Perri Anna, Vizza Donatella, Lofaro Danilo, Gigliotti Paolo, Leone Francesca, Brunelli Elvira, Malivindi Rocco, De Amicis Francesca, Romeo Francesco, De Stefano Roberto, Papalia Teresa, Bonofiglio Renzo
Research Center Kidney and Transplantation, Annunziata Hospital Cosenza, Cosenza - Italy; and Health Center, University of Calabria, Calabria - Italy.
J Nephrol. 2013 Nov-Dec;26(6):1049-54. doi: 10.5301/jn.5000269. Epub 2013 Apr 3.
Adiponectin (ADPN) is predominantly produced by adipose tissue, and high ADPN levels have been detected in patients affected by proteinuric glomerulonephritis. In this study we investigate whether human tubular epithelial cells express and secrete ADPN.
In human proximal tubular epithelial cells, HK-2, ADPN mRNA was evaluated by real-time PCR assay, while protein expression levels were measured by Western blot analysis and immunofluorescence assay. Moreover, renal ADPN distribution was assessed by immunohistochemical analysis of kidney biopsy samples from healthy patients. Finally, by ELISA, we measured ADPN concentrations in culture media of HK-2 cells treated with 10 µg/mL lipopolysaccharide (LPS).
Our analyses revealed that HK-2 cells express ADPN both in terms of mRNA and protein. These results were confirmed by the observed cytoplasmatic HK-2 intense immunoreactivity for ADPN antibody and by immunohistochemical analysis showing a diffuse ADPN distribution in normal kidney tissue. Furthermore, we observed that tubular cells secrete ADPN in the basal condition and, more interestingly, that this secretion significantly increases (p<0.05) upon LPS treatment in a time-dependent manner. Finally, immunohistochemical analysis of kidney biopsy samples obtained from patients affected by membranous and rapidly progressive glomerulonephritis showed a similar pattern of ADPN staining to that observed in healthy controls.
Our study demonstrates for the first time that renal tubular cells express and secrete ADPN, and their concentration increases upon inflammatory stimulus. These results suggest that in renal inflammatory diseases, tubular cells may contribute to the increase in circulating ADPN levels, triggering a feedback response in order to self-mitigate the inflammatory process.
脂联素(ADPN)主要由脂肪组织产生,在蛋白尿性肾小球肾炎患者中已检测到高ADPN水平。在本研究中,我们调查人肾小管上皮细胞是否表达和分泌ADPN。
在人近端肾小管上皮细胞HK-2中,通过实时PCR测定法评估ADPN mRNA,同时通过蛋白质印迹分析和免疫荧光测定法测量蛋白质表达水平。此外,通过对健康患者肾活检样本的免疫组织化学分析评估肾脏ADPN分布。最后,通过酶联免疫吸附测定(ELISA),我们测量了用10μg/mL脂多糖(LPS)处理的HK-2细胞培养基中的ADPN浓度。
我们的分析显示,HK-2细胞在mRNA和蛋白质方面均表达ADPN。ADPN抗体在HK-2细胞质中的强烈免疫反应性以及免疫组织化学分析显示正常肾组织中ADPN呈弥漫性分布,证实了这些结果。此外,我们观察到肾小管细胞在基础条件下分泌ADPN,更有趣的是,LPS处理后这种分泌以时间依赖性方式显著增加(p<0.05)。最后,对膜性和快速进展性肾小球肾炎患者的肾活检样本进行的免疫组织化学分析显示,ADPN染色模式与健康对照中观察到的相似。
我们的研究首次证明肾小管细胞表达和分泌ADPN,并且它们的浓度在炎症刺激下增加。这些结果表明,在肾脏炎症性疾病中,肾小管细胞可能有助于循环ADPN水平的升高,触发反馈反应以自我减轻炎症过程。