Jianchao Ma

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.

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.

Cellulose Triacetate Dialyzer Reduces Platelet Loss during Continuous Veno-Venous Hemofiltration

Thrombocytopenia is a common complication in patients receiving continuous veno-venous hemofiltration (CVVH) in the intensive care unit. The hemofilter itself plays an important role in the genesis of thrombocytopenia. The present study was undertaken to test whether there were differences in platelet loss and activation during CVVH with a polysulfone (PS) hemofilter or a cellulose triacetate (CTA) dialyzer. 96 patients with thrombocytopenia and acute kidney injury requiring CVVH were randomly assigned to four groups receiving low-molecular-weight heparin (LMWH) PS (n = 24), LMWH CTA (n = 24), no anticoagulation PS (n = 24), and no anticoagulation CTA (n = 24), respectively. We found a significant decrease in platelet counts, but an increased platelet activation with the PS hemofilter in patients who received no anticoagulation. There was no significant decrease in platelet counts and activation in the CTA group. The cellulose membranes could be an effective alternative to the standard synthetic membranes in patients at high risk for thrombocytopenia during CVVH.

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.

Bovine parathyroid hormone enhances osteoclast bone resorption by modulating V-ATPase through PTH1R

The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose-dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling.

Urinary messenger RNA of the receptor activator of NF-kappaB could be used to differentiate between minimal change disease and membranous nephropathy

Abstract Podocyte damage and loss together have an important role in the pathogenesis and progression of glomerulonephritis. Glomerulonephritis patients and healthy controls were enrolled in this study. Biochemical, clinical and experimental procedures included measurement of total urinary protein, renal biopsy and gene expression analysis of the receptor activator of NF-kappaB (RANK). The urinary mRNA levels of RANK were significantly higher in the glomerulonephritis group compared to the controls. The urinary RANK level of glomerular subtypes was correlated significantly with proteinuria. The calculated area of RANK mRNA levels under the curve was 0.61 for minimal change disease (MCD), 0.97 for membranous nephropathy (MN), 0.65 for IgA nephropathy (IgAN), 0.70 for lupus nephritis (LN) and 0.70 for focal segmental glomerulosclerosis (FSGS). The urinary mRNA of RANK might be used to differentiate histologic subtypes of glomerulonephritis, particularly between MCD and MN.

RANK Deficiency Ameliorates Podocyte Injury by Suppressing Calcium/Calcineurin/ NFATc1 Signaling

Background/Aims: Podocyte injury and loss contribute to proteinuria, glomerulosclerosis and eventually kidney failure. Receptor activator of NF-κB (RANK) belongs to the TNF receptor superfamily, which plays a key role in the pathogenesis of podocyte injury. However, the mechanism underlying the effect of RANK in podocyte injury remains unclear. Here, we sought to explore the possible molecular mechanisms involved in podocyte injury caused by RANK. Methods: Immortalized mouse podocytes were treated with siRNA targeting RANK for 48 h or ionomycin for 24 h before harvest. Western blot, quantitative RT-PCR and immunofluorescence staining were used to evaluate the expression and function of RANK, nuclear factor of activated T cells c1 (NFATc1), transient receptor potential cation channel, subfamily C, member 6 (TRPC6) and calcineurin in podocytes. The Calcineurin Cellular Activity Assay kit was used to detect the phosphatase activity of calcineurin in cultured podocytes. A Ca2+ influx assay was performed to analyze alterations in Ca2+ entry under different conditions. Co-immunoprecipitation assays were used to observe the relationship between RANK and TRPC6. Results: RANK mRNA and protein expression were markedly increased in injured podocytes (ionomycin stimulation). Further study found that translocation of NFATc1 to the nucleus was significantly reduced after knocking down RANK by siRNA. Meanwhile, we also demonstrated that loss of RANK suppressed the phosphatase activity of calcineurin and attenuated the ionomycin-induced increase in Ca2+ influx. In addition, we showed that RANK knockdown in cultured podocytes decreased TRPC6 protein expression. Co-immunoprecipitation experiments suggested that RANK binds to TRPC6 and that ionomycin enhanced the binding of RANK to TRPC6. Conclusion: Our findings demonstrated that RANK deficiency ameliorates podocyte injury by suppressing calcium/calcineurin/NFATc1 signaling, which may present a promising target for therapeutic intervention.

Soluble Urokinase Plasminogen Activator Receptor is Associated with Coronary Artery Calcification and Cardiovascular Disease in Patients Undergoing Hemodialysis

Background/Aims: Cardiovascular disease (CVD) is an important cause of morbidity and mortality in hemodialysis patients. Vascular calcification is thought to play an important role in causing CVD. Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker strongly predictive of cardiovascular outcomes in the pathogenesis of diabetic patients with renal disease treated with hemodialysis. We investigated the relationship between suPAR and coronary artery calcification (CAC) in patients undergoing maintenance hemodialysis. Methods: A total of 99 adult hemodialysis patients were enrolled in this study. Plasma samples were analyzed for suPAR with an enzyme-linked immunosorbent assay and the CAC score was determined with multidetector computed tomography. The occurrence of cardiovascular events and all-cause mortality during follow-up were recorded from January 1, 2010 to June 1, 2016. Results: In 99 patients treated with maintenance hemodialysis, 91 (91.9%) had varying degrees of CAC, and suPAR correlated positively with the CAC score in a Spearman analysis. In a mean follow-up period of 33 months, 36 patients (36.4%) experienced at least one cardiovascular event. When the quartiles of suPAR concentrations were used as the cutoff points for a subgroup analysis, the incidence of CVD and all-cause mortality was much higher in the higher quartiles of suPAR. In a univariate Cox regression analysis, high suPAR was a risk factor for CVD and all-cause mortality. Conclusion: suPAR is associated with the CAC score and is a risk factor for new-onset CVD in patients undergoing hemodialysis.

High-volume continuous venovenous hemodiafiltration plus resin hemoperfusion improve severe metformin-associated toxicity

We present the case of a 42‐year‐old female patient who attempted suicide by taking approximately 100 tablets of metformin (500 mg). Laboratory tests revealed severe lactic acidosis with lactate levels of 24 mmol/L and pH of 7.09. The patient was treated with high‐volume continuous venovenous hemodiafiltration (CVVH) and resin‐sorbent hemoperfusion. Metformin concentrations were measured by high‐performance liquid chromatography during CVVH and hemoperfusion treatment. Before extracorporeal treatment, the plasma metformin concentration was 208.5 mg/L. After CVVH treatment for 24 h, the plasma metformin concentration had decreased to 13.9 mg/L. Resin‐based sorbent hemoperfusion plus CVVH treatment had reduced the metformin plasma concentration by 61.8% after 3 h. After 7 days, the patients laboratory tests and clinical syndrome were improved, and she was discharged from hospital. We provide evidence that CVVH plus hemoperfusion is effective in eliminating metformins and metabolic products.