The molecular mechanism linking aldosterone and endothelin-1 in the development of diabetic nephropathy has not been completely elucidated. Here, we provide evidence showing that streptozotocin-induced diabetic rats have significantly increased aldosterone and endothelin-1 in the kidney tissue and markedly decreased expression of Dot1a and Af9. Blocking aldosterone with spironolactone significantly reduced proteinuria, glomerulosclerosis, tubulointerstitial injury and endothelin-1 expression, and significantly increased Dot1a and Af9 expression. Increasing Dot1a and Af9 expression by spironolactone or by stable transfection led to impaired endothelin-1 expression in NRK-52 cells. In contrast, downregulation of Dot1a and Af9 by aldosterone in NRK-52E cells caused upregulation of endothelin-1. Genetic inactivation of Dot1l, which encodes Dot1a, in Aqp2-expressing principal cells of mouse kidney impaired association of Dot1a and H3 dimethyl K79 with the specific subregions of endothelin-1 promoter, and upregulates endothelin-1 mRNA and protein expression. Our data suggest that Dot1a and Af9 repress endothelin-1 in vitro and in vivo. Excessive aldosterone induces kidney injury, in part possibly by downregulating Dot1a and Af9, and thus relieving Dot1a-Af9-mediated repression to increase endothelin-1 transcription. Spironolactone ameliorates kidney injury in Streptozotocin-induced diabetic rats, possibly by restoring Dot1a-Af9-mediated repression to reduce endothelin-1 expression. Therefore, Dot1a and Af9 as aldosterone-downregulated targets are negative regulators of endothelin-1 transcription in vitro and in vivo, and may be considered as new potential therapeutic targets of kidney injury in diabetes.

BackgroundLong-term exposure to hypertonic and high glucose in peritoneal dialysis fluid can result in peritoneal fibrosis. Spleen tyrosine kinase (SYK) has a role in inflammation and fibrosis. This study aimed to investigate the role of SYK in an in vivo rat model of peritoneal fibrosis and in rat peritoneal mesothelial cells (PMCs) in vitro and to investigate the underlying mechanisms.Material/MethodsSprague-Dawley rats (N=24) were randomized into the sham control group (N=6); the peritoneal fibrosis group (N=6) treated with intraperitoneal chlorhexidine digluconate; the SYK inhibitor group (N=6), treated with chlorhexidine digluconate and fostamatinib; and the TGF-β inhibitor group (N=6), treated with chlorhexidine digluconate and LY2109761. The rat model underwent daily intraperitoneal injection with 0.5 ml of 0.1% chlorhexidine digluconate. Rat peritoneal mesothelial cells (PMCs) were cultured in vitro in high glucose. SYK expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR measured inflammatory mediators. Transforming growth factor-β1 (TGF-β1) and Smad3 were detected by Western blot. Short hairpin RNA (shRNA) was used to target the SYK gene.ResultsSYK was upregulated in the rat model of peritoneal fibrosis and was induced rat PMCs cultured in high glucose. Knockdown of SYK and inhibition of TGF-β1 significantly reduced fibrosis and inflammation. Findings in the in vivo rat model confirmed that SYK mediated peritoneal fibrosis by regulating TGF-β1/Smad3 signaling.ConclusionsIn a rat model and in rat PMCs, expression of SYK increased peritoneal fibrosis through activation of the TGF-β1/Smad3 signaling pathway.

Background Peritoneal dialysis is the most common treatment for end-stage renal disease. However, peritoneal fibrosis resulting from long-term peritoneal dialysis restricts peritoneal ultrafiltration. Previous studies have shown a role for 1,25-dihydroxyvitamin D3 (1,25[OH] 2 D3) in preventing fibrosis, but the potential mechanisms remain unknown. This study aimed to investigate the role of 1,25(OH) 2 D3 in epithelial-mesenchymal transition (EMT) and the downstream signaling pathway in HMrSV5 human peritoneal mesothelial cells in vitro . Material/Methods An in vitro cell model of peritoneal fibrosis was established using the HMrSV5 human peritoneal mesothelial cell line. High glucose and lipopolysaccharide (LPS) culture conditions, with or without 1,25(OH) 2 D3, were used. Wnt agonist 1, a Wnt signaling pathway activator, was applied. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure the vitamin D receptor (VDR) and histone deacetylase 3 (HDAC3) gene and protein expression levels, β-catenin, and EMT-associated biomarkers. Results High glucose plus LPS culture medium inhibited cell proliferation, induced cell apoptosis and promoted EMT in HMrSV5 cells, which was reversed by 1,25(OH) 2 D3 by down-regulation of HDAC3 and upregulation of VDR. HDAC3 inhibited VDR gene expression. The expression of EMT-associated biomarkers was increased by Wnt agonist 1 and inhibited by 1,25(OH) 2 D3. Conclusions In HMrSV5 human peritoneal mesothelial cells, 1,25(OH) 2 D3 reversed EMT by inhibiting the expression of HDAC3 and upregulating VDR gene expression via the Wnt/β-catenin signaling pathway.

Background. Clear cell renal cell carcinoma (ccRCC) is a cancer with abnormal metabolism. The purpose of this study was to investigate the effect of metabolism-related genes on the prognosis of ccRCC patients. Methods. The data of ccRCC patients were downloaded from the TCGA and the GEO databases and clustered using the nonnegative matrix factorization method. The limma software package was used to analyze differences in gene expression. A random forest model was used to screen for important genes. A novel Riskscore model was established using multivariate regression. The model was evaluated based on the metabolic pathway, immune infiltration, immune checkpoint, and clinical characteristics. Results. According to metabolism-related genes, kidney clear cell carcinoma (KIRC) datasets downloaded from TCGA were clustered into two groups and showed significant differences in prognosis and immune infiltration. There were 667 differentially expressed genes between the two clusters, of which 408 were screened by univariate analysis. Finally, 12 differentially expressed genes (MDK, SLC1A1, SGCB, C4orf3, MALAT1, PILRB, IGHG1, FZD1, IFITM1, MUC20, KRT80, and SALL1) were filtered out using the random forest model. The model of Riskscore was obtained by multiplying the expression levels of these 12 genes with the corresponding coefficients of the multivariate regression. We found that the Riskscore correlated with the expression of these 12 genes; the high Riskscore matched the low survival rate verified in the verification set. The analysis found that the Riskscore model was associated with most of the metabolic processes, immune infiltration of cells such as plasma cells, immune checkpoints such as PD-1, and clinical characteristics such as M stage. Conclusion. We established a new Riskscore model for the prognosis of ccRCC based on metabolism. The genes in the model provided several novel targets for the study of ccRCC.