Berberine Protects Glomerular Podocytes via Inhibiting Drp1-Mediated Mitochondrial Fission and Dysfunction.
ABSTRACT: Elevated levels of plasma free fatty acid (FFA) and disturbed mitochondrial dynamics play crucial roles in the pathogenesis of diabetic kidney disease (DKD). However, the mechanisms by which FFA leads to mitochondrial damage in glomerular podocytes of DKD and the effects of Berberine (BBR) on podocytes are not fully understood. Methods: Using the db/db diabetic mice model and cultured mouse podocytes, we investigated the molecular mechanism of FFA-induced disturbance of mitochondrial dynamics in podocytes and testified the effects of BBR on regulating mitochondrial dysfunction, podocyte apoptosis and glomerulopathy in the progression of DKD. Results: Intragastric administration of BBR for 8 weeks in db/db mice significantly reversed glucose and lipid metabolism disorders, podocyte damage, basement membrane thickening, mesangial expansion and glomerulosclerosis. BBR strongly inhibited podocyte apoptosis, increased reactive oxygen species (ROS) generation, mitochondrial fragmentation and dysfunction both in vivo and in vitro. Mechanistically, BBR could stabilize mitochondrial morphology in podocytes via abolishing palmitic acid (PA)-induced activation of dynamin-related protein 1 (Drp1). Conclusions: Our study demonstrated for the first time that BBR may have a previously unrecognized role in protecting glomerulus and podocytes via positively regulating Drp1-mediated mitochondrial dynamics. It might serve as a novel therapeutic drug for the treatment of DKD.
Project description:Excessive mitochondrial fission plays a key role in podocyte injury in diabetic kidney disease (DKD), and long noncoding RNAs (lncRNAs) are important in the development and progression of DKD. However, lncRNA regulation of mitochondrial fission in podocytes is poorly understood. Here, we studied lncRNA maternally expressed gene 3 (Meg3) in mitochondrial fission in vivo and in vitro using human podocytes and Meg3 podocyte-specific knockdown mice. Expression of lncRNA Meg3 in STZ-induced diabetic mice was higher, and correlated with the number of podocytes. Excessive mitochondrial fission of podocytes and renal histopathological and physiological parameters were improved in podocyte-specific Meg3 knockdown diabetic mice. Elongated mitochondria with attenuated podocyte damage, as well as mitochondrial translocation of dynamin-related protein 1 (Drp1), were decreased in Meg3 knockout podocytes. By contrast, increased fragmented mitochondria, podocyte injury, and Drp1 expression and phosphorylation were observed in lncRNA Meg3-overexpressing podocytes. Treatment with Mdivi1 significantly blunted more fragmented mitochondria and reduced podocyte injury in lncRNA Meg3-overexpressing podocytes. Finally, fragmented mitochondria and Drp1 mitochondrial translocation induced by high glucose were reduced following treatment with Mdivi1. Our data show that expression of Meg3 in podocytes in both human cells and diabetic mice was higher, which regulates mitochondrial fission and contributes to podocyte injury through increased Drp1 and its translocation to mitochondria.
Project description:Tangshen Formula (TSF) is a Chinese Medicine formula that has been reported to alleviate proteinuria and protect renal function in humans and animals with diabetic kidney disease (DKD). However, little is known about its mechanism in improving proteinuria. The dysregulation of podocyte cell-matrix adhesion has been demonstrated to play an important role in the pathogenesis and progression of proteinuric kidney diseases including DKD. In the present study, the underlying protective mechanism of TSF on podocytes was investigated using the murine model of type 2 DKD db/db mice in vivo and advanced glycation end products (AGEs)-stimulated primary mice podocytes in vitro. Results revealed that TSF treatment could significantly mitigate reduction of podocyte numbers and foot process effacement, reduce proteinuria, and protect renal function in db/db mice. There was a significant increase in expression of transient receptor potential canonical channel 6 (TRPC6) and a decrease in expression of talin1 in podocytes of db/db mice. The results of AGEs-stimulated primary mice podocytes showed increased cell migration and actin-cytoskeleton rearrangement. Moreover, primary mice podocytes stimulated by AGEs displayed an increase in TRPC6-dependent Ca2+ influx, a loss of talin1, and translocation of nuclear factor of activated T cell (NFATC) 2. These dysregulations in mice primary podocytes stimulated by AGEs could be significantly attenuated after TSF treatment. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, blocked the protective role of TSF on podocyte cell-matrix adherence. In conclusion, TSF could protect podocytes from injury and reduce proteinuria in DKD, which may be mediated by the regulation of the TRPC6/Talin1 pathway in podocytes.
