P0061LOSS OF UBIQUITIN SPECIFIC PROTEASE-40 CAUSES PODOCYTE DETACHMENT AND CONTRIBUTES TO THE DISEASE PROGRESSION OF GLOMERULOSCLEROSIS

Background and Aims We reported that the ubiquitin specific protease-40 (USP40) is expressed in podocytes beyond the species and plays a crucial role in glomerulogenesis in zebrafish (Takagi H, Am J Physiol Renal Physiol 2017). However, the role of USP40 in podocytes and its functional downstream after birth is largely unknown. Recent report indicated that other USP family: USP10 regulates integrin beta1 and fibrotic wound healing in myofibroblasts (Gillespie SR, J Cell Sci 2017). The present study aimed to explore the biological role of USP40 in the acquired podocyte injury and to determine the interacting partner by focusing on podocyte detachment. Method We generated USP40-knockout mice (USP40KO); however, they showed no apparent kidney abnormality. Therefore, to explore the biological role of USP40 in acquired podocyte injury, we crossed USP40KO with NEP25 mice, in which selective podocyte injury can be induced by injection with an immunotoxin, LMB2. Urinary protein was analyzed until day 9 after LMB2 injection, and then kidney tissues were subjected to light microscopy, immunohistochemistry of p57, and immunofluorescence microscopy of integrin beta1. The glomeruli were isolated and subjected to immunoblot analysis of integrin beta1. The effect of USP40 knockdown on integrin beta1 expression was studied in cultured mouse podocytes. Finally, the protein binding of integrin beta1 with USP40 was determined by immunoprecipitation using cultured mouse podocytes. Results NEP-USP40KO mice developed earlier and higher levels of proteinuria compared with NEP mice. Light microscopy and immunohistochemistry showed higher levels of kidney injury in NEP-USP40KO mice compared with NEP mice in terms of hyalinosis (p<0.01), crescent formation (p<0.01), and cell proliferation (p<0.01) in the glomeruli, podocyte loss (p<0.05), and tubulointerstitial injury (p<0.01). Immunoblot analysis and immunofluorescence revealed decreased expression of integrin beta1 in podocytes of NEP-USP40KO mice compared with NEP mice. Gene knockdown of USP40 in cultured podocytes decreased the protein expression of integrin beta1. Finally, apparent protein binding of USP40 with integrin beta1 was observed. Conclusion Integrin beta1 is known to play a crucial role as the adhesion molecule that connects the podocytes with the extracellular matrix of the glomerular basement membrane. It is also known that the lack of integrin beta1 causes podocyte detachment, which is involved in the disease progression of glomerulosclerosis. Therefore, the present study suggests that USP40 may be involved in a self-defense mechanism in podocyte injury probably through interacting with the regulatory machinery of integrin beta1 in podocytes.

16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis

Many functions of vasoinhibins have been reported, but its receptor has not been clarified yet. Vasoinhibins, 11–18 kDa N-terminal fragments of prolactin, have anti-angiogenic activity and act on endothelial cells to induce apoptosis and to inhibit migration and proliferation, which are opposite to the effects of prolactin. Although vasoinhibins bind to the prolactin receptor, its binding activity is very weak compared to prolactin. Therefore, in this study, we evaluated the binding activity between 16 kDa vasoinhibin and integrin beta1, alpha5 beta1, alpha1 beta1 and alphaV beta3 to identify a specific receptor for vasoinhibins. Moreover, we examined whether 16 kDa vasoinhibin induced apoptosis through integrin beta1 and alpha5 beta1 in endothelial cells. In this study, binding assays and co-immunoprecipitation experiments demonstrated that 16 kDa vasoinhibin could bind strongly to integrin beta1 and alpha5 beta1. Moreover, neutralizing with integrin beta1 and alpha5 beta1 antibody could inhibit 16 kDa vasoinhibin-induced apoptosis in endothelial cells. These findings suggest that vasoinhibins can act on endothelial cells through integrin alpha5 beta1 to induce apoptosis.

Transplantation of umbilical cord blood-derived mesenchymal stem cells to treat liver cirrhosis in mice: a comparison of tail and portal vein injection

Introduction: To date, there have been many studies indicating the positive effects of stem cells on treating liver cirrhosis. In this study, we used umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) for treatment in a mouse model of liver cirrhosis. Specifically, we determined and compared the effectiveness of two methods of MSC injection (tail vein versus portal vein). Methods: Liver cirrhosis in male Swiss mice (of age approximately 11 weeks or under) was induced by administration of carbon tetrachloride (CCl4; 1 ml/kg). One million UCB-MSCs were then transplanted into cirrhotic mice via the portal vein or tail vein. After 21 days, blood samples were collected for measurement of transaminase, bilirubin and albumin. The expression of fibrosis-associated genes, specifically procollagen – alpha 1 and integrin – beta1, were assessed using quantitative RT-PCR. The histopathology of the specimens was also evaluated using hematoxylin/eosin, Masson trichrome staining, and immunohistochemistry using collagen type 1 and alpha-SMA antibodies. Results: After 21 days, cirrhotic mice treated with UCB-MSCs showed recovery of bilirubin index, increase of liver albumin synthesis, inhibition of fibrosis-related gene expression (e.g. procollagen – alpha 1 and integrin – beta1), and remodeling of liver histology. From comparison of the different routes of transplantation, UCB-portal route was significantly more effective than UCB-tail route at reducing aspartate transaminase (AST) activity and bilirubin index (P<0.05), and inhibiting procollagen – alpha 1 and integrin – beta1 expression (P<0.05). UCB-MSCs from both transfusion routes showed accelerated improvement of liver histopathology. Conclusion: Therapeutic strategies using UCB-MSCs have proven to be promising for the treatment of liver cirrhosis. Injection of UCB-MSC via portal vein was more effective than tail vein for cirrhosis treatment. Peer Review Details Peer review method: Single-Blind (Peer-reviewers: 02) Peer-review policy Plagiarism software screening?: Yes Date of Original Submission: 17 August 2017 Date accepted: 30 August 2017 Peer reviewers approved by: Dr. Lili Hami Editor who approved publication: Dr. Phuc Van Pham