Nephrin Expression Research Articles

Nephrin Signaling Results in Integrin β1 Activation.

Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin β1 in cultured podocytes. Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin β1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

Protective effect of morin on doxorubicin-induced hepatorenal toxicity in rats

Although Doxorubicin (DOX) is a widespread drug used in the treatment of cancer, its clinical use is restricted due to its common side effects. In addition, administrating DOX with an antioxidant has recently become a new strategy in preventing the side effects of DOX. The protective effects of morin, a natural flavonoid, against DOX-induced liver and kidney damage in rats were investigated biochemically, immunohistochemically and histopathologically in this study. The experimental procedure was planned as 10 days, and 5 groups consisting of seven rats were formed. Morin was given orally to rats at a dose of 50 and 100 mg/kg for 10 days and DOX was given a single dose of 40 mg/kg intraperitoneally on day 8. In order to determine the protective effect of morin against oxidative stress caused by DOX, reduced glutathione (GSH) and malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzyme activities were measured in liver and kidney tissues. Liver and kidney tissue damage were determined both histopathologically and by serum alanine transaminase (ALT), aspartate transaminase (AST), urea and creatinine analysis. In order to determine the effect of DOX-induced inflammation and against the effect of morin, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor kappa B (NF-κB) levels were determined in both tissues. Liver and kidney B-cell lymphoma-2 (Bcl-2) levels were determined biochemically. In addition, Bax expression in liver tissue and aquaporin-2 (AQP-2) and nephrin expression in renal tissue were determined immunohistochemically. It was determined that oxidative damage caused by DOX decreased and improvement of liver and kidney function markers were observed in the groups that were treated with morin. In addition, pre-treatment of morin showed a regulatory effect on TNF-α, IL-1β and NF-κB levels. It prevented the increase in DOX-induced Bax expression and decrease in Bcl-2 level, AQP-2 and nephrin expression. Histopathological examination revealed that it prevented tissue damage in liver and kidney tissues.

The protective effect of the TUG1/miR‑197/MAPK1 axis on lipopolysaccharide‑induced podocyte injury.

The podocyte, a type of glomerular epithelial cell, is the key constituent of the filtration barrier layer in the kidney. Previous studies have shown that long non‑coding RNA(lncRNA)‑taurine‑upregulated gene1(TUG1) served a protective role in diabetes‑induced podocyte damage. The aim of the present study was to investigate the potential role of TUG1 in the progress of podocyte injury induced by lipopolysaccharide(LPS), and explore the underlying mechanisms. The results showed that TUG1 expression was suppressed in LPS‑induced podocytes. Enhanced TUG1 expression by exogenous recombinant vector regulated the expression of podocyte associated proteins [Nephrin, Podocin and CCAAT/enhancer‑binding protein(CHOP)]. A marked decrease was observed in the level the albumin influx in cells transfected with TUG1. Further study indicated that microRNA (miR)‑197 is a potential target of TUG1. The enhanced level of miR‑197 induced by LPS was inhibited in cells transfected with TUG1. The decreased Nephrin and Podocin expression, upregulated CHOP expression and the increased albumin influx were slightly enhanced by miR‑197 mimic transfection, while markedly suppressed by miR‑197 inhibitor transfection in LPS‑induced podocytes. Mitogen‑activated protein kinase (MAPK) protein was predicted as a potential target of miR‑197. The downregulated expression of phosphorylated‑MAPK/MAPK induced by LPS was significantly suppressed by TUG1 transfection in podocytes. In addition to this, autophagy was promoted by TUG1 transfection via the elevation of the Beclin1 and light chain (LC)3 II/LC3I levels, and suppressing p62 expression. However, the p38 MAPK inhibitor SB203580 reversed the changes that TUG1 induced in the levels of Beclin1, LC3II/LC3I and p62. Taken together, these results demonstrated that LPS‑induced podocyte injury could be alleviated by the TUG1/miR‑197/MAPK1 axis.

Anemoside B4 Protects Rat Kidney from Adenine-Induced Injury by Attenuating Inflammation and Fibrosis and Enhancing Podocin and Nephrin Expression.

