EHHADH deficiency regulates pexophagy and accelerates tubulointerstitial injury in diabetic kidney disease.

Peroxisomal L-bifunctional enzyme (EHHADH) plays a role in the classic peroxisomal fatty acid ß-oxidation pathway; however, the relationship between EHHADH expression and diabetic kidney disease has not been well understood. Here, we found that endogenous EHHADH levels were strongly correlated with the progression and severity of diabetic nephropathy in T2D patients. EHHADH knockout mice exhibited worsened renal tubular injury in diabetic mice. Furthermore, EHHADH is a modulator of pexophagy. In renal tubular epithelial cells (RTECs) in vitro, the knockdown of EHHADH induced a dramatic loss of peroxisomes. The loss of peroxisomes in EHHADH-deficient RTECs was restored by either an autophagic inhibitor 3-methyladenine or bafilomycin A1 both in vitro and in vivo. NBR1 was required for pexophagy in EHHADH-knockdown cells, where the level of reactive oxygen species (ROS) was increased, while inhibition of ROS blocked pexophagy. In summary, our findings revealed EHHADH deficiency accelerated renal injury in DKD as a modulator of pexophagy.

Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR.

BACKGROUND: Histone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). METHODS: Renal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. RESULTS: HDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. CONCLUSIONS: Collectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

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Objective: To investigate the role of histone deacetylase 6 (HDAC6) in podocyte injury in diabetic kidney disease (DKD) in mice. Methods: 1) The 8-week-old male CD-1 mice were selected to construct the model of DKD with streptozocin (STZ). After the model was established, the mice were intraperitoneally injected with HDAC6 inhibitor CAY10603 (5mg/kg/daily) or same volume vehicle as control. The mice were divided into four groups, control (CTL)+vehicle (Veh) (n=5), CLT+CAY10603 (n=3), STZ+Veh (n=9), and STZ+CAY10603 (n=7). Mice in STZ+Veh and STZ+CAY10603 groups developed DKD, while mice in the CTL+Veh and CTL+CAY10603 groups were served as normal controls. The therapeutic effect was evaluated through urine albumin-to-creatinine ratio (uACR) and renal pathology after the 2-week treatment with CAY10603. 2) Human podocytes were cultured in vitro and were divided into four groups as follows: CTL, transforming growth factor-ß (TGFß), TGFß+CAY10603 (250 nmol/L), and TGFß+CAY10603 (500 nmol/L) groups. The control group did not receive any treatment, the last three groups were given 36-h TGFß treatment at 5 ng/µL, with or without CAY10603 as indicated for an additional 12 h. Western blot was performed to determine the inhibitory effect of CAY10603 on NLRP3 inflammasome. 3) HDAC6 knockout (KO) mice were generated and used to create STZ-induced model of DKD. The mice were divided into four groups: C57BL/6J wild type (WT) (n=6), HDAC6 KO (n=6), WT+STZ (n=10), and HDAC6 KO+STZ (n=9). Samples were collected 16 weeks after successful modeling and changes in uACR and renal pathology were evaluated accordingly. Results: After 2 weeks of treatment, mice in the STZ+CAY10603 group exhibited reduction in uACR (P<0.05) and inhibition of glomerular mesangium expansion (P<0.05) compared with those of the mice in the STZ+Veh group. There was no statistically significant difference in the indicators between the CTL+Veh group and the CTL+CAY10603 group. In vitro cultured podocytes, compared with the control group, NLRP3 inflammasome activation was seen in the TGFß group. CAY10603 treatment significantly inhibited the activation of NLRP3 in the dosage-dependent manner (P<0.05). Compared with those of the WT group, the WT+STZ group showed increased uACR (P<0.05), obvious glomerulosclerosis and loss of podocytes numbers. Compared with those of the WT+STZ group, the HDAC6 KO+STZ group showed effectively reduction of uACR (P<0.05) and improvement in the renal pathological changes in mice. There was no significant difference in these aspects between the WT and HDAC6 KO groups. Conclusion: Inhibition of HDAC6 alleviates proteinuria and podocyte injury in the mouse model of DKD by suppressing the activation of NLRP3 inflammasome.

Inhibition of HDAC6 With CAY10603 Ameliorates Diabetic Kidney Disease by Suppressing NLRP3 Inflammasome.

