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The altered homeostasis of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) was closely associated with the pathological process of nervous system diseases and insulin resistance. Here, the exact implication of phosphofurin acidic cluster sorting protein 2 (PCAS-2), an anchor protein in the MAM interface, in diabetic kidney disease was investigated. In the kidneys of type 1 and type 2 diabetes mice and HG-induced HK-2 cells, a notable disruption of ER-mitochondria interactions, accompanied by a decreased PACS-2 expression in all subcellular fractions. Furthermore, PACS-2 knockout mice with diabetes displayed accelerated development of proteinuria, deterioration of kidney function, and aggravated disruption of MAM area, ER stress, mitochondrial dysfunction, renal apoptosis, and fibrosis. However, overexpression of PACS-2 effectively protected diabetic kidneys and HG-treated HK-2 cells from renal tubular impairments. Importantly, experimental uncoupling of ER-mitochondria contacts reversed the protective effects of PACS-2 restoration on HK-2 cells under HG conditions. In summary, our data indicate a pivotal role of PACS-2 in the development of diabetic renal tubular injury via the stabilization of MAM.

Growth arrest and DNA damage-inducible beta (GADD45B) is closely linked with cell cycle arrest, DNA repair, cell survival, or apoptosis in response to stress and is known to regulate the mitogen-activated protein kinase (MAPK) pathway. Here, using an RNA sequencing approach, we determined that GADD45B was significantly upregulated in diabetic kidneys, which was accompanied by renal tubular epithelial-mesenchymal transition (EMT) and apoptosis, as well as elevated MAPK pathway activation. In vitro, GADD45B expression in cultured human kidney proximal tubular epithelial cells (HK-2 cells) was also stimulated by high glucose (HG). In addition, overexpression of GADD45B in HK-2 cells exacerbated renal tubular EMT and apoptosis and increased p38 MAPK and c-Jun N-terminal kinases (JNK) activation, whereas knockdown of GADD45B reversed these changes. Notably, the activity of extracellular regulated kinase (ERK) was not affected by GADD45B expression. Furthermore, inhibitors of p38 MAPK (SB203580) and JNK (SP600125) alleviated HG‐ and GADD45B overexpression-induced renal tubular epithelial-mesenchymal transition and apoptosis. These findings indicate a role of GADD45B in diabetes-induced renal tubular EMT and apoptosis via the p38 MAPK and JNK pathways, which may be an important mechanism of diabetic kidney injury.

Background and purpose Excessive mitochondrial fission was observed in diabetic kidney disease (DKD). Phosphoglycerate mutase family member 5 (PGAM5) plays an important role in mitochondrial fission by dephosphorylating the dynamin-related protein 1 at Ser637 (DRP1S637). Whether PGAM5 participates in the mitochondrial fission in diabetic renal tubular injury is unknown. Clinical trials have observed encouraging effect of Sodium-glucose cotransporter 2 (SGLT2) inhibitors on DKD though the underling mechanisms remain unclear. Experimental approach We used KK-Ay mice as diabetic model and Empagliflozin (Empa) were administrated by oral gavage. The mitochondrial fission and the expressions of phosphorylated AMP-activated protein kinase (p-AMPK), specificityprotein1 (SP1), PGAM5 and DRP1S637 were tested. We also examined these changes in HK2 cells that cultured in normal glucose (NG), high glucose (HG) and high glucose+Empa (HG + Empa) environment. Then we verified our deduction using AMPK activator (5-aminoimidazole-4-carboximide Riboside, AICAR), inhibitor (Compound C), si-SP1 and si-PGAM5. Lastly, we testified the interaction between SP1 and the PGAM5promotor by CHIP assay. Key results The mitochondrial fission and the expression of SP1, PGAM5 increased and the expression of p-AMPK, DRP1S637 decreased in diabetic or HG environment. These changes were all reversed in Empa or AICAR treated groups. These reversal effects of Empa could be diminished by Compound C. Either si-SP1 or si-PGAM5 could alleviate the mitochondrial fission without affection on AMPK phosphorylation. Finally, the CHIP assay confirmed the interaction between SP1 and the PGAM5 promotor. Conclusions and implications The PGAM5 aggravated the development of diabetic renal tubular injury and the Empa could improve the DKD by alleviating mitochondrial fission via AMPK/SP1/PGAM5 pathway.

Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex pathway were inhibited by 50 μM atorvastatin in INS-1 cells in vitro. Moreover, 50 μM atorvastatin reduced the binding of p-CREB with deoxyribonucleic acid (DNA) in INS-1 cells in vitro. Conclusion: Atorvastatin inhibits insulin synthesis in beta cells by inhibiting the activation of the Ras complex pathway.