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The highly conserved mitochondrial protein induced in high glucose-1 (IHG-1) functions to maintain mitochondrial quality and is associated with the development of fibrosis in diabetic nephropathy. Towards identifying novel approaches to treating diabetic kidney disease, IHG-1-protein-protein interactions were investigated using epitope-tagged immunoprecipitation analyses followed by mass spectrometry. Here we show that IHG-1 is solely expressed in mitochondria and localised to the inner mitochondrial membrane, the region where mitochondrial reactive oxygen species are generated. Chaperones HSPA5 and TRAP1 and cold shock protein YBX1 were identified as IHG-1 binding partners. All three proteins are important in the cellular response to oxidative stress and play important roles in mitochondrial transcription and DNA repair. Both redox imbalance and IHG-1 stimulate TGF-β signalling. IHG-1, HSPA5 and YBX1 all show increased expression in diabetic nephropathy, chronic kidney disease and in the Unilateral Ureteral Obstruction model of kidney fibrosis. Increased IHG-1 expression in UUO correlated with loss of TRAP1 expression. IHG-1 may target TRAP1 for degradation. When IHG-1 is no longer localised to mitochondria, it retains the ability to interact with the cold shock protein YBX1, facilitating anti-fibrotic actions in the nucleus. Targeting these proteins may offer alternative treatments for fibrotic kidney disease.

Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI — encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker — E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.