Anemoside B4 (B4) isolated from Radix Pulsatilla has anti-inflammatory activities in the colon and antitumor effects. However, its role in the prevention and treatment of kidney injury has not been reported. Here, we reported the effects of B4 on chronic kidney injury (CKI) and studied its related mechanism based on an adenine-induced kidney injury model in rats. The results showed that serum BUN (blood urea nitrogen), Crea (creatinine), and urinary proteins increased significantly after oral administration of adenine. Meanwhile, the adenine contents in both renal tissue and urine increased markedly compared with those of normal rats. Moreover, IL-1β, IL-6, TNFα, and NFκB expression was upregulated in the kidney. Simultaneously, the expression of NLRP3 (the nucleotide-binding and oligomerization domain–like receptor, leucine-rich repeat and pyrin domain–containing 3) in the inflammasome, which consists of Caspase 1, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and IL-18, was significantly upregulated. B4 could significantly decrease BUN and Crea; reduce urinary proteins in rats; suppress the expression of IL-6, IL-1β, NFκB, NLRP3, Caspase 1, ASC, and IL-18; and increase urinary adenine contents and promote its excretion. In addition, B4 also upregulated the expression of podocin and nephrin, two major podocyte proteins, and reduced the fiber collagen in the renal interstitial, suggesting that B4 could protect the glomerular matrix from adenine injury in addition to its anti-inflammatory effects. The results of this study show new perspective of B4 as a potential drug against adenine-induced renal injury.

Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria.

Metabolic syndrome (MetS) is associated with nutrient surplus and kidney hyperfiltration, accelerating chronic renal failure. The potential involvement of podocyte damage in early MetS remains unclear. Mitochondrial dysfunction is an important determinant of renal damage, but whether it contributes to MetS-related podocyte injury remains unknown. Domestic pigs were studied after 16 wk of diet-induced MetS, MetS treated with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg·kg-1·day-1 sc) for the last month of diet, and lean controls (n = 6 pigs/group). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured using multidetector computed tomography, and podocyte and mitochondrial injury were measured by light and electron microscopy. Urinary levels of podocyte-derived extracellular vesicles (pEVs; nephrin positive/podocalyxin positive) were characterized by flow cytometry. Body weight, blood pressure, RBF, and GFR were elevated in MetS. Glomerular size and glomerular injury score were also elevated in MetS and decreased after ELAM treatment. Evidence of podocyte injury, impaired podocyte mitochondria, and foot process width were all increased in MetS but restored with ELAM. The urinary concentration of pEVs was elevated in MetS pigs and directly correlated with renal dysfunction, glomerular injury, and fibrosis and inversely correlated with glomerular nephrin expression. Additionally, pEV numbers were elevated in the urine of obese compared with lean human patients. Early MetS induces podocyte injury and mitochondrial damage, which can be blunted by mitoprotection. Urinary pEVs reflecting podocyte injury might represent early markers of MetS-related kidney disease and a novel therapeutic target.

Salidroside ameliorates Adriamycin nephropathy in mice by inhibiting β-catenin activity.

Salidroside is a major phenylethanoid glycoside in Rhodiola rosea L., a traditional Chinese medicine, with multiple biological activities. It has been shown that salidroside possesses protective effects for alleviating diabetic renal dysfunction, contrast‐induced‐nephropathy and other kidney diseases. However, the involved molecular mechanism was still not understood well. Herein, we examined the protective effects of salidroside in mice with Adriamycin (ADR)‐induced nephropathy and the underlying molecular mechanism. The results showed that salidroside treatment ameliorates proteinuria; improves expressions of nephrin and podocin; and reduces kidney fibrosis and glomerulosclerosis induced by ADR. Mechanistically, ADR induces a robust accumulation of β‐catenin in the nucleus and stimulates its downstream target gene expression. The application of salidroside largely abolishes the nuclear translocation of β‐catenin and thus inhibits its activity. Furthermore, the activation of β‐catenin almost completely counteracts the protective roles of salidroside in ADR‐injured podocytes. Taken together, our data indicate that salidroside ameliorates proteinuria, renal fibrosis and podocyte injury in ADR nephropathy, which may rely on inhibition of β‐catenin signalling pathway.

