Lizhong Du

Expression profile of nephrin, podocin, and CD2AP in Chinese children with MCNS and IgA nephropathy

There have been many exciting advances in our understanding of genetic causes of nephrotic syndrome since 1998 when nephrin was first found. The mRNA expressions of nephrin and CD2AP were studied by quantitative real-time polymerase chain reaction (PCR) in aspirated renal biopsy tissues from 9 subjects with minimal change nephrotic syndrome (MCNS), 6 with primary IgA nephropathy (IgAN), and 15 controls. Protein expression of nephrin, podocin, and CD2AP were analyzed by immunohistochemistry, indirect immunofluorescence, and laser confocal microscope. Compared with controls, the CD2AP mRNA level was significantly downregulated in renal samples from MCNS and IgAN patients (p=0.001 in MCNS, p=0.046 in IgAN), though no significant downregulation was found in the mRNA level of nephrin (p=0.346 in MCNS, p=0.311 in IgAN). The expression levels of protein CD2AP and nephrin were significantly reduced in MCNS and IgAN (MCNS: nephrin, p=0.034, CD2AP, p=0.005; IgAN: nephrin, p=0.021, CD2AP, p=0.025). The podocin staining did not differ significantly between controls and disease groups (p value 0.340 and 0.787, respectively). The results suggest that transcript and translation expression changes of nephrin and CD2AP may have pathogenetic roles in some patients with MCNS and IgAN in Chinese, though no correlation was found in podocin with proteinuria in this study.

Treatment of tacrolimus or cyclosporine A in children with idiopathic nephrotic syndrome

BackgroundCyclosporine A (CsA) and tacrolimus (TAC) are often alternative treatment choices for patients with nephrotic syndrome.MethodsIn this prospective study, the efficacy and safety of CsA and TAC in inducing and maintaining remission in 74 children with idiopathic nephrotic syndrome (INS) were evaluated.ResultsIn terms of short-term efficacy, TAC was more effective than CsA in children with steroid-resistant nephrotic syndrome (χ2 = 13.75, P = 0.001), although no significant difference in number of episodes of relapse were found in patients with complete remission between the two treatment groups (first year: χ2 = 0.261, P = 0.88; second year: χ2 = 2.685, P = 0.26). In patients with frequently relapsing or steroid-dependent nephrotic syndrome, no significant difference in short-term remission (χ2 = 1.908, P = 0.39) or in relapse frequency during follow-up (within first year: χ2 = 1.046, P = 0.59; within second year: χ2 = 0.587, P = 0.75) were found between the two groups. There was a difference in the rate of adverse effects between the two treatment groups [nephrotoxicity: 4/24 (CsA) vs .0/50 (TAC), P = 0.002; hirsutism: 8/24 (CsA) vs. 0/50 (TAC), P < 0.001].ConclusionsIn our pediatric patient cohort, the treatment of steroid-resistant nephrotic syndrome with tacrolimus was associated with higher efficacy and lower renal toxicity in comparison to CsA, although no favorable outcome in relapse rate during long-term follow-up was seen. On the other hand, tacrolimus was not always the better choice to replace CsA in the treatment of severe frequently relapsing or steroid-dependent nephrotic syndrome.

NPHS1 and NPHS2 Gene Mutations in Chinese Children With Sporadic Nephrotic Syndrome

Recent discoveries indicate that the molecules in glomerular podocytes and slit diaphragms may play an important role in the development of proteinuria and nephrotic syndrome. Mutational analyses of NPHS1 and NPHS2 were performed to verify this hypothesis in sporadic nephrotic syndrome (NS) patients. Clinical characteristics and DNA samples were collected from 38 Chinese children with sporadic steroid-sensitive NS, 22 with steroid-resistant NS and 30 controls. Direct sequencing was performed after PCR amplification of all 29 and 8 exons of the NPHS1 and NPHS2 genes, respectively. In NPHS1, 4 patients had heterozygous missense mutations leading to amino acid substitutions (R800C, Q453R). Furthermore, 3 known single nucleotide polymorphism (SNP) were found (T741T, V763V, S1105S). In NPHS2, 3 patients had novel heterozygous allelic variants leading to amino acid substitutions (S206I, E188D), while 1 patient was found to carry a novel nonsense mutation leading to a truncated protein product (Glu237STOP). Two known polymorphisms were also found (A318A, L346L). The results demonstrate that NPHS1 and NPHS2 mutations are also present in Chinese sporadic NS patients, suggesting that genetic changes of nephrin and podocin may play pathogenetic roles in some patients with sporadic steroid resistant NS.

