Search

Podocyte injury leads to impaired filtration barrier function of the kidney that underlies the pathophysiology of idiopathic nephrotic syndrome (INS), the most common NS occurring in children. The heat shock protein 90 (Hsp90) is involved in the regulation of apoptosis in a variety of cell types, however, little is known about its role in podocytes and whether it associated with NS. Here, we show that Hsp90 is upregulated in glomeruli podocytes from mice with adriamycin (ADR)-induced nephropathy, and that it is also upregulated in an immortalized podocyte cell line treated with ADR in vitro, together suggesting an association of Hsp90 upregulation in podocytes with NS pathogenesis. Functionally, Hsp90 inhibition with PU-H71 aggravates ADR-induced podocyte apoptosis and worsens the impairment of filtration barrier function. Mechanistically, Hsp90 inhibition with PU-H71 enhances the activation of intrinsic apoptotic pathway, and moreover, blockade of podocyte apoptosis with zVAD-fmk (aVAD), a pan-caspase inhibitor, abrogates effects of Hsp90 inhibition on filtration barrier function of ADR-treated podocytes, thus demonstrating that Hsp90 inhibition aggravates ADR-induced podocyte injury through intrinsic apoptosis pathway. In sum, this study reveals a detrimental role of Hsp90 inhibition in podocyte injury, which may offer it as a potential therapeutic target in NS therapy., Competing Interests: Declaration of competing interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020. Published by Elsevier Inc.)

Diabetic nephropathy (DN), the primary cause of end-stage renal disease (ESRD), is often accompanied by dyslipidemia, which is closely related to the occurrence and development of DN and even the progression to ESRD. Mitophagy, the selective degradation of damaged and dysfunctional mitochondria by autophagy, is a crucial mitochondrial quality control mechanism, and largely regulated by PINK1 (PTEN-induced putative kinase 1)/Parkin signaling pathway. In the present study, we demonstrated that PA induced mitochondrial damage and excessive mitoROS generation in podocytes. We also found PA treatment resulted in the activation of mitophagy by increasing co-localization of GFP-LC3 with mitochondria and enhancing the formation of mitophagosome, stabilization of PINK1 and mitochondrial translocation of Parkin, which indicated that PINK1/Parkin pathway was involved in PA-induced mitophagy in podocytes. Furthermore, inhibition of mitophagy by silencing Parkin dramatically aggravated PA-induced mitochondrial dysfunction, mitoROS production, and further enhanced PA-induced apoptosis of podocytes. Finally, we showed that PINK1/Parkin pathway were up-regulated in kidney of high fat diet (HFD)-induced obese rats. Taken together, our results suggest that PINK1/Parkin mediated mitophagy plays a protective role in PA-induced podocytes apoptosis through reducing mitochondrial ROS production and that enhancing mitophagy provides a potential therapeutic strategy for kidney diseases with hyperlipidemia, such as DN., Competing Interests: Declaration of competing interest There is no conflict of interest among all authors. We also confirm that all the listed authors have participated actively in the study and approved the submitted manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Podocytes, a type of highly specialized epithelial cells, require substantial levels of energy to maintain glomerular integrity and function, but little is known on the regulation of podocytes' energetics. Lack of metabolic analysis during podocyte development led us to explore the distribution of mitochondrial oxidative phosphorylation and glycolysis, the two major pathways of cell metabolism, in cultured podocytes during in vitro differentiation. Unexpectedly, we observed a stronger glycolytic profile, accompanied by an increased mitochondrial complexity in differentiated podocytes, indicating that mature podocytes boost both glycolysis and mitochondrial metabolism to meet their augmented energy demands. In addition, we found a shift of predominant energy source from anaerobic glycolysis in immature podocyte to oxidative phosphorylation during the differentiation process. Furthermore, we identified a crucial metabolic regulator for podocyte development, pyruvate kinase M2. Pkm2-knockdown podocytes showed dramatic reduction of energy metabolism, resulting in defects of cell differentiation. Meanwhile, podocyte-specific Pkm2-knockout (KO) mice developed worse albuminuria and podocyte injury after adriamycin treatment. We identified mammalian target of rapamycin (mTOR) as a critical regulator of PKM2 during podocyte development. Pharmacological inhibition of mTOR potently abrogated PKM2 expression and disrupted cell differentiation, indicating the existence of metabolic checkpoint that need to be satisfied in order to allow podocyte differentiation.

Podocyte loss and proteinuria are both key features of human diabetic nephropathy (DN). The leptin-deficient BTBR mouse strain with the ob/ob mutation develops progressive weight gain, type 2 diabetes, and diabetic nephropathy that has many features of advanced human DN, including increased mesangial matrix, mesangiolysis, podocyte loss, and proteinuria. Selective antagonism of the endothelin-1 type A receptor (ET A R) by atrasentan treatment in combination with renin-angiotensin-aldosterone system inhibition with losartan has been shown to have the therapeutic benefit of lowering proteinuria in patients with DN, but the underlying mechanism for this benefit is not well understood. Using a similar therapeutic approach in diabetic BTBR ob/ob mice, this treatment regimen significantly increased glomerular podocyte number compared with diabetic BTBR ob/ob controls and suggested that parietal epithelial cells were a source for podocyte restoration. Atrasentan treatment alone also increased podocyte number but to a lesser degree. Mice treated with atrasentan demonstrated a reduction in proteinuria, matching the functional improvement reported in humans. This is a first demonstration that treatment with the highly selective ET A R antagonist atrasentan can lead to restoration of the diminished podocyte number characteristic of DN in humans and thereby underlies the reduction in proteinuria in patients with diabetes undergoing similar treatment. The benefit of ET A R antagonism in DN extended to a decrease in mesangial matrix as measured by a reduction in accumulations of collagen type IV in both the atrasentan and atrasentan + losartan-treated groups compared with untreated controls.

We evaluated alterations in the structural configurations of channels and activation of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome formation in apolipoprotein L1 (APOL1) risk and nonrisk milieus. APOL1G1- and APOL1G2-expressing podocytes (PD) displayed enhanced K + efflux, induction of pyroptosis, and escalated transcription of interleukin (IL)-1β and IL-18. APOL1G1- and APOL1G2-expressing PD promoted the transcription as well as translation of proteins involved in the formation of inflammasomes. Since glyburide (a specific inhibitor of K + efflux channels) inhibited the transcription of NLRP3, IL-1β, and IL-18, the role of K + efflux in the activation of inflammasomes in APOL1 risk milieu was implicated. To evaluate the role of structural alterations in K + channels in plasma membranes, bioinformatics studies, including molecular dynamic simulation, were carried out. Superimposition of bioinformatics reconstructions of APOL1G0, G1, and G2 showed several aligned regions. The analysis of pore-lining residues revealed that Ser342 and Tyr389 are involved in APOL1G0 pore formation and the altered conformations resulting from the Ser342Gly and Ile384Met mutation in the case of APOLG1 and deletion of the Tyr389 residue in the case of APOL1G2 are expected to alter pore characteristics, including K + ion selectivity. Analysis of multiple membrane (lipid bilayer) models of interaction with the peripheral protein, integral membrane protein, and multimer protein revealed that for an APOL1 multimer model, APOL1G0 is not energetically favorable while the APOL1G1 and APOL1G2 moieties favor the insertion of multiple ion channels into the lipid bilayer. We conclude that altered pore configurations carry the potential to facilitate K + ion transport in APOL1 risk milieu., (© 2019 Federation of European Biochemical Societies.)

