RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling.

BACKGROUND: Receptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. METHODS: We analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. RESULTS: RIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05-4.98]) and incident chronic kidney disease (HR 4.08 [1.10-15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. CONCLUSIONS: The study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

Loss of Cutibacterium is responsible for chronic kidney disease-associated pruritus in patients on dialysis.

Background: Chronic kidney disease (CKD)-associated pruritus is a severe distressing condition that frequently occurs in patients undergoing dialysis. In this study, the profile of the skin microbiome was analyzed to understand the underlying etiology and potential treatments. Methods: Seventy-six end-stage kidney disease (ESKD) patients (hemodialysis, 40; peritoneal dialysis, 36) and 15 healthy controls were enrolled and swabbed at three sites: back, antecubital fossa, and shin. The pruritus severity of the enrolled subjects was validated by the Worst Itch Numeric Rating Scale (WI-NRS), 5-D itch scale, and Uremic Pruritus in Dialysis Patients (UP-Dial). The 16S gene-based metagenomics method was applied to skin microbiome analysis. Results: In the comparison of bacterial communities of ESKD patients and the control group, there was a significant difference on back. Specifically, the average composition ratio of the Cutibacterium in the back samples was significantly lower in ESKD patients than in healthy controls (p < 0.01). In further analysis of ESKD patients, Cutibacterium was significantly lower in the high pruritus group than in the low pruritus group (p < 0.05), even though other clinical parameters such as age, calcium-phosphorus product, and intact parathyroid hormone showed no significance difference between the groups. Conclusion: In ESKD patients, the skin microbiome of the back was significantly altered, and the severity of itching was related to the reduction of Cutibacterium. This research reveals the relationship between skin microbiota and CKD-associated pruritus in multiple skin sites for the first time. The results of this study suggest a potential data basis for the diagnosis and treatment of CKD-associated pruritus.

Protective effects of klotho on palmitate-induced podocyte injury in diabetic nephropathy.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.

In vivo silencing of amphiregulin by a novel effective Self-Assembled-Micelle inhibitory RNA ameliorates renal fibrosis via inhibition of EGFR signals.

Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-ß1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-ß1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.

Paclitaxel Ameliorates Palmitate-Induced Injury in Mouse Podocytes.

BACKGROUND Palmitate, a common saturated free fatty acid, is increased in patients with diabetic nephropathy (DN). Excessive palmitate in kidney is known to cause proteinuria and fibrosis. Several studies have demonstrated that paclitaxel has anti-fibrotic and anti-inflammatory effects on kidney disease. However, whether paclitaxel can relieve podocyte injury is unclear. MATERIAL AND METHODS Immortalized mouse podocytes were used as an in vitro system. Palmitate was used to induce podocyte injury. Podocytes were divided into 4 groups: bovine serum albumin, palmitate, palmitate+1 nM paclitaxel, and palmitate+5 nM paclitaxel. The effects of paclitaxel on palmitate-induced podocyte injury were analyzed by western blot and real-time PCR. Intracellular reactive oxygen species (ROS) generation and podocyte cytoskeletons were analyzed using CM-H2DCF-DA and phalloidin staining. RESULTS Paclitaxel restored downregulated expression of nephrin and synaptopodin and upregulated VEGF expression after injury induced by palmitate. Remarkably, palmitate-induced actin cytoskeleton rearrangement in podocytes was repaired by paclitaxel. Four endoplasmic reticulum stress markers, ATF-6alpha, Bip, CHOP, and spliced xBP1, were significantly increased in palmitate-treated podocytes compared with control podocytes. Such increases were decreased by paclitaxel treatment. Palmitate-induced ROS generation was ameliorated by paclitaxel. Elevated Nox4 expression was also improved by paclitaxel. Paclitaxel alleviated the expression levels of the antioxidant molecules, Nrf-2, HO-1, SOD-1, and SOD-2. The paclitaxel effects were accompanied by inhibition of the inflammatory cytokines, MCP-1, TNF-alpha, TNF-R2, and TLR4, as well as attenuation of the apoptosis markers, Bax, Bcl-2, and Caspase-3. Furthermore, paclitaxel suppressed the palmitate-induced fibrosis molecules, fibronectin and TGF-ß1. CONCLUSIONS This study suggests that paclitaxel could be a therapeutic agent for treating palmitate-induced podocyte injury in DN.

Angiotensin II-mediated MYH9 downregulation causes structural and functional podocyte injury in diabetic kidney disease.

MYH9, a widely expressed gene encoding nonmuscle myosin heavy chain, is also expressed in podocytes and is associated with glomerular pathophysiology. However, the mechanisms underlying MYH9-related glomerular diseases associated with proteinuria are poorly understood. Therefore, we investigated the role and mechanism of MYH9 in diabetic kidney injury. MYH9 expression was decreased in glomeruli from diabetic patients and animals and in podocytes treated with Ang II in vitro. Ang II treatment and siRNA-mediated MYH9 knockdown in podocytes resulted in actin cytoskeleton reorganization, reduced cell adhesion, actin-associated protein downregulation, and increased albumin permeability. Ang II treatment increased NOX4 expression and ROS generation. The Ang II receptor blocker losartan and the ROS scavenger NAC restored MYH9 expression in Ang II-treated podocytes, attenuated disrupted actin cytoskeleton and decreased albumin permeability. Furthermore, MYH9 overexpression in podocytes restored the effects of Ang II on the actin cytoskeleton and actin-associated proteins. Ang II-mediated TRPC6 activation reduced MYH9 expression. These results suggest that Ang II-mediated MYH9 depletion in diabetic nephropathy may increase filtration barrier permeability by inducing structural and functional podocyte injury through TRPC6-mediated Ca2+ influx by NOX4-mediated ROS generation. These findings reveal a novel MYH9 function in maintaining urinary filtration barrier integrity. MYH9 may be a potential target for treating diabetic nephropathy.