Structure and function of glomerular epithelial cells in experimental lupus nephritis

Abstract

The slit diaphragms (SD) that cover the slit pores among adjacent foot processes (FP) of the podocytes are crucial constituent of the primary barrier for ultrafiltration of plasma in renal glomeruli. A putative distraction of the SDs, of its main components, nephrin and podocin is involved in the immune mediated glomerulonephritis with proteinuria. We undertook an animal and human study to determine the expression of nephrin and podocin in the various forms of lupus nephritis (LN), a prototype of autoimmune disease with a various histological and clinical picture with proteinuria been a major clinical manifestation. The kidneys were examined by light microscopy (LM), and IgG immunofluorescence in order to determine the LN characteristics. Electron microscopy was also performed. In view of electron microscopy findings, we next assessed the glomerular expression of total nephrin and podocin at the protein level by western blot analysis in normal (n=11), MMLN: mild mesangial hyperplasia (n=5), FPLN: focal (n=6) and DPLN: diffuse (n=4) proliferative nephritis in NZB/W females and of age and sex matched C57BL/6 controls (n=24) as well as in NZB (n=5) and NZW (n=4) mice. Nephrin expression was also examined by immunofluorescence in kidney biopsy specimens. To determine whether alterations to protein expression of nephrin and podocin are due to altered transcription of the corresponding genes, we performed quantitative real-time PCR in the same kidney samples. In order to verify our findings in murine LN we performed immunohistochemistry for nephrin expression in renal tissue from patients with various classes of LN: class II (n=5), IV (n=4), V (n=7) and healthy controls (n=2). Data are expressed as the mean ± standard error of the mean (SEM) value. Comparisons were performed with the Mann–Whitney U-test (numerical data) or the Fisher?s exact test (categorical data). The SD alterations determined by electron microscopy correlate with histology in NZB/W F1 mice. The severity of mesangial, subendothelial and subepithelial deposits, as well as FP effacement findings were significantly associated with 24-hr proteinuria (ρ = 0.85, p<0.003) 6 and IgG intensity (ρ = 0.90, p<0.001). Western blot and immunofluorescence showed significantly reduced nephrin protein expression in mice with FPLN or DPLN (p<0.05 for all pair-wise comparisons). Podocin protein expression was also significantly decreased in the same groups (p<0.05). Both proteins were significantly reduced in NZB/W mice with extensive FP effacement (p<0.05 for both). Glomerular nephrin and podocin mRNA levels were significantly reduced in DPLN - but not in FPLN - (p=0.034). We found a good correlation between glomerular nephrin and podocin mRNA levels in all NZB/W mice (ρ = 0.77, p<0.001). One NZB mouse showed focal proliferative nephritis while the rest NZB mice and all the NZW mice showed normal LM findings however, they exhibit significant 24-hr proteinuria. Podocin protein was reduced in the NZB and in the NZW as compared to NZB/W with normal LM findings (p= 0.024, p=0.030 respectively) and with MMLN (p= 0.034 for both NZB and NZW). Significantly more patients with DPLN had reduced nephrin expression as compared to patients with class II or V LN (p < 0.05). Our study represents the first comprehensive analysis of podocin and nephrin expression at protein and mRNA level coupled with immunofluorescence findings in murine and human LN. Glomerular nephrin protein levels were reduced by almost 50% in mild LN (MMLN) and became diminished at more advanced stages (FPLN/DPLN). Podocin is affected only in the most serious forms of the disease (FPLN/DPLN). Nephrin and podocin protein expression correlate with the severity of the disease histology. This coupled with the significant reduction of nephrin and podocin glomerular mRNA only in the most advanced stages (DPLN) may imply that the SD alterations at the protein level precede the alterations in the mRNA level. The mRNA level is finally affected only in the most sever forms of the disease. The previously regarded healthy ancestors of the NZB/W, especially the males NZW were found with ultrastructural SD alterations regarding reduced podocin which might is a co-factor contributing to NZB/W predisposition to renal disease. We found significantly reduced nephrin expression in patients with class IV proliferative disease as compared to class V patients, despite both groups having comparable levels of proteinuria. These results confirm our findings in murine LN, suggesting that nephrin expression correlates better with the histological class than with the level of proteinuria. Our findings suggest a novel role of podocytes and their structures in immune-mediated nephritis