Even so, the metal nanoparticles (metal NPs) have attractive optical and biomedical applications, the translation of metal NPs into the clinical practice remains a challenge due to their severe accumulation in the body. Active targeting to renal podocytes opens the door for enhancing kidney targeting and clearance. The goal of this study was to assess the excretion of larger particle size through kidney podocyte via active targeting. To reach this goal, PEGylated quantum dots (QDs) were coated with vapreotide (VAP) for selectively reaching somatostatin receptors (SSTRs) expressed in the podocyte cells. This QDs-VAP was tested on isolated primary podocytes, while the flow cytometry (FACS), confocal microscopy (CLSM), and inductively coupled plasma mass spectrometry (ICP-MS) were used to confirm this hypothesis. The results showed highly specific interactions with podocyte cells as detected by FACS, and CLSM. Moreover, ICP-MS demonstrated higher amount of QDs in the podocyte cells one-hour post-incubation (67.99% ID/g tissue), while the unmodified QDs did not accumulate. This study confirmed that QDs-VAP can target the podocyte's SSTRs then can be cleared via podocyte cells. Moreover, these results are considered as a highly promising approach for future therapy, targeting, clearance, and diagnosis of podocyte-associated diseases.

The clinical effectiveness of adrenocorticotropin in inducing remission of steroid-resistant nephrotic syndrome points to a steroidogenic-independent anti-proteinuric activity of melanocortins. However, which melanocortin receptors (MCR) convey this beneficial effect and if systemic or podocyte-specific mechanisms are involved remain uncertain. In vivo, wild-type (WT) mice developed heavy proteinuria and kidney dysfunction following Adriamycin insult, concomitant with focal segmental glomerulosclerosis (FSGS) and podocytopathy, marked by loss of podocin and synaptopodin, podocytopenia and extensive foot process effacement on electron microscopy. All these pathologic findings were prominently attenuated by NDP-MSH, a potent non-steroidogenic pan-MCR agonist. Surprisingly, MC1R deficiency in MC1R-null mice barely affected the severity of Adriamycin-elicited injury. Moreover, the beneficial effect of NDP-MSH was completely preserved in MC1R-null mice, suggesting that MC1R is likely non-essential for the protective action. A direct podocyte effect seems to contribute to the beneficial effect of NDP-MSH, because Adriamycin-inflicted cytopathic signs in primary podocytes prepared from WT mice were all mitigated by NDP-MSH, including apoptosis, loss of podocyte markers, de novo expression of the podocyte injury marker desmin, actin cytoskeleton derangement and podocyte hypermotility. Consistent with in vivo findings, the podoprotective activity of NDP-MSH was fully preserved in MC1R-null podocytes. Mechanistically, MC1R expression was predominantly distributed to glomerular endothelial cells in glomeruli but negligibly noted in podocytes in vivo and in vitro, suggesting that MC1R signaling is unlikely involved in direct podocyte protection. Ergo, melanocortin therapy protects against podocyte injury and ameliorates proteinuria and glomerulopathy in experimental FSGS, at least in part, via a podocyte-specific non-MC1R-mediated melanocortinergic signaling., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Angiopoietin-like-4 (ANGPTL4) is reported to mediate proteinuria in some types of glomerulonephropathy. However, the mechanism underlying the effect on podocytes of ANGPTL4 under pathologic conditions in diabetic nephropathy (DN) is unclear. We investigated the role of ANGPTL4 in the pathogenesis of DN. In DN rats, elevated ANGPTL4 expression was associated with increased proteinuria, glomerular hypertrophy, and ultrastructural changes in podocytes. In vitro, hyperglycemia induced the upregulation of ANGPTL4, which led to activation of integrin-β1/FAK signaling with increased apoptosis of podocytes and actin cytoskeleton derangement. These pathological changes were reversed by transfection with a lentivirus expressing short hairpin RNA against integrin-β1 or an ANGPTL4-neutralizing antibody in vitro. Furthermore, supplementation with the sialic acid precursor ManNAc reversed these pathological changes and conferred renoprotection in a mouse model of DN. Our findings suggest that ANGPTL4 mediates high glucose-induced loss of podocytes by modulating their detachment and apoptosis in vivo and in vitro. This study deepens our understanding of the mechanisms of podocyte loss in DN and shows targeting ANGPTL4-related signaling has therapeutic potential for DN., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Combined angiotensin receptor--neprilysin inhibition (ARNI) reduces glomerulosclerosis better than single angiotensin receptor blockade (ARB) in diabetic, hypertensive rats. The renoprotective mechanism remains unknown, but may depend on superior blood pressure control, improved renal hemodynamics, suppressed renal inflammation or prevention of podocyte loss., Methods: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic for 12 weeks and treated with vehicle (n = 10), valsartan (ARB; n = 7) or sacubitril/valsartan (ARNI; n = 8) for the final 3 weeks. Arterial pressure was measured via radiotelemetry., Results: Sacubitril/valsartan lowered mean arterial pressure by -50 ± 4 mmHg and valsartan by -43 ± 4 mmHg (P = 0.3). Both treatments lowered albuminuria, but only sacubitril/valsartan maintained high urinary atrial natriuretic peptide, improved glycemic control and protected podocyte integrity, reflected by increased nephrin expression and suppression of transient receptor potential canonical 6 and regulator of calcineurin 1. This resulted in markedly reduced glomerulosclerosis (P < 0.05 vs. control and valsartan). Despite higher effective renal plasma flow and glomerular filtration rates, sacubitril/valsartan did neither improve filtration fraction nor renal immune cell infiltration., Conclusion: Sacubitril/valsartan offers drug-class-specific renoprotection in a preclinical model of diabetes and hypertension. Renoprotection is unrelated to antihypertensive efficacy, renal hemodynamics or inflammation, but may be related to protective effects of natriuretic peptides on podocyte integrity.

It is indisputable that immunosuppressive therapy and pathological diagnosis of renal biopsy have greatly improved the prognosis of childhood nephrotic syndrome. Unfortunately, there is no "one-size-fits-all" approach for precise patient stratification and treatment when facing the huge challenges posed by steroid-resistant nephrotic syndrome (SRNS). But genomic medicine has brought a glimmer of light, and the cognition of SRNS has entered a new stage. Based on this, identification of single genetic variants of SRNS has recognized the key role of podocyte injury in its pathogenesis. Targeted treatment of podocyte injury is paramount, and immunosuppressant with podocyte-targeted therapy seems to be more suitable as the first choice for SRNS, that is, we need to pay attention to their additional non-immunosuppressive effects. In the same way, other effect factors of nephrotic syndrome and the related causes of immunosuppressive therapy resistance require us to select reasonable and targeted non-immunosuppressive therapies, instead of only blindly using steroids and immunosuppressants, which may be ineffective and bring significant side effects. This article provides a summary of the clinical value of identification of genetic variants in podocytes and non-immunosuppressive therapy for nephrotic syndrome in children.

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca 2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca 2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca 2+ influx activated 5' AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca 2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diabetes-induced podocyte apoptosis is considered to play a critical role in the pathogenesis of diabetic kidney disease (DKD). We proposed that hyperglycaemia can induce podocyte apoptosis by inhibiting the action of podocyte survival factors, thus inactivating the cellular effects of insulin signalling. In this study, we aimed to determine the effects of linagliptin on high glucose-induced podocyte apoptosis. Linagliptin reduced the increase in DNA fragmentation as well as the increase in TUNEL-positive cells in podocytes induced by high-glucose condition. Furthermore, linagliptin improved insulin-induced phosphorylation of insulin receptor substrate 1 (IRS1) and Akt, which was inhibited in high-glucose conditions. Adenoviral vector-mediated IRS1 overexpression in podocytes partially normalised DNA fragmentation in high-glucose conditions, while downregulation of IRS1 expression using small interfering RNA increased DNA fragmentation even in low-glucose conditions. Because reactive oxygen species inhibit glomerular insulin signalling in diabetes and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important intrinsic antioxidative systems, we evaluated whether linagliptin increased Nrf2 in podocytes. High-glucose condition and linagliptin addition increased Nrf2 levels compared to low-glucose conditions. In summary, linagliptin offers protection against DKD by enhancing IRS1/Akt insulin signalling in podocytes and partially via the Keap1/Nrf2 pathway. Our findings suggest that linagliptin may induce protective effects in patients with DKD, and increasing IRS1 levels could be a potential therapeutic target in DKD.