Effect of Baoshenfang Formula on Podocyte Injury via Inhibiting the NOX-4/ROS/p38 Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a serious kidney-related complication of type 1 and type 2 diabetes. The Chinese herbal formula Baoshenfang (BSF) shows therapeutic potential in attenuating oxidative stress and apoptosis in podocytes in DN. This study evaluated the effects of BSF on podocyte injury in vivo and in vitro and explored the possible involvement of the nicotinamide adenine dinucleotide phosphate-oxidase-4/reactive oxygen species- (NOX-4/ROS-) activated p38 pathway. In the identified compounds by mass spectrometry, some active constituents of BSF were reported to show antioxidative activity. In addition, we found that BSF significantly decreased 24-hour urinary protein, serum creatinine, and blood urea nitrogen in DN patients. BSF treatment increased the nephrin expression, alleviated oxidative cellular damage, and inhibited Bcl-2 family-associated podocyte apoptosis in high-glucose cultured podocytes and/or in diabetic rats. More importantly, BSF also decreased phospho-p38, while high glucose-mediated apoptosis was blocked by p38 mitogen-activated protein kinase inhibitor in cultured podocytes, indicating that the antiapoptotic effect of BSF is p38 pathway-dependent. High glucose-induced upexpression of NOX-4 was normalized by BSF, and NOX-4 siRNAs inhibited the phosphorylation of p38, suggesting that the activated p38 pathway is at least partially mediated by NOX-4. In conclusion, BSF can decrease proteinuria and protect podocytes from injury in DN, in part through inhibiting the NOX-4/ROS/p38 pathway.

A KDM6A–KLF10 reinforcing feedback mechanism aggravates diabetic podocyte dysfunction

Diabetic nephropathy is the leading cause of end‐stage renal disease. Although dysfunction of podocytes, also termed glomerular visceral epithelial cells, is critically associated with diabetic nephropathy, the mechanism underlying podocyte dysfunction still remains obscure. Here, we identify that KDM6A, a histone lysine demethylase, reinforces diabetic podocyte dysfunction by creating a positive feedback loop through up‐regulation of its downstream target KLF10. Overexpression of KLF10 in podocytes not only represses multiple podocyte‐specific markers including nephrin, but also conversely increases KDM6A expression. We further show that KLF10 inhibits nephrin expression by directly binding to the gene promoter together with the recruitment of methyltransferase Dnmt1. Importantly, inactivation or knockout of either KDM6A or KLF10 in mice significantly suppresses diabetes‐induced proteinuria and kidney injury. Consistent with the notion, we also show that levels of both KDM6A and KLF10 proteins or mRNAs are substantially elevated in kidney tissues or in urinary exosomes of human diabetic nephropathy patients as compared with control subjects. Our findings therefore suggest that targeting the KDM6A–KLF10 feedback loop may be beneficial to attenuate diabetes‐induced kidney injury.

Contribution of Membrane Raft Redox Signaling to Visfatin‐Induced Inflammasome Activation and Podocyte Injury

Recently we have shown that adipokine visfatin‐induced NLRP3 inflammasome activation contributes to podocyte injury. However, the molecular mechanisms of how visfatin‐induces the Nlrp3 inflammasome activation and podocyte damage still unknown. The present study tested whether ceramide and membrane raft (MR) redox signalling pathway plays a central role in visfatin‐induced inflammasomes formation and activation in podocytes. Using confocal microscopy, visfatin was found to increase MRs clustering in the membrane of podocytes in a dose dependent manner. Upon visfatin stimulation an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91(phox) and p47(phox) was observed in the MR clusters, forming a MR redox signaling platform. The formation of this signalling platform was blocked by prior treatment with MR disruptor MCD, NOX inhibitor DPI, ASMase inhibitor amitriptyline. In addition, electron spin resonance (ESR) spectrometry analysis showed that visfatin treatment significantly increased the superoxide (O2·−) production compared to control cells. However, prior treatment with ASMase inhibitor amitriptyline significantly attenuated the visfatin‐induced superoxide production. Furthermore, immunofluorescence analysis demonstrated that visfatin treatment significantly decreased the podocin and nephrin expression (podocyte damage), and prior treatments with DPI, WEHD (inflammasome inhibitor), MCD and amitriptyline attenuated this visfatin‐induced podocin reduction. In conclusion, our results demonstrate that visfatin‐induced the ceramide production via ASMase and thereby stimulates membrane raft clustering in the membrane of podocytes to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits, enhancing O2·− production and leading to Nlrp3 inflammasome formation and activation in podocytes and ultimate glomerular injury (supported by NIH grant, DK104031).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tetramethylpyrazine reduces the consequences of nitric oxide inhibition in pregnant rats.