Project description:Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.
Project description:Diabetic nephropathy (DN), a microvascular complication of diabetes, is the leading cause of end-stage renal disease worldwide. Multiple studies have shown that podocyte dysfunction is a central event in the progression of the disease. Beside chronic hyperglycemia, dyslipidemia can induce insulin resistance and dysfunction in podocytes. However, the exact mechanisms of free fatty acid (FFA)-induced podocyte insulin unresponsiveness are poorly understood. We used a type 2 diabetic mouse model (db/db) and mouse podocytes exposed to palmitic acid for 24 h followed by an insulin stimulation. Renal function and pathology were evaluated at 25 weeks of age to confirm the DN development. Our results demonstrate that saturated FFA activated the serine/threonine kinases I?B kinase (IKK)?/I?B? and mTORC1/S6K1, but not protein kinase C and c-jun N-terminal kinase, in podocytes and glomeruli of db/db mice. Activation of both kinases promoted serine 307 phosphorylation of IRS1, a residue known to provoke IRS1 inhibition. Using IKK, mTORC1 and ceramide production inhibitors, we were able to blunt IRS1 serine 307 phosphorylation and restore insulin stimulation of Akt. In conclusion, our results indicate that FFA and diabetes contribute to insulin resistance through the activation of IKK? and S6K1 leading to podocyte dysfunction and DN.
Project description:Mitochondrial injury is uniformly observed in several murine models as well as in individuals with diabetic kidney disease (DKD). Although emerging evidence has highlighted the role of key transcriptional regulators in mitochondrial biogenesis, little is known about the regulation of mitochondrial cytochrome c oxidase assembly in the podocyte under diabetic conditions. We recently reported a critical role of the zinc finger Krüppel-like factor 6 (KLF6) in maintaining mitochondrial function and preventing apoptosis in a proteinuric murine model. In this study, we report that podocyte-specific knockdown of Klf6 increased the susceptibility to streptozotocin-induced DKD in the resistant C57BL/6 mouse strain. We observed that the loss of KLF6 in podocytes reduced the expression of synthesis of cytochrome c oxidase 2 with resultant increased mitochondrial injury, leading to activation of the intrinsic apoptotic pathway under diabetic conditions. Conversely, mitochondrial injury and apoptosis were significantly attenuated with overexpression of KLF6 in cultured human podocytes under hyperglycemic conditions. Finally, we observed a significant reduction in glomerular and podocyte-specific expression of KLF6 in human kidney biopsies with progression of DKD. Collectively, these data suggest that podocyte-specific KLF6 is critical to preventing mitochondrial injury and apoptosis under diabetic conditions.
