Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-β responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-β. In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-β–induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-β stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-β to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-β/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-β signaling and renal fibrogenesis.

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain–containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways.

Epigenetic regulation of oxidative stress is emerging as a critical mediator of diabetic nephropathy. In diabetes, oxidative damage occurs when there is an imbalance between reactive oxygen species generation and enzymatic antioxidant repair. Here, we investigated the function of the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) in attenuating oxidative injury in podocytes, focusing on its regulation of the endogenous antioxidant inhibitor thioredoxin interacting protein (TxnIP). Pharmacologic or genetic depletion of EZH2 augmented TxnIP expression and oxidative stress in podocytes cultured under high-glucose conditions. Conversely, EZH2 upregulation through inhibition of its regulatory microRNA, microRNA-101, downregulated TxnIP and attenuated oxidative stress. In diabetic rats, depletion of EZH2 decreased histone 3 lysine 27 trimethylation (H3K27me3), increased glomerular TxnIP expression, induced podocyte injury, and augmented oxidative stress and proteinuria. Chromatin immunoprecipitation sequencing revealed H3K27me3 enrichment at the promoter of the transcription factor Pax6, which was upregulated on EZH2 depletion and bound to the TxnIP promoter, controlling expression of its gene product. In high glucose–exposed podocytes and the kidneys of diabetic rats, the lower EZH2 expression detected coincided with upregulation of Pax6 and TxnIP. Finally, in a gene expression array, TxnIP was among seven of 30,854 genes upregulated by high glucose, EZH2 depletion, and the combination thereof. Thus, EZH2 represses the transcription factor Pax6, which controls expression of the antioxidant inhibitor TxnIP, and in diabetes, downregulation of EZH2 promotes oxidative stress. These findings expand the extent to which epigenetic processes affect the diabetic kidney to include antioxidant repair.

Focal segmental glomerulosclerosis (FSGS) is an important cause of nondiabetic chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibition (SGLT2i) therapy attenuates the progression of diabetic nephropathy, but it remains unclear whether SGLT2i provides renoprotection in nondiabetic CKD such as FSGS. The primary aim of this pilot study was to determine the effect of 8 wk of dapagliflozin on glomerular filtration rate (GFR) in humans and in experimental FSGS. Secondary end points were related to changes in renal hemodynamic function, proteinuria, and blood pressure (BP). GFR (inulin) and renal plasma flow (para-aminohippurate), proteinuria, and BP were measured in patients with FSGS ( n = 10), and similar parameters were measured in subtotally nephrectomized (SNx) rats. In response to dapagliflozin, changes in GFR, renal plasma flow, and 24-h urine protein excretion were not statistically significant in humans or rats. Systolic BP (SBP) decreased in SNx rats (196 ± 26 vs. 165 ± 33 mmHg; P < 0.001), whereas changes were not statistically significant in humans (SBP 112.7 ± 8.5 to 112.8 ± 11.2 mmHg, diastolic BP 71.8 ± 6.5 to 69.6 ± 8.4 mmHg; P = not significant), although hematocrit increased (0.40 ± 0.05 to 0.42 ± 0.05%; P = 0.03). In archival kidney tissue from a separate patient cohort, renal parenchymal SGLT2 mRNA expression was decreased in individuals with FSGS compared with controls. Short-term treatment with the SGLT2i dapagliflozin did not modify renal hemodynamic function or attenuate proteinuria in humans or in experimental FSGS. This may be related to downregulation of renal SGLT2 expression. Studies examining the impact of SGLT2i on markers of kidney disease in patients with other causes of nondiabetic CKD are needed.

To contend with the deleterious effects of accumulating misfolded protein aggregates or damaged organelles cells rely on a system of quality control processes, among them the autophagy-lysosome pathway. This pathway is itself controlled by a master regulator transcription factor termed transcription factor EB (TFEB). When TFEB localizes to the cell nucleus it promotes the expression of a number of genes involved in protein clearance. Here, we set out to determine (1) whether TFEB expression is altered in chronic kidney disease (CKD); (2) whether inhibition of the cytosolic deacetylase histone deacetylase 6 (HDAC6) affects TFEB acetylation and nuclear localization; and (3) whether HDAC6 inhibition, in turn, alters the natural history of experimental CKD. TFEB mRNA and protein levels were observed to be diminished in the kidneys of humans with diabetic kidney disease, accompanied by accumulation of the protein aggregate adaptor protein p62 in tubule epithelial cells. In cultured NRK-52E cells, HDAC6 inhibition with the small molecule inhibitor Tubastatin A acetylated TFEB, increasing TFEB localization to the nucleus and attenuating cell death. In a rat model of CKD, Tubastatin A prevented the accumulation of misfolded protein aggregates in tubule epithelial cells, attenuated proteinuria progression, limited tubule cell death and diminished tubulointerstitial collagenous matrix deposition. These findings point to the common occurrence of dysregulated quality control processes in CKD and they suggest that TFEB downregulation may contribute to tubule injury in CKD. They also identify a regulatory relationship between HDAC6 and TFEB. HDAC6 inhibitors and TFEB activators both warrant further investigation as treatments for CKD.