Pre-eclampsia (PE) is closely associated with perinatal morbidity and mortality and we want to investigate tetramethylpyrazine (TMP)'s effects on PE. Pregnant Sprague-Dawley rats were randomly divided into five groups: normal pregnant (PC), PE, PE+TMP 20 mg/kg, PE+TMP 40 mg/kg, and PE+TMP 60 mg/kg group. The PE rat model was established via L-NAME treatment. Systolic blood pressures (SBP) and urinary protein concentration were detected via the tail-cuff method and CBB kit, respectively. mRNA levels of key genes were analyzed via quantitative PCR and protein levels of key genes were measured by ELISA or western blot. TMP decreased SBP and urinary protein concentration of PE rats. TMP inhibited L-NAME-induced decrease in pups alive ratio, pups weight, and the ratio of pups/placenta weight and reversed L-NAME induced changes in placental histology, whereas it had little effect on placental weight. Urinary nephrin and podocin expressions were enhanced and serum placental growth factor level was decreased in PE rats, whereas TMP inhibited the above phenomena. TMP suppressed L-NAME-induced sFlt-1 upregulation in serums and kidneys of PE rats, whereas it downregulated IL-6 and MCP-1 expression in PE rats' serums, placentas and kidneys. TMP also suppressed the increase in placental sFlt-1 and vascular endothelial growth factor level caused by L-NAME. In addition, TMP inhibited CHOP and GRP78 expressions and decreased the ratio of p-elF2α/elF2α in PE rats. TMP attenuated the consequences of NO inhibition in pregnant rats.

NLRP3 Inflammasome as a Novel Target to Abrogate Nicotine‐Induced Podocyte Injury

Recent in vivo and in vitro studies have established the role of nicotine in chronic kidney diseases. However, the molecular mechanisms of how nicotine induces chronic kidney disease are still unclear. The present study tested whether nicotine induces NLRP3 inflammasome formation and activation and thereby contributes to podocyte injury and dysfunction. Confocal microscopic analysis showed that nicotine treatment increased the colocalization of NLRP3 with Asc in podocytes compared to control cells. Pretreatment with caspase‐1 inhibitor, WEHD abolished the nicotine‐induced colocalization of NLRP3 with Asc, suggesting the formation of Nlrp3 inflammasomes. Correspondingly, nicotine treatment significantly increased the caspase‐1 activity and IL‐1β production compared to control cells. The prior treatment with WEHD significantly attenuated the nicotine‐induced caspase‐1 activity and IL‐1β production. 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, prior treatment with ROS inhibitor, NAC significantly attenuated the nicotine‐induced caspase‐1 activity, IL‐1β production and podocin and nephrin reduction in podocytes. Based on these results, it is concluded that nicotine‐induced the NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury and dysfunction (supported by NIH grant, DK104031).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Active vitamin D3 plays a protective role in podocytes of diabetic rats by regulating autophagy activity