Ghrelin ameliorates hypoxia-induced pulmonary hypertension via phospho-GSK3β/β-catenin signaling in neonatal rats

Effective treatment and/or prevention strategies for neonatal persistent pulmonary hypertension of the newborn (PPHN) have been an important topic in neonatal medicine. However, mechanisms of impaired pulmonary vascular structure in hypoxia-induced PPHN are poorly understood and consequently limit the development of effective treatment. In this study, we aimed to explore the molecular signaling cascades in the lungs of a PPHN animal model and used primary cultured rat pulmonary microvascular endothelial cells to analyze the physiological benefits of ghrelin during the pathogenesis of PPHN. Randomly selected newborn rats were exposed to hypoxia (10-12%) or room air and received daily s.c. injections of ghrelin (150 μg/kg) or saline. After 2 weeks, pulmonary hemodynamics and morphometry were assessed in the rats. Compared with the control, hypoxia increased pulmonary arterial pressure, right ventricle (RV) hypertrophy, and arteriolar wall thickness. Ghrelin treatment reduced both the magnitude of PH and the RV/(left ventricle+septum (Sep)) weight ratio. Ghrelin protected neonatal rats from hypoxia-induced PH via the upregulation of phosphorylation of glycogen synthase kinase 3β (p-GSK3β)/β-catenin signaling and associated with β-catenin translocation to the nucleus in the presence of growth hormone secretagogue receptor-1a. Our findings suggest that s.c. administration of ghrelin improved PH and attenuated pulmonary vascular remodeling after PPHN. These beneficial effects may be mediated by the regulation of p-GSK3β/β-catenin expression. We propose ghrelin as a novel potential therapeutic agent for PPHN.

Myo1e Impairment Results in Actin Reorganization, Podocyte Dysfunction, and Proteinuria in Zebrafish and Cultured Podocytes

Background Podocytes serve as an important constituent of the glomerular filtration barrier. Recently, we and others identified Myo1e as a key molecular component of the podocyte cytoskeleton. Results Myo1e mRNA and protein was expressed in human and mouse kidney sections as determined by Northern blot and reverse transcriptase PCR, and its expression was more evident in podocytes by immunofluorescence. By specific knock-down of MYO1E in zebrafish, the injected larvae exhibited pericardial edema and pronephric cysts, consistent with the appearance of protein in condensed incubation supernate. Furthermore, specific inhibition of Myo1e expression in a conditionally immortalized podocyte cell line induced morphological changes, actin cytoskeleton rearrangement, and dysfunction in cell proliferation, migration, endocytosis, and adhesion with the glomerular basement membrane. Conclusions Our results revealed that Myo1e is a key component contributing to the functional integrity of podocytes. Its impairment may cause actin cytoskeleton re-organization, alteration of cell shape, and membrane transport, and podocyte drop-out from the glomerular basement membrane, which might eventually lead to an impaired glomerular filtration barrier and proteinuria.

Efficacy of triptolide for children with moderately severe Henoch-Schonlein purpura nephritis presenting with nephrotic range proteinuria: a prospective and controlled study in China.

Objective. To observe the clinical efficacy of the Chinese herb, Triptolide, in children with moderately severe Henoch-Schönlein purpura nephritis (HSPN). Methods. From January 2007 to December 2011, 56 HSPN children manifested by nephrotic range proteinuria with normal kidney function and <50% crescents or sclerosing lesions on biopsy were hospitalized in the Childrens Hospital of Zhejiang University School of Medicine. They were divided into two groups: the treatment group (n = 42; Triptolide at a dosage of 1 mg/kg·d, combined with prednisone at a dosage of 2 mg/kg·d, within a course of medium-to-long-term therapy of 6 to 9 months) and the control group (n = 14; prednisone alone, with the same procedure). Results. Short-term remission was observed in 95% of patients from treatment group and in 72% of patients from control group, respectively. There was a significant difference between both groups (χ 2 = 6.222, P = 0.029) for short-term effects. Meanwhile, no significant difference, as proteinuria, hematuria, hypertension, and decreased eGFR, was observed between the two groups in long-term followup (χ 2 = 3.111, P = 0.097). The Kaplan-Meier plot analysis also revealed no significant difference (χ 2 = 2.633, P = 0.105). Conclusion. Triptolide is effective in relieving short-term symptoms for moderately severe HSPN children, though its long-term effects need to be observed further.

