Endoplasmic reticulum stress and proteasome pathway involvement in human podocyte injury with a truncated COL4A3 mutation.

BACKGROUND: Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury. METHODS: Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. RESULTS: Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group. CONCLUSIONS: We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation.

PPAR-gamma agonists inhibit TGF-beta1-induced chemokine expression in human tubular epithelial cells.

AIM: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has a wide range of biological functions, including anti-inflammation. In this study, we investigated the inhibitory effects of PPAR-gamma on transforming growth factor beta1 (TGF-beta1)-induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) expression in renal tubular epithelial cells (HK-2). METHODS: HK-2 cells were pretreated with 15d-PGJ2 or troglitazone (TGL) and then treated with TGF-beta1. Expression of MCP-1 and IL-8 was measured using real-time PCR and ELISA. RESULTS: Treatment with 5 ng/mL TGF-beta1 for 24 h increased both MCP-1 and IL-8 mRNA and protein levels in HK-2 cells. Both 15d-PGJ2 at 2.5 and 5 micromol/L and TGL at 2.5 micromol/L exhibited inhibitory effects on TGF-beta1-induced MCP-1 expression. Additionally, 15d-PGJ2 at 2.5 and 5 micromol/L and TGL at 2.5 micromol/L inhibited TGF-beta1-induced expression of IL-8. CONCLUSION: PPAR-gamma agonists (15d-PGJ2 and TGL) could inhibit the TGF-beta1-induced expression of chemokines in HK-2 cells. Our results suggest that PPAR-gamma agonists have the potential to be used as a treatment regimen to reduce inflammation in renal tubulointerstitial disease.