Objective To investigate the protective effect and mechanism of active vitamin D3 on podocyte injury in type 1 diabetic rats. Methods Animals were randomly divided into normal control group (NC group), diabetic nephropathy group (DN group), diabetes nephropathy plus active vitamin D3 group (DN+ VD group). Random tail vein blood glucose was measured and 24 hours of urine was collected every 3 weeks to observe the dynamic changes of blood glucose and 24-hour urine volume and urinary albumin. Rats were sacrificed at the end of 18th week, the kidney weight to body weight ratio, serum creatinine, blood urea nitrogen, serum calcium, and serum phosphorus levels were measured. Pathological in glomeruli were observed by PAS staining. Immunohistochemistry and Western blotting were used to observe the expression of slit diaphragms proteins including Nephrin, Podocin, and vitamin D receptor protein VDR. The mRNA level of autophagy-related protein P62 was detected by realtime quantitative PCR, and expression of autophagy-related protein including LC3B/A, Beclin1, and P62 were detected by Western blotting. Ultrastructure of podocytes and autophagosomes in podocytes were observed by electron microscopy. Results Levels of serum creatinine, blood urea nitrogen, and blood glucose in diabetic rats were higher than those in NC group (P 0.05). Compared with the DN group, the urinary protein and kidney weight to body weight ratio in the DN+ VD group were significantly lower (P<0.05). Mesangial matrix hyperplasia and basement membrane thickening were improved, and podocyte fusion and shedding were partially reversed. The expressions of Nephrin, Podocin, VDR, LC3B/A and Beclin1 were increased, and P62 mRNA and protein were down-regulated (P<0.05). The number of autophagosomes in podocytes increased. Besides, positive correlations were found between Nephrin and Beclin1 (r=0.939 8, P<0.05), as well as Nephrin and VDR (r=0.948 3, P<0.05), and Beclin1 and VDR (r=0.909 3, P<0.05). Conclusion Active vitamin D3 inhibits the injury of diabetic nephropathy podocytes by up-regulating VDR expression and enhancing autophagy activity, thereby reducing proteinuria and delaying the development of diabetic nephropathy. Key words: Active vitamin D3; Diabetic nephropathy; Podocytes; Autophagy

The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat

ObjectiveDiabetic nephropathy (DN) is characterized by glomerular and tubulointerstitial injury, proteinuria and remodeling. Here we examined whether the combination of an inhibitor of neprilysin (sacubitril), a natriuretic peptide-degrading enzyme, and an angiotensin II type 1 receptor blocker (valsartan), suppresses renal injury in a pre-clinical model of early DN more effectively than valsartan monotherapy.MethodsSixty-four male Zucker Obese rats (ZO) at 16 weeks of age were distributed into 4 different groups: Group 1: saline control (ZOC); Group 2: sacubitril/valsartan (sac/val) (68 mg kg−1 day−1; ZOSV); and Group 3: valsartan (val) (31 mg kg−1 day−1; ZOV). Group 4 received hydralazine, an anti-hypertensive drug (30 mg kg−1 day−1, ZOH). Six Zucker Lean (ZL) rats received saline (Group 5) and served as lean controls (ZLC). Drugs were administered daily for 10 weeks by oral gavage.ResultsMean arterial pressure (MAP) increased in ZOC (+ 28%), but not in ZOSV (− 4.2%), ZOV (− 3.9%) or ZOH (− 3.7%), during the 10 week-study period. ZOC were mildly hyperglycemic, hyperinsulinemic and hypercholesterolemic. ZOC exhibited proteinuria, hyperfiltration, elevated renal resistivity index (RRI), glomerular mesangial expansion and podocyte foot process flattening and effacement, reduced nephrin and podocin expression, tubulointerstitial and periarterial fibrosis, increased NOX2, NOX4 and AT1R expression, glomerular and tubular nitroso-oxidative stress, with associated increases in urinary markers of tubular injury. None of the drugs reduced fasting glucose or HbA1c. Hypercholesterolemia was reduced in ZOSV (− 43%) and ZOV (− 34%) (p < 0.05), but not in ZOH (− 13%) (ZOSV > ZOV > ZOH). Proteinuria was ameliorated in ZOSV (− 47%; p < 0.05) and ZOV (− 30%; p > 0.05), but was exacerbated in ZOH (+ 28%; p > 0.05) (ZOSV > ZOV > ZOH). Compared to ZOC, hyperfiltration was improved in ZOSV (p < 0.05 vs ZOC), but not in ZOV or ZOH. None of the drugs improved RRI. Mesangial expansion was reduced by all 3 treatments (ZOV > ZOSV > ZOH). Importantly, sac/val was more effective in improving podocyte and tubular mitochondrial ultrastructure than val or hydralazine (ZOSV > ZOV > ZOH) and this was associated with increases in nephrin and podocin gene expression in ZOSV (p < 0.05), but not ZOV or ZOH. Periarterial and tubulointerstitial fibrosis and nitroso-oxidative stress were reduced in all 3 treatment groups to a similar extent. Of the eight urinary proximal tubule cell injury markers examined, five were elevated in ZOC (p < 0.05). Clusterin and KIM-1 were reduced in ZOSV (p < 0.05), clusterin alone was reduced in ZOV and no markers were reduced in ZOH (ZOSV > ZOV > ZOH).ConclusionsCompared to val monotherapy, sac/val was more effective in reducing proteinuria, renal ultrastructure and tubular injury in a clinically relevant animal model of early DN. More importantly, these renoprotective effects were independent of improvements in blood pressure, glycemia and nitroso-oxidative stress. These novel findings warrant future clinical investigations designed to test whether sac/val may offer renoprotection in the setting of DN.