Triple immunosuppressive therapy in steroid-resistant nephrotic syndrome children with tacrolimus resistance or tacrolimus sensitivity but frequently relapsing.

The treatment strategy for steroid‐resistant nephrotic syndrome remains uncertain at present, especially in those with calcineurin inhibitor resistance or intolerance. To date, few studies have been published using multiple combination therapy of immunosuppressive reagents for children with calcineurin inhibitor‐resistant or ‐intolerant nephrotic syndrome.

Overexpression of Myo1e in Mouse Podocytes Enhances Cellular Endocytosis, Migration, and Adhesion

Podocytes are a terminally differentiated and highly specialized cell type in the glomerulus that forms a crucial component of the glomerular filtration barrier. Recently, Myo1e was identified in the podocytes of glomeruli. Myo1e podocyte‐specific knockout mice exhibit proteinuria, podocyte foot process effacement, glomerular basement membrane disorganization, signs of chronic renal injury, and kidney inflammation. After overexpression of Myo1e in a conditionally immortalized mouse podocyte cell line (MPC5), podocyte migration was evaluated via transwell assay, endocytosis was evaluated using FITC‐transferrin, and adhesion was evaluated using a detachment assay after puromycin aminonucleoside treatment. Myo1e overexpression significantly increased the adherence of podocytes. ANOVA analysis indicated significant differences for cell adhesion between the overexpression and control groups (overexpression vs. control, t = 11.3199, P = 0.005; overexpression vs. negative control, t = 12.0570, P = 0.0006). Overexpression of Myo1e inhibited puromycin aminonucleoside‐induced podocyte detachment, and the number of cells remaining on the bottom of the culture plate increased. Cell migration was enhanced in Myo1e‐overexpressing podocytes in the transwell migration assay. Internalization of FITC‐transferrin also increased in Myo1e‐overexpressing podocytes relative to control cells. Overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. J. Cell. Biochem. 115: 410–419, 2014.

Effects of Long-Term High-Fat/High-Energy and High-Protein Diets on Insulin and Ghrelin Expression in Developing Rats

Objective. This study investigated the long-term effects of high-fat/high-energy and high-protein diets on insulin secretion and ghrelin expression. Methods. Dams of Sprague-Dawley rats were fed a standard, high-fat/high–energy, or high-protein diet during pregnancy and lactation, and their pups were defined as control, high-fat and high-energy, and high-protein groups, respectively. The pups were fed the same diet as their dams after weaning. Plasma glucose, ghrelin, and insulin were analyzed on the first, third, seventh, and tenth postnatal days and at the end of second, third, fourth, eighth, and twelfth weeks. Ghrelin and insulin expression in the pancreas was measured using radioimmunoassay, double-staining immunohistochemistry, and confocal microscopy. Results. Fasting blood glucose, plasma insulin concentrations, and homeostasis model assessment-insulin resistance index increased with age. Total plasma ghrelin concentrations decreased with age. Plasma ghrelin concentrations were negatively correlated with glucose levels in all three groups. Plasma ghrelin was negatively correlated with plasma insulin only in the high-fat and high-energy group. Insulin secretion in the high-protein and high-fat and high-energy groups and pancreatic ghrelin content, pancreatic ghrelin-positive cells, and beta cells in all groups decreased with age. The percentage of ghrelin-positive cells correlated with the percentage of beta cells in all groups. Conclusion. Insulin and ghrelin expression in the plasma and pancreas was adversely affected by long-term high-fat/high-energy and high-protein diets.

Familial steroid-sensitive idiopathic nephrotic syndrome: seven cases from three families in China

OBJECTIVES: Familial steroid-sensitive idiopathic nephrotic syndrome is rare, and only approximately 3% of patients have affected siblings. METHODS: Herein, we report seven cases of patients with steroid-sensitive idiopathic nephrotic syndrome from three Chinese families. Mutational screening of the Nphs2 gene was performed in all the patients. RESULTS: All seven of the familial steroid-sensitive idiopathic nephrotic syndrome cases in our sample exhibited minimal change disease, and one case also presented with mesangial proliferative glomerulonephritis, according to the renal pathology. No significant was associations were found between Nphs2 gene mutations and the onset of proteinuria and nephrotic syndrome in these familial cases. CONCLUSIONS: The presence of minimal change disease is important, but it is not an unusual finding in patients with familial steroid-sensitive idiopathic nephrotic syndrome, which appears to be clinically benign and genetically distinct from other types of nephrosis.