Project description:Inherited and acquired mitochondrial defects have been associated with podocyte dysfunction and chronic kidney disease (CKD). Peroxisome proliferator-activated receptor ? coactivator-1? (PGC1?) is one of the main transcriptional regulators of mitochondrial biogenesis and function. We hypothesized that increasing PGC1? expression in podocytes could protect from CKD. We found that PGC1? and mitochondrial transcript levels are lower in podocytes of patients and mouse models with diabetic kidney disease (DKD). To increase PGC1? expression, podocyte-specific inducible PGC1?-transgenic mice were generated by crossing nephrin-rtTA mice with tetO-Ppargc1a animals. Transgene induction resulted in albuminuria and glomerulosclerosis in a dose-dependent manner. Expression of PGC1? in podocytes increased mitochondrial biogenesis and maximal respiratory capacity. PGC1? also shifted podocytes towards fatty acid usage from their baseline glucose preference. RNA sequencing analysis indicated that PGC1? induced podocyte proliferation. Histological lesions of mice with podocyte-specific PGC1? expression resembled collapsing focal segmental glomerular sclerosis. In conclusion, decreased podocyte PGC1? expression and mitochondrial content is a consistent feature of DKD, but excessive PGC1? alters mitochondrial properties and induces podocyte proliferation and dedifferentiation, indicating that there is likely a narrow therapeutic window for PGC1? levels in podocytes.
Project description:Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identified SMPDL3b as a modulator of insulin signaling and demonstrated that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD. Overall design: RNA sequence data in whole cell extracts of differentiated human podocytes RNA from Ctrl and SMPDL3b OE podocytes was isolated and analyzed for differences in the mRNA expression profile RNA extraction, Preparation and sequencing RNA libraries, Illumina Sequencing RNA Data Analysis
Project description:The bioactive peptides of the apelinergic system and its receptor APJ have been shown to play a protective role in experimental cardiovascular and diabetic kidney disease (DKD). Mechanisms of this renoprotective effect remain to be elucidated. In this study, we examined the localization of APJ within the normal kidney and its kidney expression in the db/db model of DKD. The effect of hyperglycemia and angiotensin II on APJ was examined in cultured podocytes. In the glomerulus, APJ colocalized with podocyte but not endothelial cell markers. In podocytes stimulated with Pyr1 Apelin-13, a change in the phosphorylation status of the signaling proteins, AKT, ERK, and p70S6K, was observed with an increase 15 min after stimulation. Apelin-13 decreased activity of Caspase-3 in podocytes after high glucose treatment reflecting an antiapoptotic effect of APJ stimulation. In podocytes, APJ mRNA was downregulated in high glucose, when compared to normal glucose conditions and exposure to angiotensin II led to a further significant decrease in APJ mRNA. APJ and preproapelin mRNA levels in kidneys from db/db mice were markedly decreased along with decreased tubular APJ protein by western blotting and immunostaining when compared to db/m controls. In conclusion, the apelinergic system is decreased in kidneys from db/db mice. Within the glomerulus, APJ is mainly localized in podocytes and in this cell type its activation by Apelin-13 abolishes the proapoptotic effect of high glucose, suggesting a potential therapeutic role of apelin and emerging agonists with extended half-life for therapy of DKD.
Project description:Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.
Project description:AbstractMitochondria play important roles in the development of diabetic kidney disease (DKD). The SS peptide is a tetrapeptide that is located and accumulated in the inner mitochondrial membrane; it reduces reactive oxygen species (ROS) and prevents mitochondrial dysfunction. Podocytes are key cellular components in DKD progression. However, whether the SS peptide can exert renal protection through podocytes and the mechanism involved are unknown. In the present study, we explored the mechanisms of the SS peptide on podocyte injury in vivo and in vitro. Compared with the control group, the glomerular podocyte number and expression of WT1 were significantly reduced and TUNEL-positive podocytes were significantly increased in renal tissues in the diabetic rat. These effects were further exacerbated by hypochlorite-modified albumin (HOCl-alb) challenge but prevented by SS-31. In vitro, SS-31 blocked apoptosis in podocyte cell line induced by HOCl-alb. SS-31 prevented oxidative stress and mitochondria-dependent apoptosis signalling by HOCl-alb in vivo and in vitro, as evidenced by the release of cytochrome c (cyt c), binding of apoptosis activated factor-1 (Apaf-1) and caspase-9, and activation of caspases. These data suggest that SS-31 may prevent podocyte apoptosis, exerting renal protection in diabetes mellitus, probably through an apoptosis-related signalling pathway involving oxidative stress and culminating in mitochondria.