Chronic kidney diseases (CKDs) lead to end-stage renal diseases (ESRD) which are characterized by glomerulosclerosis, tubular injury, anemia, inflammation, and interstitial fibrosis. Vitamin D is known to have renal protective effects. However, its effects relate to low and high doses of Vitamin D in CKD model is still unknown. CKD was performed using 5/6 subtotal nephrectomy procedure in male Sprague Dawley rats (3 months old, 200-300 grams, SN group; n=6), then rats were sacrificed on day 14 after operation. Sham operation was used for control (SO group; n=6). Calcitriol was administered in two doses : 0.01 µg/mL/100 gramsBW/day (SND1 group; n=6) and 0.05 µg/mL/100 gramsBW/day (SND2 group; n=6) intraperitoneally for 14 days. Glomerulosclerosis and tubular injury score were examined using PAS staining, meanwhile, interstitial fibrosis area fraction was assessed with Sirius Red staining. RT-PCR was performed for assessing nephrin, podocin, IL-6, CD68, Collagen-1, and TGF-β1 mRNA expressions. Immunostaining (IHC) was carried out to observe macrophage (CD68) and myofibroblast (α-SMA). SN demonstrated CKD condition with higher tubular injury, glomerulosclerosis, interstitial fibrosis, and inflammation compared to SO. Calcitriol-treated group (especially SND2) demonstrated significant lower tubular injury, glomerulosclerosis, and interstitial fibrosis compared to SN. SND2 group showed not only significantly lower CD68, IL-6, Collagen-1, and TGF-β1 mRNA expressions, but also higher mRNA expressions of nephrin and podocin. SND2 group also demonstrated reduction of macrophages infiltration and myofibroblasts expansion based on its histopathological appearance. Vitamin D may have a renoprotective effect on 5/6 subtotal nephrectomy model by attenuating podocytopathy, tubular injury, inflammation and interstitial fibrosis.

Ethnopharmacological Relevance: Curcumin, a phenolic compound extracted from the rhizome of turmeric (Curcuma longa L.), has been reported to have broad biological functions including potent antioxidant and renoprotective effects. It has been reported that Curcumin has a certain protective effect on the kidney. However, its mechanism of action needs further study., Aim of the Study: The present research aims at investigating the therapeutic effects and its underlying mechanism of curcumin on NS., Materials and Methods: The conditionally immortalized mouse podocyte cell line was utilized to evaluate the podocyte-protective effect of curcumin and its effects on NF-κB pathway and Nrf2/ARE pathway in podocyte in vitro. Furthermore, the DOX-induced NS rats were utilized to investigate the therapeutic effects and its underlying mechanism of curcumin against NS in vivo., Results: The consequences of this study revealed that curcumin activated Nrf2, inhibited NF-κB pathway and up-regulated podocin in DOX-induced podocyte. Further research results showed that curcumin can considerably alleviate proteinuria and improve hypoalbuminemia in NS rats, and lower blood lipid levels to alleviate hyperlipidemia in NS rats, indicating that curcumin has significant therapeutic effects on rat NS. Further observation by electron microscopy and detection showed that curcumin can improve renal function and podocyte injury, which may be related to the repairment of mRNA expression and podocin protein. Interestingly, the results of the blood rheology test showed that curcumin can effectively reduce whole blood viscosity (WBV) and plasma viscosity (PV), and reduce hematocrit (Hct). In addition, the oxidative stress state of kidney in NS rats was considerably reversed by curcumin, which may be achieved by activating Nrf2 and increasing the expression of antioxidant enzymes HO-1, NQO-1. We also found that NF-κB pathway is activated in the kidney of NS rats, and curcumin can inhibit the activation of NF-κB by down-regulating the expression of NF-κB p65, reducing the level of p-IκBα and up-regulating the expression of IκBα., Conclusion: These findings suggest that curcumin, as a multifunctional agent, exerts a protective effect on DOX-induced nephrotic syndrome in rats, which provides a pharmacological basis for the further development of curcumin and also provides a basis for the advantages of multi-targeted drugs in the processing of NS., (Copyright © 2019. Published by Elsevier B.V.)

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K + channels, but only APOL3 exhibits Ca 2+ -dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent studies have shown that serum secretory phospholipase A2 group IB (sPLA2-IB) is associated with proteinuric kidney diseases and plays a pivotal role in podocyte injury via its natural receptor. Arachidonic acid (AA), as a major metabolite of sPLA2-IB, regulates the actin bungling remodeling and contributes to the podocyte injury. However, the underlying mechanism of AA in the regulation of podocyte actin remodeling and human podocyte injury is unclear. Here, we reported that AA induced F-actin cytoskeletal ring formation and promoted protein kinase A (PKA), nephrin and c-Abl phosphorylation. Moreover, AA promoted c-Abl translocation from the nucleus to the cytoplasm and increased the recruitment of c-Abl to p-nephrin by the interaction between them. H89 (PKA inhibitor) provided protection against AA-induced F-actin bunching remodeling, down-regulated nephrin phosphorylation, and suppressed the c-Abl translocation and activation. STI571 (c-Abl inhibitor) also improved the AA associated F-actin bunching remodeling. In addition, H89 and STI571 both alleviated apoptosis and adhesion damage of podocyte. These results indicate that an excess of AA treatment is detrimental to the podocyte actin cytoskeleton and promotes podocyte injury due to the activation of PKA-c-Abl signaling., Competing Interests: Declaration of competing interests All authors declare that there are no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiotensin II (Ang II) is a key contributor to glomerular disease by predominantly resulting in podocyte injury, whereas the underlying molecular mechanisms has not been fully understood. This study aimed to investigate if and how ADP-ribosylation factor 6 (Arf6), a small GTP-binding protein, involves Ang II-induced cellular injury in cultured human podocytes. Cellular injury was evaluated with caspase 3 activity, reactive oxygen species (ROS) level and TUNEL assay. Arf6 activity was measured using an Arf6-GTP Pull-Down Assay. Ang II significantly enhanced Arf6 expressions accompanied by increase of Arf6-GTP. The TUNEL-positive cells as well as activated caspase 3, NADPH oxidase 4 protein (Nox4) and ROS levels were dramatically increased in Ang II-treated podocytes, which was prevented by secinH3, an Arf6 activity inhibitor. Induction of ROS by Ang II was inhibited in podocytes with Nox4 knockdown. Ang II-induced elevation of Nox4 and ROS was prevented by Arf6 knockdown. Phpspho-Erk1/2Thr202/Tyr204 levels were upregulated remarkably following Ang II treatment, and Erk inhibitor LY3214996 significantly downregulated Nox4 expression. In addition, Ang II decreased CD2AP expression. Overexpression of CD2AP prevented Ang II-induced upregulation of Arf6-GTP. Our data demonstrated that Ang II promotes ROS production and podocytes injury through activation of Arf6-Erk1/2-Nox4 signaling. We also provided evidence that Ang II activates Arf6 by degradation of CD2AP., Competing Interests: The authors have declared that no competing interests exist.

