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The mitotic arrest deficient protein MAD2B is a well-defined anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase zeta. It is critical for mitotic control and DNA repair. However, the pathological role of MAD2B in kidney diseases has not been fully elucidated. In the present study, we aim to explore the role of MAD2B in the pathogenesis of renal tubulointerstitial fibrosis (TIF) and the underlying mechanism. By immunofluorescence and immunohistochemistry, we found an obvious MAD2B enhancement in tubular area of TIF patients and unilateral ureteral obstruction (UUO) mice. In vitro, transforming growth factor-β1 (TGF-β1) induced a time-dependent MAD2B accumulation prior to tubular epithelial-to-mesenchymal transition (EMT) in a rat proximal tubular epithelial cell line, NRK-52E. Knocking down MAD2B using siRNA dramatically inhibited TGF-β1-induced tubular EMT process and subsequent extracellular matrix (ECM) production. We also found that Skp2, a confirmed APC/C-CDH1 substrate and E-cadherin destroyer, was increased in TGF-β1-treated proximal tubular epithelial cells, which could be blocked by MAD2B depletion. In addition, Skp2 expression was also found to be increased in the renal tubular area of UUO mice. Locally knocking down MAD2B expression in the renal cortex using lentiviral transfection inhibited Skp2 expression, tubular EMT, and subsequent ECM accumulation. Taken together, our data suggests a pro-fibrotic role of MAD2B in the pathogenesis of tubular EMT and TIF by inducing Skp2 expression. MAD2B might be a potential target of promising interventions for renal TIF. Key messages: [ABSTRACT FROM AUTHOR]

Progressive renal tubulointerstitial fibrosis is a common final pathway of nearly all forms of chronic kidney disease. Many efforts have been done to arrest or prevent renal tubulointerstitial fibrosis but with little progress. Nowadays, few therapeutic agents are available in clinical use. Norcantharidin (NCTD) is of great benefit in anticancer treatment, by inducing cell apoptosis, inhibiting cell proliferation, in addition, blocking tumor metastasis and angiogenesis in cancer, whereas little attention is given to its relationship with other diseases. Our recent studies demonstrated that NCTD was protective against renal tubulointerstitial fibrosis both in vivo and in vitro. The underlying mechanisms may include modulation of TGF-β1/Smad signal cascade, inhibition of protein serine/threonine phosphatases (PPP) as well as NF-κB. NCTD may be a promising therapeutic agent for renal tubulointerstitial fibrosis. In the present article, we will review the action of NCTD in renal tubulointerstitial fibrosis and discuss its possible mechanisms. [ABSTRACT FROM AUTHOR]

In order to explore the role of connective tissue growth factor (CTGF) in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction (UUO) group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor- β1 (TGF- β1), collagen I (col I), and plasminogen activator inhibitor-1 (PAI-1) were detected using reverse transcriptional-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to evaluate the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF- β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO ( P<0.01). With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index ( r=0.62, P<0.01), the expression of TGF- β1 ( r=0.85, P<0.01), col I ( r=0.78, P<0.01), and PAI-1 ( r=0.76, P<0.01). Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course ( P<0.01, as compared with sham-operated group). It is concluded that CTGF can be induced by TGF- β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix (ECM) synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis. [ABSTRACT FROM AUTHOR]

The Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse is a hereditary model animal for nephrotic syndrome with chronic renal tubulointerstitial fibrosis. In most fibrotic diseases, myofibroblastic differentiation is considered to play crucial roles in pathogenesis of fibrosis and is dominantly regulated by the transforming growth factor (TGF)-β1 signaling system. To reveal the pathogenic mechanism of chronic renal fibrosis in ICGN mice, we examined the expression and localization of TGF-β1 signal transducer proteins (TGF-β receptor-I and -II, Smad2/3 and Smad4) in kidney sections and in primarily cultured tubulointerstitial fibroblasts (TIFs). In kidneys of ICGN mice, many tubulointerstitial cells were differentiated to myofibroblastic cells and were α-smooth muscle actin (αSMA)-positive. The numbers of αSMA-positive TIFs prepared from kidneys of ICGN mice (ICGN-TIFs), but not those of ICR control mice (ICR-TIFs), increased during cell culture. No significant differences in production or activation of TGF-β1 between ICGN-TIFs and ICR-TIFs were seen by enzyme-linked immunosorbent assay. In vitro transcriptional reporter assay for TGF-β1 and Western immunoblotting for TGF-β1 signal transducers showed no notable differences in the expression levels of TGF-β receptor-I or -II or Smad2/3 between these TIFs. However, augumented cytoplasmic Smad4 protein in ICGN-TIFs, but not ICR-TIFs, seemed to cause hypersensitivity against TGF-β1, and the eventual nuclear localization of Smad2/3-Smad4 complex was increased in ICGN-TIFs. Thus, the abnormal cytoplasmic augmentation of Smad4 induces acceleration of TGF-β1 signaling in the renal tubulointerstitial cells of ICGN mice. [ABSTRACT FROM AUTHOR]