RESUMO
Objective:To study the changes in long non-coding RNA C2dat1 expression in kidney tissues of rats at different stages of diabetic kidney disease (DKD) and its relationship with renal interstitial fibrosis.Methods:Forty-eight male SD rats were randomly divided into two groups with 24 rats in each group: control group and DKD group. The rats in the control group were fed with ordinary diet, while those in the DKD group were fed with high-fat diet and drank water freely. After eight weeks of feeding, the rats were fasted for 12 h with free access to water. Then, the DKD group was given a one-time intrabitoneal injection of streptozotocin and the control group was given an equal dose of sodium citrate buffer. After 72 h, the random peripheral blood glucose concentration (≥ 16.7 mmol/L for three consecutive days) and urine sugar (positive) were tested to assess the establishment of the diabetes model. Urine, blood and kidney samples were collected at 3, 6, 9 and 12 weeks. The urinary protein excretion rate within 24 h, urinary creatinine and serum total cholesterol were measured by automatic biochemical apparatus. Pathological changes in kidney tissues were observed by HE staining. The expression of calcium/calmodulin-dependent protein kinase Ⅱ delta (CaMK2D), p65, p50, α-SMA and E-cardherin was detected by immunohistochemistry. Quantitative real-time PCR (qPCR) was used to detect the expression of lncRNA C2dat1 and CaMK2D. The relationship of lncRNA C2dat1 with α-SMA, E-cardherin and CaMK2D was analyzed by correlation analysis. In in vitro experiment, renal tubular epithelial cells HK-2 were induced by high glucose. The expression of lncRNA C2dat1 and CaMK2D in HK-2 cells was detected by qPCR after 24, 48 and 72 h of intervention. Results:The rats in the DKD group showed typical symptoms such as polydipsia, polyphagia, significant weight loss and increased blood glucose as compared with the rats in the control group. Results of the biochemical tests revealed that compared with the control group, the DKD group had increased 24 h excretion rate of urinary protein, decreased urinary creatinine and up-regulated total cholesterol. HE staining showed that the rats in the control group had intact glomeruli, normal basement membrane and no mesangial hyperplasia or inflammatory cell infiltration. However, enlarged glomeruli and evenly thickened basement membrane were observed in the DKD group. Immunohistochemistry indicated that the expression of CaMK2D, p50 and α-SMA was higher in the DKD group than in the control group, while the expression of E-cardherin was lower in the DKD group. qPCR results showed that the expression of lncRNA C2dat1 and CaMK2D at mRNA level was higher in the DKD group than in the control group. In in vitro experiment, the expression of lncRNA C2dat1 and CaMK2D at mRNA level was also higher in HK-2 cells induced by high glucose than in the control group. Correlation analysis indicated that lncRNA C2dat1 was positively correlated with α-SMA and CaMK2D, but negatively correlated with E-cardherin. Conclusions:During the progression of DKD, the high expression of lncRNA C2dat1 might promote diabetic renal interstitial fibrosis by regulating the expression of CaMK2D to activate the NF-κB signaling pathway.
RESUMO
Objective This study aimed to investigate the renal expression change of high mobility of nucleosome binding protein 1 ( HMGN1) in epithelia-mesenchymal transition ( EMT) process, and to study the effect of HMGN1 on renal fibrosis in the diabetic nephropathy mice model. Methods 20 C57BL/6 mice were randomly divided into control group, model group, benazepril group, and insulin group. After 8 weeks of drug intervention, blood, urine and kidney tissue samples were taken from mice. The routine physiological and biochemical indexes were detected. Renal structure and fibrosis were detected by HE and Sirius red staining, respectively. Immunohistochemistry and in situ hybridization were used to investigate the protein and mRNA expression levels of HMGN1, CD68, F4/80,α-smooth muscle actin (α-SMA) , and E-cadherin in renal tissue. Results Blood glucose, renal index, and urinary albumin to creatinine ratio ( UACR) were significantly higher in the model group than those in the normal group. In the model group, HE staining showed glomerular hypertrophy and interstitial inflammatory cell infiltration, and Sirius red showed collagen deposition in the renal tissue. Compared with normal group, HMGN1, CD68, F4/80 positive cell counts andα-SMA protein expression were all increased, while E-cadherin protein expression was downregulated in the model group ( all P<0.05) . The above indexes were not improved significantly in the benazepril group. And after intervention of insulin, the expression levels of CD68 positive cell count andα-SMA protein were decreased and the expression levels of E-cadherin protein were increased compared with the model group ( all P<0.05) . The correlation analysis showed that the level of HMGN1 was correlated with CD68, F4/80, α-SMA, E-cadherin and collagen protein, while CD68 and f4/80 were correlated withα-SMA, collagen protein and blood glucose, respectively ( all P<0. 05 ) . Conclusion HMGN1 is involved in the progression of diabetic nephropathy fibrosis, and its underlying mechanism might be related to the macrophage-mediated EMT process.