Podocytes have foot processes that comprise an important cellular layer of the glomerular barrier involved in regulating glomerular permeability. The disturbance of podocyte function plays a central role in the development of proteinuria in diabetic nephropathy. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes, and these channels are involved in disturbing the glomerular filtration barrier in diabetes. Metformin is an anti-diabetic drug widely used for treating patients with type 2 diabetes. Recent studies have suggested that the therapeutic effect of metformin might be mediated by AMPK. The precise function of metformin on cellular function and intracellular signaling in podocytes under diabetic conditions is not fully understood. In this study, we demonstrated that metformin normalized TRPC6 expression via AMPKα1 activation in podocytes exposed to high glucose concentrations. A quantitative analysis showed that metformin increased the colocalization of TRPC6 and AMPKα1 subunits from 42% to 61% in standard glucose (SG) medium and from 29% to 52% in high glucose (HG) medium. AMPK activation was also necessary for maintaining appropriate levels of Rho-family small GTPase activity in HG conditions. Moreover, metformin through AMPK activation remodeled cytoskeleton dynamics, and consequently, reduced filtration barrier permeability in diabetic conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aims/introduction: Amelioration of renal impairment is the key to diabetic nephropathy (DN) therapy. The progression of DN is closely related to podocyte dysfunction, but the detailed mechanism has not yet been clarified. The present study aimed to explore the renal impairment amelioration effect of berberine and related mechanisms targeting podocyte dysfunction under the diabetic state., Materials and Methods: Streptozotocin (35 mg/kg) was used to develop a DN rat model together with a high-glucose/high-lipid diet. Renal functional parameters and glomerular ultrastructure changes were recorded. The alterations of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and phosphorylated Akt in the kidney cortex were determined by western blot. Meanwhile, podocyte dysfunction was induced and treated with berberine and LY294002. After that, podocyte adhesion functional parameters, protein biomarker and the alterations of the PI3K-Akt pathway were detected., Results: Berberine reduces the increased levels of biochemical indicators, and significantly improves the abnormal expression of PI3K, Akt and phosphorylated Akt in a rat kidney model. In vitro, a costimulating factor could obviously reduce the podocyte adhesion activity, including decreased expression of nephrin, podocin and adhesion molecule α3β1 levels, to induce podocyte dysfunction, and the trends were markedly reversed by berberine and LY294002 therapy. Furthermore, reduction of PI3K and phosphorylated Akt levels were observed in the berberine (30 and 60 μmol/L) and LY294002 (40 μmol/L) treatment group, but the Akt protein expression showed little change., Conclusions: Berberine could be a promising antidiabetic nephropathy drug through ameliorating renal impairment and inhibiting podocyte dysfunction in diabetic rats, and the underlying molecular mechanisms might be involved in the regulation of the PI3K-Akt signaling pathway., (© 2019 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Background: Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo ., Methods: To study the effects of AngII on podocyte calcium levels in vivo , we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3., Results: In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2 , the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease., Conclusions: Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker., (Copyright © 2020 by the American Society of Nephrology.)

Aims: To observe the effects of saxagliptin on the expression of mitogen-activated protein kinase 38 (p38MAPK), nephrin and podocin in renal tissue in type 2 diabetic (T2DM) rats, and to explore the possible mechanism of its renal protection., Methods: Forty-eight male Sprague-Dawley rats were used for the study and divided into four different groups: normal controls (Group NC), DM controls (Group DM), DM + glibenclamide (Group Su) and DM + saxagliptin (Group Sa). The day drug administration started was defined as week 0. After 12 weeks, hemoglobin A1c (HbA1c), total cholesterol (TC), triglyceride (TG), urea nitrogen (BUN) and creatinine (Cr) in serum were detected, simultaneously albumin and creatinine in urine were measured, respectively, and then urinary albumin/creatinine ratio (UACR) was calculated. The pathological morphology of kidney tissue in different groups was observed, and the expression of nephrin and podocin mRNA and protein in kidney tissue were detected., Results: (1) After 12 weeks, FBG and HbA1c in Group Su and Group Sa were significantly lower than those in Group DM (both P < 0.05), while there was no significant difference between Group Su and Group Sa. TC, TG and UACR in Group Sa were significantly decreased than those in Group DM. (2) When compared with Group DM, the kidney weight/body weight ratios, the average width of glomerular basement membrane and foot process fusion ratio were all improved in Group Sa after 12 weeks. (3) The expression of p38MAPK mRNA and protein was significantly decreased, while nephrin and podocin mRNA and protein were significantly higher in Group Sa than those in Group DM after 12 weeks. (4) A significant negative correlation was detected between p38MAPK mRNA and nephrin (r = - 0.421, P = 0.009) and podocin mRNA (r = - 0.570, P = 0.000), respectively., Conclusions: Saxagliptin can reduce urinary albumin excretion and exert renal protective effect, especially on podocytes in T2DM rats. The mechanism may be related to its inhibition of renal p38MAPK signaling pathway and the increase in the expression of nephrin and podocin in renal tissue, which is independent of its hypoglycemic effect.

Diabetic nephropathy (DN) is one of the serious complications of diabetes mellitus. Orientin, a major bioactive constituent of Fenugreek, has been reported to possess antihyperglycemic properties. However, its effects on DN remain unclear. Therefore, we explored the protective effect of orientin on podocytes. Here, we assessed cell viability and toxicity, level of autophagy, mitochondrial morphological changes, and podocyte apoptosis. The results indicated that high glucose (HG) induced podocyte apoptosis as well as mitochondrial injury can be partially blocked by orientin. The results showed that orientin could repair autophagy disorder induced by HG, while 3-methyladenine (3-MA) reversed the protection of orientin. Our study demonstrated the possibility of treating DN with orientin., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

Background: Podocyte migration is actively involved in the process of podocyte loss and proteinuria production, which is closely associated with the development of diabetic nephropathy (DN). Exosomes from adipose-derived stem cells (ADSCs-Exos) effectively inhibit podocyte apoptosis in the treatment of DN. However, how ADSCs-Exos affect the migration of podocytes is obscure. This study is aimed at exploring the regulatory role of ADSCs-Exos on cell migration and the underlying mechanism., Methods: ADSCs-Exo was authenticated by transmission electron microscopy (TEM), western blotting, and flow cytometry. Cell viability and migration ability of podocytes were measured by CCK8 and Transwell assays, respectively. Relative expressions of miRNAs and mRNAs were determined by qRT-PCR. The transmitting between PKH26-labeled exosome and podocytes was evaluated by IF assay. Dual luciferase reporter assay was employed to detect the relationship between miR-215-5p and ZEB2 ., Results: The exposure to serum from DN patient (hDN-serum) significantly inhibited cell viability of podocytes, but ADSCs-Exo addition notably blunts cytotoxicity induced by the transient stimulus of hDN-serum. Besides, ADSCs-Exo administration powerfully impeded high glucose- (HG-) induced migration and injury of podocyte. With the podocyte dysfunction, several miRNAs presented a significant decline under the treatment of HG including miR-251-5p, miR-879-5p, miR-3066-5p, and miR-7a-5p, all of which were rescued by the addition of ADSCs-Exo. However, only miR-251-5p was a key determinant in the process of ADSCs-Exo-mediated protective role on podocyte damage. The miR-251-5p inhibitor counteracted the improvement from the ADSCs-Exo preparation on HG-induced proliferation inhibition and migration promotion. Additionally, miR-215-5p mimics alone remarkably reversed HG-induced EMT process of podocyte. Mechanistically, we confirmed that ADSCs-Exos mediated the shuttling of miR-215-5p to podocyte, thereby protecting against HG-induced metastasis, possibly through inhibiting the transcription of ZEB2., Conclusion: ADSCs-Exo has the protective effect on HG-evoked EMT progression of podocytes thru a mechanism involving ZEB2. Potentially, the ADSCs-Exo preparation is a useful therapeutic strategy for improving podocyte dysfunction and DN symptoms clinically., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Juan Jin et al.)