Bone marrow mononuclear cell transplantation rescues the glomerular filtration barrier and epithelial cellular junctions in a renovascular hypertension model.

What is the central question of this study? Can a single bone marrow mononuclear cell (BMMC) transplant into the subcapsular region of kidney improve cellular communication and adhesion, while restoring renal tissue cytoarchitecture and function during renovascular hypertension? What is the main finding and its importance? The BMMC transplantation restored connexin40 expression and led to recovery of N- and E-cadherin levels within 15 days. It was observed, for the first time, that BMMC transplantation restores expression of nephrin, a component of the glomerular filtration barrier related to podocytes and the glomerular basal membrane. Stem cell therapy has emerged as a potential treatment for renal diseases owing to the regenerative potential of stem cells. However, a better understanding of the morphological and functional changes of damaged renal cells in the presence of transplanted stem cells is needed. The aim of this study was to investigate cell-cell communication and adhesion in renal parenchyma, with analysis of fibrosis, to evaluate renal morphology and function after bone marrow mononuclear cell (BMMC) transplantation in two-kidney-one-clip rats. The BMMC therapy significantly decreased blood pressure and renin expression, improved renal morphology and restored the glomerular filtration barrier, with remodelling of podocytes. In addition, there was a reduction in fibrosis, and connexin40 and nephrin expression were significantly increased after 7 and 15 days of transplantation. Plasma creatinine, urea and total protein levels were restored, and proteinuria was reduced. Furthermore, N- and E-cadherin expression was increased soon after BMMC therapy. Green fluorescent protein-positive BMMCs were found in the renal cortex 24 and 48h after transplantation into the renal subcapsule, and at 7 and 15 days after transplantation, these cells were observed throughout the renal medulla, indicating cellular migration. Therefore, these data suggest that transplanted BMMCs improve cell-cell communication and adhesion between damaged cells, which is accompanied by a recovery of renal morphology and function.

Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

APOL1-miR193a axis participates in the preservation of molecular phenotype of differentiated podocytes (DPDs). We examined the hypothesis that APOL1 (G0) preserves, but APOL1 risk alleles (G1 and G2) disrupt APOL1-miR193a axis in DPDs. DPDG0s displayed down-regulation of miR193a, but upregulation of nephrin expression. DPDG1s/G2s exhibited an increase in miR193a and down-regulation of the expression of adherens complex’s constituents (CD2AP, nephrin, and dendrin). DPDG0s showed decreased Cathepsin L, enhanced dynamin expressions, and the intact actin cytoskeleton. On the contrary, DPDG1s/G2s displayed an increase in Cathepsin L, but down-regulation of dynamin expressions and disorganization of the actin cytoskeleton. APOL1 silencing enhanced miR193a and Cathepsin L, but down-regulated dynamin expressions. DPDG1s/G2s displayed nuclear import of dendrin, indicating an occurrence of destabilization of adherens complexes in APOL1 risk milieu. These findings suggest that DPDG1s and DPDG2s developed disorganized actin cytoskeleton as a consequence of disrupted APOL1-miR193a axis. Interestingly, docking and co-labeling studies suggested an interaction between APOL1 and CD2AP. APOL1G1/G1 and APOL1G1/G2 transgenic mice displayed nuclear import of dendrin indicating destabilization of adherens complexes in podocytes; moreover, these mice showed a four-fold increase in urinary albumin to creatinine ratio and development of focal segmental glomerular lesions.

A20 overexpression exerts protective effects on podocyte injury in lupus nephritis by downregulating UCH-L1.

