:It has shown that the expression of Klotho protein was inhibited and the expression of Caspase-12 protein was increased in renal tubules of rats with type 2 diabetes. There is a negative correlation between the expression of Klotho and caspase-12 in rats with DN. Meanwhile, the apoptosis in renal tubules cells increased. It is proposed that apoptosis mediated by ER stress might be involved in the pathogenesis and development of DN.Key words: Type 2 diabetes mellitus,diabetic nephropathy,Klotho,Ca... |
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