Aims: This study aimed to investigate the characteristics and mechanism of autophagy on podocyte apoptosis under high glucose (HG) conditions and further explore the effect of berberine on podocyte autophagy, apoptosis and the potential mechanism., Materials and Methods: The levels of LC3II/I in podocytes stimulated with HG were detected at 0, 2, 4, 8, 12, 24, 36 and 48 h by western blotting. CCK-8 was used to detect the viability of podocytes. The level of autophagy was detected by western blotting, transmission electron microscopy and immunofluorescence. Podocyte apoptosis was analysed by using Hoechst staining, western blotting, annexin V/propidium iodide dual staining, and confocal microscopy. Then, podocytes were transfected with siRNA to silence mTOR, and the expression levels of proteins and mRNA involved in the mTOR/P70S6K/4EBP1 pathway were further investigated by western blotting and qRT-PCR., Key Findings: In this study, we found significantly reduced LC3II/LC3I and increased p62 in podocytes stimulated with HG for 24 h, and the level of autophagy reached a minimum at 24 h. Berberine restored podocyte viability and significantly attenuated HG-mediated inhibition of autophagy, as evidenced by the increased expression of LC3II/LC3I, the number of autophagosomes and the inhibition of p62. Moreover, berberine counteracted HG-induced podocyte apoptosis and injury, which was negatively correlated with the autophagy effect. Notably, silencing mTOR with siRNA augmented the inhibition of P70S6k and 4EBP1 phosphorylation, which was similar to the effect of berberine., Significance: Berberine activates podocyte autophagy by inhibiting the mTOR/P70S6K/4EBP1 signaling pathway, thereby alleviating podocyte apoptosis., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.

Introduction: Epithelial-mesenchymal transition (EMT) may contribute to podocyte dysfunction in diabetic nephropathy (DN). Aiming to identify novel therapeutic options, we investigated the protective effects of Panax notoginseng (PN) on podocyte EMT in diabetic rats and explored its mechanisms., Methods: Diabetes was induced in rats with streptozotocin (STZ) by intraperitoneal injection at 55 mg/kg. Diabetic rats were randomly divided into three groups, namely, diabetic rats, diabetic rats treated with beraprost sodium (BPS) at 0.6 mg/kg/d or PN at 0.4 g/kg/d p.o., for 12 weeks. Urinary albumin/creatinine ratio (ACR), biochemical parameters, renal histopathology, and podocyte morphological changes were evaluated. Protein expression of EMT markers (desmin, α-SMA, and nephrin) as well as components of the Wnt/β-catenin pathway (wnt1, β-catenin, and snail) was detected by immunohistochemistry and Western blot, respectively., Results: In diabetic rats, severe hyperglycemia and albuminuria were detected. Moreover, mesangial expansion and podocyte foot process effacement were found markedly increased in diabetic kidneys. Increased protein expression of wnt1, β-catenin, snail, desmin, and α-SMA, as well as decreased protein expression of nephrin was detected in diabetic kidneys. All these abnormalities found in DN rats were partially restored by PN treatment., Conclusion: PN ameliorated albuminuria and podocyte EMT in diabetic rats partly through inhibiting Wnt/β-catenin signaling pathway. These findings provide experimental arguments for a novel therapeutic option in DN., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Xie et al.)

Background: PM2.5 is associated closely with an increased risk of membranous nephropathy (MN), however, whether PM2.5 could induce podocytes injury, the underlying pathology for MN, has not be thoroughly studied. Triptolide, an active component in Tripterygium wilfordii Hook F, is frequently used to treat MN in China, but its effects on PM2.5-induced podocytes injury is still largely unknown. Therefore, we evaluated the effects of PM2.5 on podocytes, and explored whether triptolide could improve PM2.5-induced podocytes injury and the possible underlying mechanisms., Results: Podocytes were incubated with PM2.5 after being pre-treated with triptolide, viability, apoptosis rate and migratory capacity of podocytes were determined by CCK-8 assay, flow cytometry and Transwell assay, respectively. Additionally, the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) in podocytes, the cytoskeleton of podocytes, the protein expressions of nephrin, podocin, Bcl-2, Bax, nuclear factor kappa-B/p65 (NF-κB/p65) and phospho-inhibitor of NF-κB (p-IκBα) were measured. Our data showed that PM2.5 treatment significantly increased the disorganization of F-actin stress fibers, the damaged structural integrity of nucleus, the deranged and dissociated cytoskeleton in podocytes, increased the podocytes apoptosis rate, the levels of MDA and LDH, markedly up-regulated the protein expression of Bax, NF-κB/p65 and p-IκBα, down-regulated the protein expression of nephrin, podocin and Bcl-2, and significantly decreased the level of SOD, the migration rate and the viability of podocytes, compared with those of the untreated podocytes. These effects of PM2.5 on podocytes, however, were reversed by triptolide administration., Conclusion: These results suggest that triptolide could prevent against PM2.5-induced podocytes injury via suppressing NF-κB signaling pathway.

Several recent studies have reported that gold nanoparticles (AuNPs) attenuate hyperglycemia in diabetic animal models without any observed side effects. The present study was intended to provide insight into the effects of 50-nm AuNPs on diabetic kidney disease. Adult male rats were divided into three groups (n = 7/group): control (non-diabetic, ND), diabetic (D), and diabetic treated intraperitoneally with 50-nm AuNPs (AuNPs + D; 2.5 mg/kg/day) for 7 weeks. Diabetes was induced by a single-dose injection of 55 mg/kg streptozotocin. The result showed that AuNP treatment prevented diabetes-associated increases in the blood glucose level. Reduction in 24-h urinary albumin excretion rate, glomerular basement membrane thickness, foot process width, and renal oxidative stress markers was also demonstrated in the AuNP-treated group. In addition, the results showed downregulation effect of AuNPs in renal mRNA or protein expression of transforming growth factor β1 (TGF-β 1 ), fibronectin, collagen IV, tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor-A (VEGF-A). Moreover, the protein expression of nephrin and podocin, podocyte markers, in glomeruli was increased in the AuNPs + D group compared with the D group. These results provide evidence that 50-nm AuNPs can ameliorate renal damage in experimental models of diabetic nephropathy through improving the renal function and downregulating extracellular matrix protein accumulation, along with inhibiting renal oxidative stress and amelioration of podocyte injury.

Nephrotic syndrome (NS), a common chronic kidney disease, embraces a variety of kidney disorders. Though Glucocorticoids (GCs) are generally used in the treatment of NS, their mechanism of action is poorly understood. A plethora of evidence indicates that podocytes are considered as the main target cells for the therapeutic strategies to prevent NS. GCs regulate the transactivation and transrepression of genes in podocytes that affect their morphological and cytoskeletal features, motility, apoptosis and survival rate. Moreover, they prevent protein leakage through the glomerular barrier membrane by affecting the synthesis, trafficking and posttranslational modifications of slit diaphragms components, podocytes' intercellular junctions. The response to the treatment is variable among different ethnics and populations and resistance to the steroids is detected in almost 50% of adult patients. Not only do pharmacokinetics and pharmacogenetics of steroids play a role in GC resistance but also the genetic variations in one or more podocyte related genes are connected with the steroid resistance in cases with NS. The focus of this review is to explain the underlying cellular and molecular mechanisms of GCs in podocytes. Understanding the mechanisms by which the GCs and GCs receptors in podocytes regulate the gene expression network and crosstalk with other molecular pathways would guarantee an optimum therapeutic benefit of steroid treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target., Methods: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR., Results: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells., Conclusions: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)

Objective: Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca 2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra , with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6., Methods: Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 μM) and tetrandrine (0-40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca 2+ concentration were measured., Results: Maximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca 2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca 2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6., Conclusion: Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca 2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage., Competing Interests: The authors declare that they have no competing interests., (© 2020 Yu et al.)

