| Proteinuria, a common clinical feature of glomerular diseases, has not only been considered as a marker of renal injury, but also activate directly renal proximal tubular epithelial cells (PTECs), leading to tubulointerstitial lesion which is followed by kidney fibrosis. Several studies in recent years have shown that albumin overload is to much extent involved in the process of PTECs phenotype change and inflammatory cell infiltration. However, its mechanism has not been hitherto completely uncovered. Cubilin, a novel multifunctional endocytic receptor expressed in the apical membranes of PTECs, is responsible for proteins reabsorption, such as albumin and high-density lipoprotein, from the glomerular filtrate. It was found that both cubilin gene mutation patients (called Imerslund-Gr?sbeck syndrome ) and cubilin gene knock-out dogs present heavy albuminuria, but normal kidney structure microscopically and renal function in their lifetime, which were distinguished from the characteristics of proteinuria patients with glomerular diseases. Based on the above findings, we hypothesized that cubilin may mediate renal injury associated with proteinuria. To test this hypothesis, we firstly examined albumin deposition and chemokines expression in renal tissues from the patients with nephrotic syndrome, then constructed antisense cubilin plasmid and observed its inhibitory role in the PTECs activation induced by albumin overload.Methods 1. Thirteen proteinuria patients with nephrotic syndrome and five patients with occulting hematuria were collected into our study. The plasma and urinary biochemical parameters, including serum albumin, lipoprotein, Scr and urinary protein levels, were analyzed by routine biochemical method. The histologic changes were examined by HE and PAS staining. Albumin deposition and MCP-1 and RANTES expression in renal tissues were detected by immunohistochemistry. 2. The cubilin gene fragment containing regulatory region was amplified by RT-PCR, and cloned into the pGEM-T Easy vector. The plasmids were digested with EcoR I restriction endonuclease, and the fragments were subcloned into the eukaryotic expression vector pCDNA3.1(+). By means of digestion with Hind III and DNA sequencing, the senseor antisense cubilin plasmid was identified and named pCDNA-ACUB or pCDNA-CUB. These two plasmids as well as pCDNA3.1(+) were transfected into HK2 cells with lipofectin DOTAP, respectively. Albumin uptake by HK2 cells were determined with fluorescence microscope and flow cytometer(FCM).3. After incubation with albumin or DMEM (albumin free) for 24h, HK2 cells were harvested to analyze the expression of cubilin mRNA by RT-PCR and the protein abundance of MCP-1 and RANTES by ELISA or Western blot. Results1. Under light microscopy, the patients with occulting hematuria were shown normal kidney structure; a small quantity of albumin deposition, basis expression of MCP-1 and RANTES within the PTECs cytoplasm, but few positive signals in the glomerulus and interstitium. On the contrary, the patients with nephrotic syndrome presented significant renal histologic changes including PTECs degeneration, atrophy, inflammatory cell infiltration, interstitial edema and fibrosis. A large amount of albumin deposition was displayed within the PTECs cytoplasm, moderate deposition in the glomerulus and interstitium, and very strong expression of MCP-1 and RANTES was only found within the PTECs.2. By digestion with restriction endonucleases and sequencing analysis, pCDNA-ACUB and pCDNA-CUB were identified, and the insert fragments were completely homogenous to cubilin cDNA sequence in GenBank. HSA-FITC uptake in HK2 cells was inhabited by pCDNA-ACUB transfection when comparing with the cells harboring pCDNA3.1(+) or pCDNA-CUB.3. Cubilin mRNA expression in PTECs was up-regulated by albumin with a dose-dependent manner. Both MCP-1 and RANTES expression were significantly increased in HK2 cells after incubation with 10g/L albumin, while was obviously suppressed by pCDNA-ACUB trasnfection.Conclu...
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