ROBO2-mediated RALDH2 signaling is required for common nephric duct fusion with primitive bladder.

Congenital anomalies of the urinary tract are a significant cause of morbidity in infancy, and many congenital anomalies are linked to ureter development; however, the mechanism by which congenital anomalies control ureter development remains unknown. The loss of Robo2 can cause ureter defects and vesicoureteral reflux. However, how Robo2 impacts ureter development is unclear. We found that ROBO2 is expressed in the common nephric duct (CND) and primitive bladder, and impacts CND migration and fusion with the primitive bladder via its novel binding partner retinaldehyde dehydrogenase-2 (RALDH2). Delayed apoptosis that is due to the failure of CND fusion with the primitive bladder in the Robo2-/-embryo results in an abnormal ureter connection to the CND, which is required for ureter development. We define a novel pathway in which the CND is remodeled by ROBO2 and retinoic acid rescued the ureter anomalies in the Robo2-/-embryo. These findings may be relevant to diverse disease conditions that are associated with altered signaling in the primitive bladder.

Effect of a low-protein diet supplemented with keto-acids on autophagy and inflammation in 5/6 nephrectomized rats.

Ketoacids (KA) are known to preserve muscle mass among patients with chronic kidney disease (CKD) on a low-protein diet (LPD). The present study was to compare the effects of KA supplemented diet therapy in autophagy and inflammation in CKD rats' skeletal muscle. Rats with 5/6 nephrectomy were randomly divided into three groups and fed with either 11 g/kg/day protein [normal-protein diet (NPD)], 3 g/kg/day protein (LPD) or 3 g/kg/day protein which including 5% protein plus 1% KA (LPD + KA) for 24 weeks. Sham-operated rats with NPD intake were used as control. LPD could improve body weight, gastrocnemius muscle mass, as well as gastrocnemius muscle cross-sectional area, with the effect being more obvious in the LPD + KA group. The autophagy marker LC3 (microtubule-associated protein 1 light chain 3), p62, Parkin and PTEN induced putative kinase 1 (PINK1) were significantly attenuate in LPD + KA group than LPD group. LPD + KA group had the lower total mtDNA (mitochondiral DNA) and cytosol mtDNA, NACHT-PYD-containing protein 3 (NALP3) inflammasome than LPD group, but its reactive oxygen species (ROS), caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) level was higher. Immunoblotting showed IL-1ß (interleukin-1-beta) was lower in LPD and LPD + KA group than the NPD group, but IL-18 showed no significant difference among control and CKD group; toll-like receptor signalling-dependent IL-6 was higher in LPD + KA group than LPD group, but tumor necrosis factor-α (TNF-α) was not significantly changed between LPD + KA and LPD group. Systematic changes of the four cytokines were different from that of the tissue. Although LPD + KA could further ameliorate-activated autophagy than LPD, its effect on the activated inflammation state in CKD was not distinctly. Further study is still required to explore the method of ameliorating inflammation to provide new therapeutic approaches for CKD protein energy wasting (PEW).

Inhibition of the tubular epithelial-to-mesenchymal transition in vivo and in vitro by the Uremic Clearance Granule ().

OBJECTIVE: To investigate the effect of the Uremic Clearance Granule (UCG, ), a Chinese patent medicine, on tubular epithelial-to-mesenchymal transition (EMT) in a unilateral ureteral obstruction (UUO) model in vivo and transforming growth factor (TGF)-ß1 induced EMT of HK-2 cells in vitro. METHODS: In vivo study, 50 Sprague Dawley rats were divided into three groups: a sham operation group (n=10), a UUO group (n=20), and a UUO with UCG treatment group (n=20). The UCG was given at a dose of 4.5 g/kg body weight per day by gavage after surgery. In vitro study, HK-2 cells were cultured in 10% fetal bovine serum (FBS), 10% healthy rat serum, 10% FBS and TGF-ß1 (10 ng/mL), 10% healthy rat serum and TGF-ß1, or 10% rat serum containing the uremic clearance granule and TGF-ß1. The expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in kidney tissues and HK-2 cells were investigated by Western blot analysis and immunofluorescence staining. RESULTS: The rats of the UUO group showed obvious tubulointerstitial fibrosis, compared with the sham operation group rats. Tubulointerstitial fibrosis score was reduced by 17.5%±1.1% at day 7 and by 20.0%±1.2% at day 14 in the UCG-treated group, compared with the UUO group. The UCG could maintained expression of E-cadherin and suppressed expression of vimentin and α-SMA in kidney tissues of UUO rats at days 7 and 14, as determined by Western blot analysis and immunofluorescence staining. Rat serum containing the UCG partially inhibited TGF-ß1-induced fibroblast phenotype of HK-2 cells and maintained the epithelial morphology of HK-2 cells in vitro. This occurred partially through a reduction of vimentin expression and an increase of E-cadherin expression. CONCLUSION: These results suggest that the UCG prevents tubular EMT and may be a promising agent for treating tubulointerstitial fibrosis.

Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6.

BACKGROUND: Mesothelial cell injury plays an important role in peritoneal fibrosis. Present clinical therapies aimed at alleviating peritoneal fibrosis have been largely inadequate. Mesenchymal stem cells (MSCs) are efficient for repairing injuries and reducing fibrosis. This study was designed to investigate the effects of MSCs on injured mesothelial cells and peritoneal fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Rat bone marrow-derived MSCs (5 × 10(6)) were injected into Sprague-Dawley (SD) rats via tail vein 24 h after peritoneal scraping. Distinct reductions in adhesion formation; infiltration of neutrophils, macrophage cells; number of fibroblasts; and level of transforming growth factor (TGF)-ß1 were found in MSCs-treated rats. The proliferation and repair of peritoneal mesothelial cells in MSCs-treated rats were stimulated. Mechanically injured mesothelial cells co-cultured with MSCs in transwells showed distinct increases in migration and proliferation. In vivo imaging showed that MSCs injected intravenously mainly accumulated in the lungs which persisted for at least seven days. No apparent MSCs were observed in the injured peritoneum even when MSCs were injected intraperitoneally. The injection of serum-starved MSCs-conditioned medium (CM) intravenously reduced adhesions similar to MSCs. Antibody based protein array of MSCs-CM showed that the releasing of TNFα-stimulating gene (TSG)-6 increased most dramatically. Promotion of mesothelial cell repair and reduction of peritoneal adhesion were produced by the administration of recombinant mouse (rm) TSG-6, and were weakened by TSG-6-RNA interfering. CONCLUSIONS/SIGNIFICANCE: Collectively, these results indicate that MSCs may attenuate peritoneal injury by repairing mesothelial cells, reducing inflammation and fibrosis. Rather than the engraftment, the secretion of TSG-6 by MSCs makes a major contribution to the therapeutic benefits of MSCs.

Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis.

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.

Characteristics and influence factors of pathologic transformation in the subclasses of class IV lupus nephritis.

The study explored the characteristics and correlation factors of transformation in subclasses of class IV lupus nephritis. Patients with class IV lupus nephritis were subjected to repeat biopsies after 6 months of induction treatment. Transformation rate between two subclasses, class IV-S and class IV-G, was compared. Influence Factors of transformation were evaluated. Class IV-G had more severe hypertension and higher score of immunofluorescence index, glomerular active lesions, tubular and vascular lesions. Class IV-S had a higher percentage of glomerular fibrinoid necrosis. Class IV-S appeared a higher rate of transformation to class II than class IV-G (57% vs. 27%). In each subclass, active lesion also showed a higher rate of transformation to class II than active/chronic lesion (IV-G: 41.2% vs. 12.5%; IV-S: 71.4% vs. 42.8%). Patients who maintained class IV had higher blood pressure, obvious proteinuria, declined kidney function, and lower C3 level. Immunosuppressive therapy, urine protein, and vascular lesions were independent risk factors for the pathologic transformation. The rate of transformation in class IV-S was higher than that in class IV-G. The transformation is most likely to benefit from immunosuppressive therapy. Urine protein and vascular lesions are correlated with the transformation in class IV lupus nephritis.