Wenjian Wang

1,25-Dihydroxyvitamin D(3) prevents puromycin aminonucleoside-induced apoptosis of glomerular podocytes by activating the phosphatidylinositol 3-kinase/Akt-signaling pathway.

Background: Accumulating evidence suggests that vitamin D and its analogs reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that vitamin D protects podocytes from undergoing apoptosis. Methods: A rat model of podocyte apoptosis was created by a single intravenous injection of 100 mg·kg–1 puromycin aminonucleoside (PAN) and received either solvent or 1,25(OH)2D3 treatment. Proteinuria, podocyte apoptosis, the expression of nephrin protein and mRNA, TGF-β/Smad and phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway were evaluated, respectively. Results: PAN induced massive proteinuria, serum creatinine elevation and podocyte apoptosis in PAN nephropathy rats, which was associated with the loss of nephrin, an adhesion molecule specific for the glomerular slit and the reduced of p-Akt/Akt ratio. Moreover, PAN induced foot process retraction, redistribution of nephrin and the activation of TGF-β/Smad-signaling pathway. Compared with PAN nephropathy rats, 1,25(OH)2D3 significantly prevented loss of nephrin, foot process retraction and podocyte apoptosis by stimulating Akt phosphorylation and suppressing TGF-β/Smad-signaling pathway. Conclusion: 1,25(OH)2D3 reduced the PAN-induced podocyte apoptosis and loss of nephrin in PAN nephropathy rat. The anti-apoptotic effects of 1,25(OH)2D3 on podocytes may be partly attributable to activation of a PI3K/Akt survival pathway.

The calcineurin–NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury

Circulating and podocyte-bound urokinase receptor (uPAR) is a mediator of podocyte injury, proteinuria, and focal segmental glomerulosclerosis (FSGS) allowing pathological activation of the uPAR-β3 integrin signaling axis. Clinically, calcineurin inhibitors (e.g., cyclosporine A, CsA) are known to suppress T cells, yet are also being used to reduce proteinuria in FSGS, suggesting the possibility of signal cross talk between uPAR and calcineurin. Calcineurin is known to facilitate the nuclear translocation of the nuclear factor of activated T cells (NFAT). Accordingly, in vivo conditional NFATc1 activation in podocytes leads to proteinuria in mice, yet the downstream targets of NFAT remain unclear. Here, we show that inducible podocyte-specific expression of constitutively active NFATc1 increased podocyte uPAR expression by binding to the Plaur gene promoter (encoding uPAR) in chromatin immunoprecipitation assays. Pathological uPAR signals in podocytes are independent of T cells and affect cell motility via activation, but not expression, changes of the β3 integrin and can be blocked by CsA, NFAT-siRNA, or the cell-permeable NFAT inhibitor (11R-VIVIT) using rodent models of glomerular disease (LPS; 5/6 nephrectomized rats). Taken together, these findings identify podocyte uPAR as a downstream target of NFAT and provide further insights into the pathogenesis of FSGS.Circulating and podocyte-bound urokinase receptor (uPAR) is a mediator of podocyte injury, proteinuria, and focal segmental glomerulosclerosis (FSGS) allowing pathological activation of the uPAR-β3 integrin signaling axis. Clinically, calcineurin inhibitors (e.g., cyclosporine A, CsA) are known to suppress T cells, yet are also being used to reduce proteinuria in FSGS, suggesting the possibility of signal cross talk between uPAR and calcineurin. Calcineurin is known to facilitate the nuclear translocation of the nuclear factor of activated T cells (NFAT). Accordingly, in vivo conditional NFATc1 activation in podocytes leads to proteinuria in mice, yet the downstream targets of NFAT remain unclear. Here, we show that inducible podocyte-specific expression of constitutively active NFATc1 increased podocyte uPAR expression by binding to the Plaur gene promoter (encoding uPAR) in chromatin immunoprecipitation assays. Pathological uPAR signals in podocytes are independent of T cells and affect cell motility via activation, but not expression, changes of the β3 integrin and can be blocked by CsA, NFAT-siRNA, or the cell-permeable NFAT inhibitor (11R-VIVIT) using rodent models of glomerular disease (LPS; 5/6 nephrectomized rats). Taken together, these findings identify podocyte uPAR as a downstream target of NFAT and provide further insights into the pathogenesis of FSGS.

Necrostatin-1 Attenuates Ischemia Injury Induced Cell Death in Rat Tubular Cell Line NRK-52E through Decreased Drp1 Expression

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In addition, we also investigated the mechanism of Nec-1 attenuates cell death in this renal ischemia model. The NRK-52E cells were incubated with TNF-α + antimycinA (TA) for 24 h with or without Nec-1. Cell death was observed under fluorescent microscope and quantified by flow cytometry. Cell viabilities were detected by MTT assay. The protein expression of dynamin-related protein 1 (Drp1) was detected by Western blotting and immunofluorescence assay. Increased cell death in simulated ischemia injury of NRK-52E cells were markedly attenuated in the Nec-1 pretreated ischemia injury group. Meanwhile, cell viability was significantly improved after using Nec-1. In addition, we also observed that the protein expression of Drp1, a mediator of mitochondrial fission, was significantly increased in simulated ischemia injury group. Increased Drp1 expression in the ischemia injury group can be abolished by Nec-1 or Drp1-knock down, accompanied with decreased cell death and improved cell viabilities. These results suggest that Nec-1 may inhibit cell death induced by simulated ischemia injury in the rat tubular cell line NRK-52E through decreased Drp1 expression.

NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice.

Podocyte injury plays a key role in the development of diabetic nephropathy (DN). We have recently shown that 11R‐VIVIT, an inhibitor of cell‐permeable nuclear factor of activated T‐cells (NFAT), attenuates podocyte apoptosis induced by high glucose in vitro. However, it is not known whether 11R‐VIVIT has a protective effect on DN, especially podocyte injury, under in vivo diabetic conditions. Hence, we examined the renoprotective effects of 11R‐VIVIT in diabetic db/db mice and the possible mechanisms underlying its protective effects on podocyte injury in vivo and in vitro.

NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

BACKGROUND Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. METHODS Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca(2+) was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. RESULTS HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500nM) or 11R-VIVIT (100nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca(2+)]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. CONCLUSION Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway, which may present a promising target for therapeutic intervention.

Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury

Background Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK - RANKL is a receptor - ligand complex for podocytes responding to injury. Methodology/Principal Findings In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA. Conclusions/Significance These findings suggest the increase in RANK–RANKL expression is a response to podocyte injury, and RANK–RANKL may be a novel receptor–ligand complex for the survival response during podocyte injury.

1,25-Dihydroxyvitamin D(3) Inhibits Podocyte uPAR Expression and Reduces Proteinuria

Background Accumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury. Methodology/Principal Findings In this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3. Conclusions/Significance Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria.

Spironolactone inhibits podocyte motility via decreasing integrin β1 and increasing integrin β3 in podocytes under high-glucose conditions

Integrin β1 and β3 expression by podocytes is required to maintain glomerular structural integrity. Previous studies have shown that aldosterone (ALD) is involved in glomerular podocyte injury, and mineralocorticoid receptor (MR) blocker spironolactone effectively reduces proteinuria in patients with diabetic nephropathy. The present study was designed to observe the effects of spironolactone on β1 and β3 integrin expression and podocyte motility under in vitro diabetic conditions. Immortalized mouse podocytes were cultured in media containing normal glucose (NG) levels, high glucose (HG) or HG plus spironolacton. The expression of β1 and β3 integrin in podocytes was detected by reverse transcription quantitative polymerase chain reaction, immunofluorescence and western blot analyses. The effects of spironolacton on podocyte motility was further evaluated using a wound healing assay. HG stimulation markedly decreased mRNA and protein expression of integrin β1, and significantly increased mRNA and protein expression of integrin β3 in cultured podocytes. However, simultaneous treatment with spironolacton (10‑7 mol/l) significantly attenuated HG-mediated increases in integrin β3 and decreases in integrin β1 expression. Furthermore, the migration of podocytes induced by HG was abrogated by concomitant treatment with spironolacton. In conclusion, the present study suggested that HG decreased the expression of integrin β1 in cultured podocytes, accompanied with an increase of integrin β3. Spironolactone inhibited cell motility and stabilized podoctyes treated with HG, probably through partly normalizing the expression of integrin β1 and decreasing the expression of integrin β3.

Time-averaged serum potassium levels and its fluctuation associate with 5-year survival of peritoneal dialysis patients: two-center based study.

The time-averaged serum potassium was more comprehensive to reflect the all-time changes of serum potassium levels during peritoneal dialysis (PD). However, the association of fluctuation of time-averaged serum potassium level with long-time survival of PD patients remains unknown. In this retrospective study, we included 357 incident PD patients in 2 centers from January 1, 2007 to October 31, 2012 with follow-up through October 31, 2014. Our data demonstrated that it was the lower time-averaged serum potassium level rather than baseline of serum potassium level that was associated with high risk of death. Patients with higher standard deviation (SD) had significantly poorer all-cause (p = 0.016) and cardiovascular mortality (p = 0.041). Among the patients with time-averaged serum potassium levels below 4.0 mEq/L, a lower mean value was more important than its SD to predict death risk. In contrast, the patients with time-averaged serum potassium levels above 4.0 mEq/L, those with serum potassium SD < 0.54 mEq/L, exhibited a higher 3-year and 5-year survival rate for both all-cause and cardiovascular mortality compared to the control groups. Our data clearly suggested both time-averaged serum potassium and its fluctuation contributed disproportionately to the high death risk in PD patients.

Probucol combined with valsartan in immunoglobulin A nephropathy: A multi‐centre, open labelled, randomized controlled study

Angiotensin receptor antagonists (ARBs) and anti‐oxidants reduce urinary protein excretion and delay progression of immunoglobulin A (IgA) nephropathy. We investigated the efficacy and safety of probucol (an anti‐oxidant) combined with valsartan (an ARB) on the progression of IgA nephropathy.