Lupus nephritis (LN), an autoimmune kidney disease caused by systemic lupus erythematosus (SLE), is the inflammation of the kidney. Although the treatment of LN is still a therapeutic challenge for many practitioners, the present study aims to provide a new insight for the treatment and management. The study aims to explore the effect of A20 on LN in relation to the nuclear factor-kappa B (NF-κB) signaling pathway. MRL/lpr mice were used as the LN mouse model. Next, A20, UCH-L1, and NF-κB expression in LN patients and MRL/lpr mice was determined. A20 was upregulated in podocytes to assess biological functions of A20 in LN. Furthermore, to further investigate the pivotal role of the NF-κB pathway in LN, the NF-κB pathway was blocked in podocytes. Next, UCH-L1 was downregulated in MRL/lpr mice to assess biological functions of UCH-L1 in LN. A20 was downregulated, whereas UCH-L1 was upregulated in LN. Overexpressed A20 declined NF-κB, UCH-L1 expression, and the extent of p65 phosphorylation. A20 overexpression or UCH-L1 inhibition increased expression of synaptoporin and nephrin but decreased desmin expression and ubiquitin accumulation level in podocytes. Moreover, A20 overexpression or UCH-L1 inhibition increased the podocyte number but decreased protein level of cleaved caspase-3, podocyte lesion improvement, decreased foot process width, glomerulus basement membrane, and foot process fusion rate. In addition, urine protein, blood urea nitrogen, serum creatinine, and ds-DNA antibody levels decreased with elevated A20 or depleted UCH-L1. Collectively, it could be concluded that A20 protects against podocyte injury in LN via UCH-L1 by inactivating the NF-κB signaling pathway.

Resveratrol Reduces Oxidative Stress and Apoptosis in Podocytes via Sir2-Related Enzymes, Sirtuins1 (SIRT1)/Peroxisome Proliferator-Activated Receptor γ Co-Activator 1α (PGC-1α) Axis.

BackgroundPGC-1α can be activated by deacetylation reactions catalyzed by SIRT1. Resveratrol is currently known as a potent activator of SIRT1. However, it is unknown whether the renal-protective effect of resveratrol is further related to activation of the podocyte SIRT1/PGC-1α pathway.Material/MethodsHigh glucose was used to stimulate mouse podocytes. Resveratrol and PGC-1α siRNA transfection were used to perform co-intervention treatments. The protein and mRNA expression levels of SIRT1, PGC-1α, NRF1, and TFAM were detect by immunofluorescence, Western blot analysis, and qRT-PCR in the podocytes, respectively. DCHF-DA and MitoSOX™ staining were used to monitor the total ROS and mitochondrial ROS levels, respectively. The specific activities of complexes I and III were measured using Complex I and III Assay Kits. Mitochondrial membrane potential and cell apoptosis were measured using JC-1 staining and Annexin V-FITC/PI double-staining, respectively.ResultsWe found that high-glucose stimulation results in time-dependent decreases in the expression of SIRT1, PGC-1α, and its downstream genes NRF1 and mitochondrial transcription factor A (TFAM) for mouse podocytes, and increases ROS levels in cells and mitochondria. Moreover, the expression of nephrin was downregulated and the cell apoptotic rate was increased. Resveratrol treatment can improve abnormalities caused by high-glucose stimulation. In addition, it can also reduce the release of mitochondrial cytochrome C and DIABLO proteins to the cytoplasm and increase respiratory chain complex I and III activity and mitochondrial membrane potential.ConclusionsResveratrol can reduce the oxidative damage and apoptosis of podocytes induced by high-glucose stimulation via SIRT1/PGC-1α-mediated mitochondrial protection.

The associations of endothelial and podocyte injury in proliferative lupus nephritis: from observational analysis to in vitro study.