Background: Diabetic nephropathy (DN) is one of the leading causes of chronic kidney disease (CKD) worldwide, tubular injury is the driving force during the pathogenesis and progression of DN. Thus, we aim to utilize the connectivity map (CMap) with renal tubulointerstitial transcriptomic profiles of biopsy-proven DN to identify novel drugs for treating DN. Methods: We interrogated the CMap profile with tubulointerstitial transcriptomic data from renal biopsy-proven early- and late-stage DN patients to screen potential drugs for DN. Therapeutic effects of candidate drug were assessed in Murine model of diabetic kidney disease (STZ-induced CD-1 mice), and HK-2 cells and immortalized bone marrow-derived macrophages (iBMDMs). Results: We identified CAY10603, a specific inhibitor of histone deacetylase 6 (HDAC6), as a potential drug that could significantly reverse the altered genes in the tubulointerstitial component. In DN patients and mice, upregulation of HDAC6 was mainly observed in renal tubular cells and infiltrated macrophages surrounding the diluted tubules. In both early- and late-onset diabetic mice, daily CAY10603 administration effectively alleviated renal dysfunction and reduced macrophage infiltration, tubular injury and tubulointerstitial fibrosis. Mechanistically, CAY10603 suppressed NLRP3 activation in both HK-2 cells and iBMDMs. Conclusion: CAY10603 exhibited therapeutic potential for DN by suppressing NLRP3 inflammasome activation in both tubular cells and macrophages.

Body Mass Index and Risk of Diabetic Nephropathy: A Mendelian Randomization Study.

CONTEXT: The associations of obesity and diabetic nephropathy (DN) in type 2 diabetes are inconsistent in observational studies, and causality remains unclear. OBJECTIVE: To explore the causal effect of body mass index (BMI) on DN, estimated glomerular filtration rate (eGFR), and proteinuria in type 2 diabetes by a 2-sample Mendelian randomization (MR) analysis. METHODS: A total of 56 genetic variants were selected as instrumental variables for BMI in 158 284 participants from BioBank Japan, and their effects on DN risk, eGFR, and proteinuria were estimated in 3972 individuals with type 2 diabetes. Then, sex-stratified MR analysis was performed between BMI and DN. We selected generalized summary MR analysis as the primary method and 6 other robust methods to test MR assumptions. RESULTS: One SD increase in BMI was causally associated with higher DN risk [odds ratio (OR) 3.76, 95% CI 1.88-7.53, P < 0.001] and lower eGFR level (OR 0.71, 95% CI 0.59-0.86, P < 0.001). However, BMI was not causally associated with proteinuria (P = 0.22). Sex-stratified analyses indicated the causal effect of BMI on DN was stronger in women (OR 14.81, 95% CI 2.67-82.05, P = 0.002) than in men (OR 3.48, 95% CI 1.18-10.27, P = 0.02). Sensitivity analyses did not show evidence for violation of the MR assumptions. CONCLUSIONS: Genetic evidence showed that higher BMI levels were causally associated with increased risk of DN and decreased eGFR levels. Moreover, the increase in BMI level had a greater impact on DN risk in women.

Nuclear Receptor Interacting Protein-2 Mediates the Stabilization and Activation of ß-Catenin During Podocyte Injury.

Objective: Activation of ß-catenin causes podocyte injury and proteinuria, but how ß-catenin signalling is regulated during podocyte injury remains elusive. Nuclear receptor interacting protein 2 (NRIP2) modulates the Wnt pathway in colorectal cancer-initiating cells, but the role of NRIP2 in podocyte injury has not yet been investigated. We aimed to examine the interaction between NRIP2 and ß-catenin signalling. Materials and Methods: Knockdown or overexpression of NRIP2 and ß-catenin and chemical treatments were performed in cultured human podocytes. Immunoprecipitation, immunoblotting and immunofluorescence assays were used to assess protein interactions and expression. Data from the GEO dataset and kidney tissues from patients with focal segmental glomerulosclerosis (FSGS) and surgical nephrectomy were examined. An adriamycin (ADR) nephropathy model was established in NRIP2 knockout mice. Results: NRIP2 knockdown accelerated ß-catenin degradation, which was reversed by MG132; specifically, NRIP2 bound ß-catenin and stabilized it to prevent its degradation through the ubiquitin proteasomal pathway. Overexpression of NRIP2 led to ß-catenin activation and Snail1 induction, and these effects were attenuated by ß-catenin knockdown. NRIP2 knockdown blocked ADR-stimulated ß-catenin activation. In ADR mice, genetic knockout of Nrip2 ameliorated podocyte injury and loss, glomerulosclerosis, and proteinuria by inhibiting ß-catenin activation. Moreover, NRIP2 was significantly upregulated in podocytes of FSGS patients and colocalized with nuclear ß-catenin. Conclusion: These results established NRIP2 as a stabilizer of ß-catenin activation through the ubiquitin proteasomal pathway in podocyte injury.