Dysregulation of apoptotic and autophagic function are characterized as the main pathogeneses of diabetic nephropathy (DN). It has been reported that Karyopherin Alpha 2 (KPNA2) contributes to apoptosis and autophagy in various cells, but its role in DN development remains unknown. The purpose of present study was to explore the function and underling mechanisms of KPNA2 in development of DN. In this study, 30 mM high glucose (HG)-evoked podocytes were used as DN model. The expression of KPNA2 was detected by qRT-PCR and Western blot assays. The cell viability was tested by CCK-8 kit, the apoptosis was measured using flow cytometry assay, the apoptotic and the autophagy related genes was detected by Western blot. Our results indicated that KPNA2 was significantly increased after HG stimulation. Knockdown of KPNA2 inhibited apoptosis, and promoted cell viability and autophagy in HG-treated podocytes. In addition, silencing of KPNA2 deactivated mTORC1/p70S6K pathway activation via regulating SLC1A5. Further results demonstrated that activating mTORC1/p70S6K pathway strongly ameliorated the effect of KPNA2 on cell viability, apoptosis and autophagy. Therefore, our study suggested that knockdown of KPNA2 rescued HG-induced injury via blocking activation of mTORC1/p70S6K pathway by mediating SLC1A5., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diabetic kidney disease (DKD) is a major cause of end-stage renal disease (ESRD), and therapeutic strategies for delaying its progression are limited. Loss of podocytes by apoptosis characterizes the early stages of DKD. To identify novel therapeutic options, we investigated the effects of Xuesaitong (XST), consisting of total saponins from Panax notoginseng, on podocyte apoptosis in streptozotocin- (STZ-) induced diabetic rats. XST (5 mg/kg·d) or Losartan (10 mg/kg·d) was given to diabetic rats for 12 weeks. Albuminuria, renal function markers, and renal histopathology morphological changes were examined. Podocyte apoptosis was determined by triple immunofluorescence labelling including a TUNEL assay, WT1, and DAPI. Renal expression of Nox4, miRNA-214, PTEN, PDK1, phosphorylated Akt, mTOR, and mTORC1 was detected. In diabetic rats, severe hyperglycaemia and albuminuria developed, and apoptotic podocytes were markedly increased in diabetic kidneys. However, XST attenuated albuminuria, mesangial expansion, podocyte apoptosis, and morphological changes of podocytes in diabetic rats. Decreased expression of PTEN, as well as increased expression of Nox4, miRNA-214, PDK1, phosphorylated Akt, mTOR, and mTORC1, was detected. These abnormalities were partially restored by XST treatment. Thus, XST ameliorated podocyte apoptosis partly through modulating the PTEN-PDK1-Akt-mTOR pathway. These novel findings might point the way to a natural therapeutic strategy for treating DKD., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020 Rui Xue et al.)

Krüppel-like factor 15 (KLF15) is a well-known transcription factor associated with podocyte injury and fibrosis. Recently, hypertensive nephropathy was discovered to be closely related to podocyte injury and fibrosis. However, methods to stimulate hypertension in vitro are lacking. Here, we constructed an in vitro model mimicking hypertension using a rotational force device to identify the role of KLF15 in fibrosis due to mechanically induced hypertensive injury. First, we found that KLF15 expression was decreased in patients with hypertensive nephropathy. Then, an in vitro study of hypertension due to rotational force was conducted, and an increase in fibrosis markers and decrease in KLF15 levels were determined after application of 4 mmHg pressure in primary cultured human podocytes. KLF15 and tight junction protein levels increased with retinoic acid treatment. siRNA-mediated inhibition of KLF15 exacerbated pressure-induced fibrosis injury, and KLF15 expression after treatment with angiotensin II was similar to that observed after treatment with the blood pressure modeling device. Furthermore, the reduced KLF15 levels after mechanical pressure application were restored after the administration of an antihypertensive drug. KLF15 expression was also low in vivo. We confirmed the protective role of KLF15 in fibrosis using a mechanically induced in vitro model of hypertensive injury., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Podocytes are specialized cells with a limited capacity for cell division that do not regenerate in response to injury and loss. Insults that compromise the integrity of podocytes promote proteinuria and progressive renal disease. The aim of this study was to evaluate the potential renoprotective and regenerative effects of mesenchymal stromal cells (mSC) in a severe form of the podocyte injury model induced by intraperitoneal administration of puromycin, aggravated by unilateral nephrectomy. Bone derived mSC were isolated and characterized according to flow cytometry analyses and to their capacity to differentiate into mesenchymal lineages. Wistar rats were divided into three groups: Control, PAN, and PAN+ mSC, consisting of PAN rats treated with 2 × 10 5 mSC. PAN rats developed heavy proteinuria, hypertension, glomerulosclerosis and significant effacement of the foot process. After 60 days, PAN rats treated with mSC presented a significant amelioration of all these abnormalities. In addition, mSC treatment recovered WT1 expression, improved nephrin, podocin, synaptopodin, podocalyxin, and VEGF expression, and downregulated proinflammatory Th1 cytokines in the kidney with a shift towards regulatory Th2 cytokines. In conclusion, mSC administration induced protection of podocytes in this experimental PAN model, providing new perspectives for the treatment of renal diseases associated with podocyte damage.

Background/aims: Recent studies have shown that nicotine induces podocyte damage. However, it remains unknown how nicotine induces podocyte injury. The present study tested whether nicotine induces NLRP3 inflammasomes activation and thereby contributes to podocyte injury., Results: Nicotine treatment significantly increased the colocalization of NLRP3 with Asc, caspase-1 activity, IL-β production, cell permeability in podocytes compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD significantly abolished the nicotine-induced colocalization of NLRP3 with Asc, caspase-1 activity, IL-1β production and cell permeability in podocytes. Immunofluorescence analysis showed that nicotine treatment significantly decreased the podocin and nephrin expression compared to control cells. However, prior treatment with WEHD attenuated the nicotine-induced podocin and nephrin reduction. In addition, we found that nicotine treatment significantly increased the O 2 .- production compared to control cells. However, prior treatment with WEHD did not alter the nicotine-induced O 2 .- production. Furthermore, prior treatment with ROS scavenger, NAC significantly attenuated the nicotine-induced caspase-1 activity, IL-1β production, podocin and nephrin reduction in podocytes., Conclusions: Nicotine-induced the NLRP3 inflammasome activation in podocytes and thereby results in podocyte injury., Methods: Inflammasome formation and immunofluorescence expressions were quantified by confocal microscopy. Caspase-1 activity, IL-1β production and O 2 .- production were measured by ELISA and ESR.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). The ROS-mediated PI3K/AKT pathway plays a key role in podocyte apoptosis and DN progression. Our previous study demonstrated that Baoshenfang (BSF) can decrease proteinuria and attenuate podocyte injury. However, the effects of BSF on podocyte apoptosis induced by the ROS-mediated PI3K/AKT pathway remain unclear. Herein, in vivo and in vitro studies have been performed. In our in vivo study, BSF significantly decreased 24-h urinary protein, serum creatinine, and blood urea nitrogen levels in DN mice. Meanwhile, BSF significantly inhibited oxidative stress and podocyte apoptosis in our in vivo and in vitro studies. Moreover, BSF significantly decreased the inhibition of the PI3K/AKT pathway induced by HG in DN. More importantly, the effects of BSF on podocyte apoptosis were reversed by PI3K siRNA transfection. In conclusion, BSF can decrease proteinuria and podocyte apoptosis in DN, in part through regulating the ROS-mediated PI3K/AKT pathway., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Fang-qiang Cui et al.)

Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms' tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.

Numerous studies have reported that angiotensin (Ang) II, nephrin, and podocin serve pivotal roles in podocyte injury, and thus can lead to the occurrence of proteinuria and the progression of kidney diseases. This study aimed to investigate the effects of Ang II on the production of nephrin and podocin, and their relationship with podocyte injury. We also aimed to determine whether nephrin, podocin and caspase‑9 production depends on the PI3K/Akt/nuclear factor (NF)‑κB signaling pathway in cultured mouse podocytes. We treated mouse podocytes with different doses of Ang II (10‑9, 10‑8, 10‑7 and 10‑6 mol/l) for 12, 24, and 48 h to analyse cell viability, and at 10‑6 mol/l Ang II for 12, 24, and 48 h to evaluate cell apoptosis. Cells were treated with 10‑6 mol/l of Ang II and/or LY294002 (inhibitor of Akt) or 740Y‑P (activator of PI3K) for 48 h to detect Akt, phosphorylated (phospho)‑Akt, p65 NF‑κB, and phospho‑p65 NF‑κB, nephrin, podocin and caspase‑9 expression, and podocyte apoptosis. Treatment with Ang II suppressed the viability and promoted the apoptosis of podocytes in a dose‑ and time‑dependent manner. Ang II decreased phospho‑Akt, phospho‑p65 NF‑κB, nephrin, and podocin and increased caspase‑9 expression, while podocyte apoptosis was promoted. LY294002 further enhanced Ang II‑induced downregulation of Akt and p65 NF‑κB activation, as well as upregulation of caspase‑9 mRNA and protein, and promoted the apoptosis of podocytes. Of note, 740Y‑P restored Ang II‑induced downregulation of Akt and p65 NF‑κB activation, and upregulation of caspase‑9, and decreased podocyte apoptosis. Interestingly, LY294002 and 740Y‑P were determined to have no notable effects on the expression of nephrin and podocin. The data suggested that Ang II could regulate the expression of nephrin, podocin and caspase‑9. Collectively, our findings suggested that the PI3K/Akt/NF‑κB survival axis may serve a pivotal role in podocyte injury.

Mitochondria 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.

Context: Curcumin could ameliorate diabetic nephropathy (DN), but the mechanism remains unclear. Objective: The efficacy of curcumin on epithelial-to-mesenchymal transition (EMT) of podocyte and autophagy in vivo and in vitro was explored. Materials and methods: Thirty male Sprague-Dawley rats were divided into the normal, model and curcumin (300 mg/kg/d, i.g., for 8 weeks) groups. Rats received streptozotocin (50 mg/kg, i.p.) and high-fat-sugar diet to induce DN. Biochemical indicators and histomorphology of renal tissues were observed. In addition, cultured mouse podocytes (MPC5) was induced to EMT with serum from DN rats, and then exposed to curcumin (40 µM) with or without fumonisin B1, an Akt specific activator or 3BDO, the mTOR inducer. Western blot analysed the levels of EMT and autophagy associated proteins. Results: Administration of curcumin obviously reduced the levels of blood glucose, serum creatinine, urea nitrogen and urine albumen (by 28.4, 37.6, 33.5 and 22.4%, respectively), and attenuated renal histomorphological changes in DN rats. Podocytes were partially fused and autophagic vacuoles were increased in curcumin-treated rats. Furthermore, curcumin upregulated the expression of E-cadherin and LC3 proteins and downregulated the vimentin, TWIST1, p62, p-mTOR, p-Akt and P13K levels in DN rats and MPC5 cells. However, fumonisin B1 or 3BDO reversed the effects of curcumin on the expression of these proteins in cells. Discussion and conclusions: The protection against development of DN by curcumin treatment involved changes in inducing autophagy and alleviating podocyte EMT, through the PI3k/Akt/mTOR pathway, providing the scientific basis for further research and clinical applications of curcumin.

Background/Aim: Ginsenoside Rg1 exerts a beneficial effect in many kidney diseases. But little work has been done to confirm whether ginsenoside Rg1 could also exert a protective effect on anti-glomerular basement membrane (anti-GBM) glomerular nephritis (GN). We aimed to explore the role of ginsenoside Rg1 in attenuating anti-GBM GN in vitro and in vivo and investigate the mechanism under its action. Methods: Interleukin-1β (IL-1β) treated podocytes were used as a cell model. A total of 20 mice were used to build the Anti-GBM GN mice model. Real-time PCR analysis (RT-PCR), Western blot analysis and ELISA assay were conducted to detect related indicators in this study. The statistical analysis was performed using GraphPad Prism software 6.0. Results: Ginsenoside Rg1 attenuates IL-1β-induced inflammation and apoptosis in podocytes. NRF2 expression can be inhibited by IL-1β, whereas reversed by ginsenoside Rg1. NRF2 inhibitor ML385 can significantly reverse the effects of ginsenoside Rg1 on inflammation and apoptosis induced by IL-1β, and block the inhibitory effect of ginsenoside Rg1 on IL-1β activated MAPK pathway. In addition, ginsenoside Rg1 could improve anti-GBM GN injury in vivo . Conclusion: Ginsenoside Rg1 inhibits the anti-GBM GN damage through regulating NRF2 pathway in vitro and in vivo . Our findings provide new insight and mechanism of ginsenoside Rg1 to prevent anti-GBM GN.

The aim of this paper was to explore the effects of triptolide( TP),the effective component of Tripterygium wilfordii on improving podocyte epithelial-mesenchymal transition( EMT) induced by high glucose( HG),based on the regulative mechanisms of Nod-like receptor protein 3( NLRP 3) inflammasome in the kidney of diabetic kidney disease( DKD). The immortalized podocytes of mice in vitro were divided into the normal( N) group,the HG( HG) group,the low dose of TP( L-TP) group,the high dose of TP( HTP) group and the mannitol( MNT) group,and treated by the different measures,respectively. More specifically,the podocytes in each group were separately treated by D-glucose( DG,5 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) + TP( 5 μg·L~(-1))or HG( 30 mmol·L~(-1)) + TP( 10 μg·L~(-1)) or DG( 5 mmol·L~(-1)) + MNT( 24. 5 mmol·L~(-1)). After the treatment of HG or TP at 24,48 and 72 h,firstly,the activation of podocyte proliferation was investigated. Secondly,the protein expression levels of the epithelial markers in podocytes such as nephrin and ZO-1,the mesenchymal markers such as collagen Ⅰ and fibronectin( FN) were detected,respectively. Finally,the protein expression levels of NLRP3 and apoptosis-associated speck-like protein( ASC) as the key signaling molecules of NLRP3 inflammasome activation,as well as the downstream effector proteins including caspase-1,interleutin( IL)-1β and IL-18 were examined,severally. The results indicated that,for the cultured podocytes in vitro,HG could cause the low protein expression levels of nephrin and ZO-1,induce the high protein expression levels of collagen Ⅰ and FN and trigger podocyte EMT. Also HG could cause the high protein expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 and induce NLRP3 inflammasome activation. On the other hand,the co-treatment of TP( L-TP or H-TP) and HG for podocytes could recover the protein expression levels of nephrin and ZO-1,inhibit the protein expression levels of collagen Ⅰ and FN and ameliorate podocyte EMT. Also the co-treatment of TP( L-TP or H-TP) and HG could down-regulate the protein expression levels of NLRP3 and ASC,inhibit NLRP3 inflammasome activation and reduce the protein expression levels of the downstream effector molecules including caspase-1,IL-1β and IL-18. On the whole,HG could activate NLRP3 inflammasome and induce podocyte EMT in vitro. TP at the appropriate dose range could inhibit NLRP3 inflammasome activation and ameliorate podocyte EMT,which may be one of the critical molecular mechanisms of TP protecting againstpodocyte inflammatory injury in DKD.

