HBV靶向受体NTCP介导足细胞感染及损伤的机制

HBV DNA was detectable in podocyte of HBV-associated glomerulonephritis (HBV-GN), It revealed that HBV might infect podocyte directly. However, It is unclear how the podocyte was infected by HBV. It suggested that sodium taurocholate cotransporting polypeptide (NTCP) is the specific receptor of HBV entry into hepatocytes, which is blocked by tacrolimus to inhibit HBV infection. Our study indicated that NTCP was also detected in human glomerulus podocyte by western blot and Immunofluorescent. We speculate that HBV entry into the podocyte and subsequent infection by binding to NTCP expressing in the podocyte. Furthermore, HBV-GN achieved the higher proteinuria remission without enhancing HBV replication due to receiving the therapy of tacrolimus. Therefore, We speculate that NTCP in the podocyte was blocked by tacrolimus to inhibit HBV infection and protect podocyte. We perform experiments to investigate the association of podocyte and HBV infection. Human NTCP overexpressing and knock-down constructs to up-regulating and down-regulating the express of NTCP receptor in cultured human podocyte. The HBV used in this study was mainly derived from the culture supernatant of HepAD38 cells. HBV infected the cultured human podocyte. We detected the marker of HBV infection in podocyte. Meanwhile, the proliferation, apoptosis and function of podocyte was detected. In addition, After NTCP of podocyte was blocked by tacrolimus and NTCP inhibitor, We observed HBV infection and function changes in podocyte. To investgate the interaction of HBV and NTCP, We futher mutate amino acids 157–165 of human podocyte NTCP. The study is expected to reveal the new pathogenesis of HBV infected and injuried podocytes, which will provide a new target and theory bases for the treatment of HBV-GN.

乙型肝炎病毒相关性肾炎（HBV-GN）的肾脏足细胞中可检测到HBV,提示HBV可能直接感染足细胞，但其机制尚不明确。研究表明：牛黄胆酸钠共转运多肽（NTCP）是HBV进入肝细胞的特异受体,且其可被免疫抑制剂他克莫司阻断。我们发现人肾脏足细胞也表达NTCP。那么，HBV是否与NTCP特异结合进入足细胞导致其感染及损伤？进一步用他克莫司治疗HBV-GN，蛋白尿缓解率高且不增加病毒复制。因此推测：他克莫司通过阻断HBV与足细胞NTCP结合,从而保护足细胞。本项目拟采用体外培养人足细胞，正、反干预NTCP及NTCP抑制剂、他克莫司阻断NTCP受体，应用源于HepAD38细胞的HBV感染足细胞，检测足细胞HBV感染及足细胞功能变化情况。并进一步突变NTCP157-165位点明确HBV与NTCP结合具体机制。课题顺利开展有望揭示HBV感染足细胞的新机制，为HBV-GN治疗提供新靶点及理论依据。

乙型肝炎病毒相关性肾炎(Hepatitis B virus associated glomerulo- nephritis,HBV-GN)为我国常见继发性肾小球肾炎之一，HBV-GN病理类型常表现为膜性肾病样改变，其主要累及肾小球足细胞, 尽管该病进展缓慢，但是仍有约30%成年患者可进展至终末期肾病，且其中10%患者需要肾脏替代治疗。因此，明确HBV-GN发病机制并予以特异靶点治疗非常重要。.项目主要以人乙型肝炎病毒相关性肾炎患者的肾足细胞及体外培养的人肾足细胞为研究对象，首次通过体内外实验明确乙型肝炎病毒相关性肾炎的发病机制之一：乙型肝炎病毒直接感染肾脏足细胞。研究发现：（1）通过免疫组化及免疫荧光共定位方法证实人肾组织足细胞中存在NTCP受体表达，进一步在体外培养的人肾足细胞中应用Western-blot方法检测到NTCP受体表达。（2）构建人NTCP过表达质粒并转染足细胞，HBV感染足细胞后，ELISA法检测感染1、3、5、7、9天后足细胞的HBsAg、HBeAg水平，PCR方法检测感染后足细胞HBV DNA、HBV cccDNA水平均较正常对照组显著升高。（3）构建人NTCP-ShRNA1，2质粒并转染足细胞，HBV感染足细胞能力受抑，HBsAg、HBeAg水平及HBV DNA、HBV cccDNA水平均较对照组显著降低。（4）他克莫司及NTCP抑制剂可以显著抑制HBV感染足细胞。（5）突变人足细胞NTCP157-165位点后，HBV感染足细胞能力显著受抑，明确了人足细胞NTCP157-165位点为HBV感染的主要位点。这些结果表明NTCP可介导HBV感染肾脏足细胞，他克莫司可通过阻断NTCP受体进而抑制HBV感染足细胞，为乙型肝炎病毒相关性肾炎提供新治疗靶点。