Our study aims to evaluate the endothelial cell-podocyte crosstalk in proliferative lupus nephritis (LN). The semi-quantification scores of glomerular endothelial cell injury and the foot process width (FPW) were processed in 110 proliferative LN patients. Podocytes were stimulated with LN-derived IgG. Glomerular endothelial cells were treated with podocyte-conditioned medium (PCM), and then podocytes were incubated with endothelial cell-conditioned medium (ECM). The levels of vascular endothelial growth factor-A (VEGF-A) in PCM and endothelin-1 in ECM were analyzed, and the injury of podocyte and glomerular endothelial cells were further evaluated. The pathological score of glomerular endothelial cell injury was correlated with FPW in LN complicated with thrombotic microangiopathy. In vitro study showed the following: 1. Stimulation of podocytes by IgG from LN led to decline in the expression of nephrin with cytoskeleton rearrangement, and reduction of VEGF-A levels. 2. Exposure of glomerular endothelial cells to PCM incubated with LN-derived IgG (PCM-LN) induced more endothelin-1 secretion and disruption of intercellular tight junction. 3. Exposure of podocytes to ECM stimulated with PCM-LN could induce cytoskeleton redistribution with decrease of nephrin. In conclusion, the pathological glomerular endothelial cell lesions were associated with FPW and the VEGF-endothelin-1 system might play a critical role in the endothelial cell-podocyte crosstalk in LN.

Amelioration of Early Markers of Diabetic Nephropathy by Linagliptin in Fructose-Streptozotocin-Induced Type 2 Diabetic Rats

Background: Early prediction and clinical intervention are extremely important in order to delay or hinder diabetic nephropathy (DN) progression. Objectives: This study aimed to detect early signs of DN and study the potential ameliorating effect of the dipeptidyl peptidase-4 inhibitor, linagliptin, on some early markers for DN in fructose-streptozotocin (Fr-STZ)-induced diabetic rats. Method: Fr-STZ rats were treated with either linagliptin (3 mg/kg p.o. daily), metformin (350 mg/kg p.o. daily), or their combination for 6 weeks. Results: Fr-STZ DN rats exhibited obvious tubular renal damage and glomerular podocyte injury as confirmed by renal kidney ­injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and vanin-1 mRNA, as well as urinary N-acetyl-β-D-glucosaminidase elevation and nephrin mRNA suppression, associated with the appearance of marked renal interstitial fibrosis and glomerulosclerosis despite the absence of microalbuminuria. Initiation of oxidative, inflammatory, fibrotic, and apoptotic reactions was evident in the settings of renal hyperglycemia. Linagliptin significantly modulated the aforementioned renal tubular injury makers and restored glomerular nephrin expression as well as reversed renal histopathological alterations. Oxidative, inflammatory, fibrotic and apoptotic processes were also alleviated. Conclusions: This study suggests that linagliptin exerts renoprotection against early features of DN in rats probably by inhibition of high glucose-induced renal tubular and glomerular injury thereby hampering KIM-1 and NGAL as well as vanin-1 associated with renal inflammation, fibrosis and oxidative stress.

Vitamin D protects podocytes from autoantibodies induced injury in lupus nephritis by reducing aberrant autophagy

SubjectThe aim of this study was to investigate whether vitamin D plays a protective role in podocyte injury induced by autoantibodies purified from the serum of patients with lupus nephritis (LN) via reducing aberrant autophagy.MethodsAutophagic activities of renal tissues of patients with LN were evaluated under transmission electronic microscope (TEM). Immunoglobulin G (IgG) from patients with LN was purified to induce human podocyte injury, and the role of vitamin D in injury was observed. Podocytes were observed under TEM, autophagic activity was evaluated by western blot analysis and quantitative real-time polymerase chain reaction, and mRFP-GFP-LC3B adenovirus was infected into human podocytes in vitro.ResultsSignificantly higher autophagic levels were observed in patients with LN (P <0.05), and apparently greater autophagic levels in podocytes were shown (P <0.05). Among different classifications of LN, class V (n = 5), III + V (n = 5), and IV + V (n = 5) gained higher autophagic levels than class III (n = 5) and IV (n = 5). Induced autophagy, which was evident by increased LC3B-II and Beclin 1 level, caused consumption of p62, more autophagosomes observed under TEM, and more LC3B dots observed under confocal microscope in the IgG group, along with decreased nephrin expression, which suggests podocyte injury. Reduction of autophagy as well as alleviated podocyte injury was observed in the IgG+ vitamin D group.ConclusionThis study demonstrates that vitamin D plays a protective role in podocyte injury induced by autoantibodies from patients with LN and appears to be a novel therapy target in LN.