Background: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β -arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis., Methods: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII., Results: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR., Conclusions: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier., (Copyright © 2019 by the American Society of Nephrology.)

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca 2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes.

Hyperglycemia-caused podocyte injury plays a crucial role in the progress of diabetic kidney disease. Podoplanin, one of the podocyte-associated molecules, is closely related to the integrity of the glomerular filtration barrier. A number of studies demonstrate that berberine could ameliorate renal dysfunction in diabetic mice with nephropathy, but the molecular mechanisms have not been fully elucidated. In this study, we explored the relationship between the renoprotective effect of berberine and podoplanin expression in streptozotocin (STZ)-induced diabetic mice as well as mouse podocytes (MPC5 cells) cultured in high glucose (HG, 30 mM) medium. We found that the expression levels of podoplanin were significantly decreased both in the renal glomerulus of STZ-induced diabetic mice and HG-cultured MPC5 cells. We also demonstrated that NF-κB signaling pathway was activated in MPC5 cells under HG condition, which downregulated the expression level of podoplanin, thus leading to increased podocyte apoptosis. Administration of berberine (100, 200 mg/kg every day, ig, for 8 weeks) significantly improved hyperglycemia and the renal function of STZ-induced diabetic mice and restored the expression level of podoplanin in renal glomerulus. In high glucose-cultured MPC5 cells, treatment with berberine (30-120 μM) dose-dependently decreased the apoptosis rate, increased the expression of podoplanin, and inhibited the activation of NF-κB signaling pathway. When podoplanin expression was silenced with shRNA, berberine treatment still inhibited the NF-κB signaling pathway, but its antiapoptotic effect on podocytes almost disappeared. Our results suggest that berberine inhibits the activation of NF-κB signaling pathway, thus increasing the podoplanin expression to exert renoprotective effects.

Proteinuria is one of the most important clinical features of nephrotic syndrome (NS). Injury of podocyte has been proved to contribute to the occurrence of proteinuria. This study explored the effects of geniposide (GEN) on lipopolysaccharide (LPS)-caused murine kidney podocyte MPC5 apoptosis and autophagy. Viability and apoptosis of MPC5 cells were respectively detected with the help of CCK-8 assay and Guava Nexin assay. 3-Methyladenine (3-MA) was used as an autophagy inhibitor, while rapamycin as autophagy activator. Si-Beclin-1 was transfected in MPC5 cells to down-regulate the expression of Beclin-1. We found that LPS stimulation significantly caused MPC5 cell viability reduction, apoptosis and autophagy (P < .05 or P < .01). GEN treatment remarkably alleviated the LPS-caused MPC5 cell viability reduction and apoptosis, but promoted cell autophagy (P < .05). Moreover, 3-MA incubation or si-Beclin-1 transfection notably weakened the effects of GEN on LPS-caused MPC5 cell apoptosis and autophagy (P < .05), while rapamycin had opposite effects (P < .05). Furthermore, GEN activated Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells (P < .05). In conclusion, this research verified the protective effects of GEN on podocytes damage. GEN alleviates LPS-caused apoptosis of murine kidney podocytes by activating Ras/Raf/MEK/ERK-mediated cell autophagy. Highlights: LPS causes podocyte MPC5 apoptosis and autophagy. GEN alleviates LPS-caused MPC5 cell apoptosis, but promotes cell autophagy. 3-MA or si-Beclin-1 weakens the effects of GEN on LPS-treated MPC5 cells. Rapamycin strengthens the effects of GEN on LPS-treated MPC5 cells. GEN activates Ras/Raf/MEK/ERK pathway in LPS-treated MPC5 cells.

Aims: Podocytes play an important role in the development of diabetic kidney disease (DKD). Mitochondria are the source of energy for cell survival, and mitochondrial abnormalities have been shown to contribute to podocyte injury in DKD. In high glucose (HG)-treated podocytes, mitochondrial function and dynamics are abnormal, and intracellular metabolism is often disrupted. However, the molecular mechanism is still unclear. Mitochondrial pyruvate carrier 2 (MPC2) mediates pyruvate transport from the cytoplasm to the mitochondrial matrix, which determines the cellular energy supply and cell survival. Here, we hypothesize that MPC2 damages mitochondria and induces apoptosis in HG-treated podocytes., Main Methods: We used Western blotting, immunofluorescence and immunoprecipitation to detect the expression of MPC2 in HG-treated podocytes. Pyruvate levels were measured to evaluate metabolic station. Mitochondrial membrane potential (MMP) was measured by inverted fluorescence microscopy and flow cytometry. Mitochondrial morphology was assayed by MitoTracker Red staining, and cellular apoptosis was examined by flow cytometry. Furthermore, we treated podocytes with UK5099 and MPC2 siRNA to assess the outcomes of UK5099 treatment and MPC2 knockdown., Key Findings: Intracellular pyruvate accumulated, the mitochondria were damaged and cellular apoptosis increased in podocytes cultured with HG compared to that in control podocytes. MPC2 acetylation was significantly increased in HG-treated podocytes. Furthermore, the mitochondrial morphology changed, the MMP decreased, and cellular apoptosis increased. Inhibition of MPC2 function by UK5099 or MPC2 knockdown by siRNA produced the same abnormal effects observed following treatment with HG., Significance: MPC2 may mediate mitochondrial dysfunction in HG-treated podocytes, ultimately leading to cell apoptosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: ShenYanXiaoBai granules is a traditional Chinese herbal medicine, It is used widely for the treatment of proteinuria caused by various kidney diseases., Aim of the Study: This study investigated the mechanism of Shenyan Xiaobai Granule in the treatment of nephritis proteinuria., Materials and Methods: 100 male wistar rats were divided into a blank group (n = 20) and a nephropathy group (n = 80) using random number table after 1 week adaptive feeded. Rats were injected with adriamycin (6.5 mg/kg) via the tail vein to induce nephropathy except for blank group. Every rat's urine protein was checked with urine protein dipstick test after three days that showed all rats in nephropathy group were successful modelled. Nephropathy group was divided into model group, benazepril group, ShenYanXiaoBai low dose group, ShenYanXiaoBai high dose group equally. Blank and model group were given distilled water 2 ml as control, then benazepril group received benazepril 0.90 mg/kg, ShenYanXiaoBai low dose group received ShenYanXiaoBai granules 1.80 g/kg as high dose group was given 3.60 g/kg, gavage for 6 days a week last for seven weeks. Urinary albumin/urinary creatinine were measured in seventh day every week. Three rats were randomly selected from each group to be executed in 3th and 5th weekend to detect the mRNA and protein expression level in kidney. The rest rats were as well., Conclusions: The therapeutic effect of ShenYanXiaoBai high dose group was better than the two other treated groups from the 5th week to the 7th week, the comparison had a significant difference. The therapeutic effect of benazepril group was better than the ShenYanXiaoBai low dose group in the 7 weeks and the comparison had a significant difference., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a -/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a -/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.