No abstract

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.

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.

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.

Aims/hypothesis Long non-coding RNAs (lncRNAs) are garnering increasing attention for their putative roles in the pathogenesis of chronic diseases, including diabetic kidney disease (DKD). However, much about in vivo lncRNA functionality in the adult organism remains unclear. To better understand lncRNA regulation and function in DKD, we explored the effects of the modular scaffold lncRNA HOTAIR (HOX antisense intergenic RNA), which approximates chromatin modifying complexes to their target sites on the genome. Methods Experiments were performed in human kidney tissue, in mice with streptozotocin-induced diabetes, the db/db mouse model of type 2 diabetes, podocyte-specific Hotair knockout mice and conditionally immortalised mouse podocytes. Results HOTAIR was observed to be expressed by several kidney cell-types, including glomerular podocytes, in both human and mouse kidneys. However, knockout of Hotair from podocytes had almost no effect on kidney structure, function or ultrastructure. Glomerular HOTAIR expression was found to be increased in human DKD, in the kidneys of mice with streptozotocin-induced diabetes and in the kidneys of db/db mice. Likewise, exposure of cultured mouse podocytes to high glucose caused upregulation of Hotair expression, which occurred in a p65-dependent manner. Although HOTAIR expression was upregulated in DKD and in high glucose-exposed podocytes, its knockout did not alter the development of kidney damage in diabetic mice. Rather, in a bioinformatic analysis of human kidney tissue, HOTAIR expression closely paralleled the expression of its genic neighbour, HOXC11, which is important to developmental patterning but which has an uncertain role in the adult kidney. Conclusions/interpretation Many lncRNAs have been found to bind to the same chromatin modifying complexes. Thus, there is likely to exist sufficient redundancy in the system that the biological effects of dysregulated lncRNAs in kidney disease may often be inconsequential. The example of the archetypal scaffold lncRNA, HOTAIR, illustrates how lncRNA dysregulation may be a bystander in DKD without necessarily contributing to the pathogenesis of the condition. In the absence of in vivo validation, caution should be taken before ascribing major functional roles to single lncRNAs in the pathogenesis of chronic diseases.

The therapeutic targeting of prostanoid subtype receptors may slow the development of chronic kidney disease (CKD) through mechanisms that are distinct from those of upstream COX inhibition. Here, employing multiple experimental models of CKD, we studied the effects of inhibition of the EP4 receptor, one of four receptor subtypes for the prostanoid prostaglandin E2. In streptozotocin-diabetic endothelial nitric oxide synthase knockout mice, EP4 inhibition attenuated the development of albuminuria, whereas the COX inhibitor indomethacin did not. In Type 2 diabetic db/db mice, EP4 inhibition lowered albuminuria to a level comparable with that of the ACE inhibitor captopril. However, unlike captopril, EP4 inhibition had no effect on blood pressure or hyperfiltration although it did attenuate mesangial matrix accumulation. Indicating a glucose-independent mechanism of action, EP4 inhibition also attenuated proteinuria development and glomerular scarring in non-diabetic rats subjected to surgical renal mass ablation. Finally, in vitro, EP4 inhibition prevented transforming growth factor-ß1 induced dedifferentiation of glomerular podocytes. In rodent models of diabetic and non-diabetic CKD, EP4 inhibition attenuated renal injury through mechanisms that were distinct from either broadspectrum COX inhibition or “standard of care” renin angiotensin system blockade. EP4 inhibition may represent a viable repurposing opportunity for the treatment of CKD.

Discovery of common pathways that mediate both pancreatic b-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I 2 (IP) receptor in pancreatic b-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 39,59-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 b-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet b-cell mass in diabetic mice. Determining that this preservation of b-cell function is mediated through cAMP/protein kinase A (PKA)/ nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.

The nonreceptor kinase Janus kinase 2 (JAK2) has garnered attention as a promising therapeutic target for the treatment of CKD. However, being ubiquitously expressed in the adult, JAK2 is also likely to be necessary for normal organ function. Here, we investigated the phenotypic effects of JAK2 deficiency. Mice in which JAK2 had been deleted from podocytes exhibited an elevation in urine albumin excretion that was accompanied by increased podocyte autophagosome fractional volume and p62 aggregation, which are indicative of impaired autophagy completion. In cultured podocytes, knockdown of JAK2 similarly impaired autophagy and led to downregulation in the expression of lysosomal genes and decreased activity of the lysosomal enzyme, cathepsin D. Because transcription factor EB (TFEB) has recently emerged as a master regulator of autophagosome-lysosome function, controlling the expression of several of the genes downregulated by JAK2 knockdown, we questioned whether TFEB is regulated by JAK2. In immortalized mouse podocytes, JAK2 knockdown decreased TFEB promoter activity, expression, and nuclear localization. analysis and chromatin immunoprecipitation assays revealed that the downstream mediator of JAK2 signaling STAT1 binds to the TFEB promoter. Finally, overexpression of TFEB in JAK2-deficient podocytes reversed lysosomal dysfunction and restored albumin permselectivity. Collectively, these observations highlight the homeostatic actions of JAK2 in podocytes and the importance of TFEB to autophagosome-lysosome function in these cells. These results also raise the possibility that therapeutically modulating TFEB activity may improve podocyte health in glomerular disease.