Alport syndrome (AS) is a hereditary glomerular nephritis caused by mutation in one of the type IV collagen genes α3/α4/α5 that encode the heterotrimer COL4A3/4/5. Failure to form a heterotrimer due to mutation leads to the dysfunction of the glomerular basement membrane, and end-stage renal disease. Previous reports have suggested the involvement of the receptor tyrosine kinase discoidin domain receptor (DDR) 1 in the progression of AS pathology. However, due to the similarity between DDR1 and DDR2, the role of DDR2 in AS pathology is unclear. Here, we investigated the involvement of DDR2 in AS using the X-linked AS mouse model. Mice were treated subcutaneously with saline or antisense oligonucleotide (ASO; 5 mg/kg or 15 mg/kg per week) for 8 weeks. Renal function parameters and renal histology were analyzed, and the gene expressions of inflammatory cytokines were determined in renal tissues. The expression level of DDR2 was highly elevated in kidney tissues of AS mice. Knockdown of Ddr2 using Ddr2 -specific ASO decreased the Ddr2 expression. However, the DDR2 ASO treatment did not improve the proteinuria or decrease the BUN level. DDR2 ASO also did not significantly ameliorate the renal injury, inflammation and fibrosis in AS mice. These results showed that Ddr2 knockdown by ASO had no notable effect on the progression of AS indicating that DDR2 may not be critically involved in AS pathology. This finding may provide useful information and further understanding of the role of DDRs in AS.

Background　Bardoxolone methyl activates nuclear factor erythroid 2 related factor 2 (Nrf2) via covalent binding and irreversible inhibition of Kelch-like ECH-associated protein-1 (Keap1), the negative regulator of Nrf2. Ongoing clinical trials of Bardoxolone methyl show promising effects for patients with chronic kidney disease (CKD). But the direct inhibition of Keap1-Nrf2 protein-protein interaction (PPI) as an approach to activate Nrf2 is less explored. Methods　We developed a non-covalent Nrf2 activator UBE-1099, which highly selectively inhibits Keap1-Nrf2 PPI, and evaluated its efficacy on progressive phenotype in Alport syndrome mouse model (Col4a5-G5X). Results　 Similar to Bardoxolone methyl, UBE-1099 transiently increased proteinuria and reduced plasma creatinine in Alport mice. Importantly, UBE-1099 improved the glomerulosclerosis, renal inflammation and fibrosis, and prolonged the lifespan of Alport mice. UBE-1099 ameliorated the dysfunction of Nrf2 signaling in renal tissue of Alport mice. Moreover, transcriptome analysis in glomerulus showed that UBE-1099 induced the expression of genes associated with cell cycle and cytoskeleton, which may explain its unique mechanism of improvement such as glomerular morphological change. Conclusions　UBE-1099 significantly ameliorates the progressive phenotype in Alport mice. Our results firstly revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis and presents a potential therapeutic drug for CKD.

Bardoxolone methyl is an electrophilic agent that induces Nrf2 activation by irreversibly and covalently binding to the cysteine residue of Keap1. Ongoing clinical trials of Bardoxolone methyl show promising effects for patients with chronic kidney disease (CKD). However, irreversible Keap1 inhibitors such as Bardoxolone methyl may covalently bind to other proteins in a non-specific manner and induce side effects due to off-target activities. In this study, we developed a reversible Keap1 inhibitor UBE-1099, which highly selectively and non-covalently inhibits Keap1-Nrf2 protein-protein interaction (PPI) and induces Nrf2 activation. We evaluated its efficacy on glomerulosclerosis in mouse model of CKD (Alport syndrome: Col4a5-G5X). Similar to Bardoxolone methyl, UBE-1099 transiently increased proteinuria and reduced plasma creatinine in CKD model mice. Importantly, UBE-1099 improved the glomerulosclerosis, renal inflammation and fibrosis, and prolonged the lifespan of CKD model mice. Moreover, transcriptome analysis in glomerulus showed that UBE-1099 induced the expression of genes associated with cell cycle and cytoskeleton, which may explain its unique mechanism of improvement such as glomerular morphological change. Thus, our results firstly revealed the efficacy of Keap1-Nrf2 PPI inhibitor for glomerulosclerosis and CKD.

Background Type IV collagen α3,4,5 (α345(IV)) is an obligate trimer that is secreted to form a collagen network, which is the structural foundation of basement membrane. Mutation in one of the genes (COL4A3, A4, A5) encoding these proteins underlies the progressive genetic nephropathy Alport syndrome (AS) due to deficiency in trimerization and/or secretion of the α345(IV) trimer. Thus, improving mutant α345(IV) trimerization and secretion could be a good therapeutic approach for AS. Methods Using the nanoluciferase-based platform that we previously developed to detect α345(IV) formation and secretion in HEK293T cells, we screened libraries of natural product extracts and compounds to find a candidate compound capable of increasing mutant α345(IV) secretion. Results The screening of >13,000 extracts and >600 compounds revealed that cyclosporin A (CsA) increased the secretion of mutant α345(IV)-G1244D. To elucidate the mechanism of the effect of CsA, we evaluated CsA derivatives with different ability to bind to calcineurin (Cn) and cyclophilin (Cyp). Alisporivir (ALV), which binds to Cyp but not to Cn, increased the trimer secretion of mutant α345(IV). Knockdown studies on Cyps showed that PPIF/CypD was involved in the trimer secretion-enhancing activity of CsA and ALV. We confirmed that other α345(IV) mutants are also responsive to CsA and ALV. Conclusions CsA was previously reported to improve proteinuria in AS patients, but due to its nephrotoxic effect, CsA is not recommended for treatment in AS patients. Our data raise the possibility that ALV could be a safer option than CsA. This study provides a novel therapeutic candidate for AS with an innovative mechanism of action, and reveals an aspect of the intracellular regulatory mechanism of α345(IV) that was previously unexplored.