MP077
ClC-5 AND PROTEINURIC NEPHROPATHIES

Introduction and Aims: In the human kidney, ClC-5 is primarily expressed in proximal tubular cells, located in the subapical endosomes and is involved in the endocytic reabsorption of albumin and LMW proteins. For the first time we demonstrated that ClC-5 is expressed also in podocytes and that it was overexpressed in glomeruli of some proteinuric nephropathies suggesting that proteinuria may play a role in its expression. Studying healthy human tissues, we recently discovered that in the human kidney are present 11 different CLCN5 5’UTR isoforms. In particular, isoform A is predominantly and isoforms C (variants 6 and 7) exclusively expressed in kidney. To go deepen the role of ClC-5 on proteinuric nephropathy, we investigated mRNA expression of ClCN5 gene and its different 5’UTR isoforms as well as ClC-5 protein in renal biopsies of nephropathic patients with different degree of proteinuria.

Methods: We collected 14 membranous glomerulopathies (MG) (4,65g/die), 12 SLE (3,31g/die), 12 IgA nephropathies (IgAN) (3.02 g/d), 13 diabetic nephropathies (DN) (0.5g/die) and 11 controls (Ctrl). CLCN5 translated region, 5’UTR Isoform A and Isoforms C were analyzed by quantitative comparative RT-PCR; GAPDH was used as housekeeping gene. Indirect immunohistochemistry with primary anti-human ClC-5 Ab (Sigma) was performed and ClC-5 signal was quantified by morphometric analysis using Image Pro-Plus software (Media Cybernetics) at 200X magnification in glomerular and tubular compartments. Student’s t test and Regression analysis were performed. A p value ≤0.05 was considered statistically significant.

Results: Relative mRNA expression of CLCN5 translated region was higher in MG (p=0.005) than in Ctrl and DN, and in SLE (p=0.002) than in Ctrl. Isoforms A and C (variants 6 and 7), were also significantly higher in MG and in SLE than in Ctrl. Isoform A was more abundant than Isoforms C. Moreover, a significant direct correlation was found between Isoform A and Isoform C (variant 6) (r=0,64 p=0,000) evaluating all proteinuric nephropathy together.No significant correlation was found between proteinuria/die and ClCN5, Isoforms A and C expression.The study at protein level was focused on glomerular and tubular compartments and revealed that : 1)ClC-5 was significantly higher in IgAN (p=0,000) and MG (p=0,034) then in SLE both in glomeruli and tubules; 2) tubular and glomerular ClC-5 protein expression was directly correlated considering all the nephropathies (r=0,59 p=0,000); 3) proteinuria did not correlate with ClC-5 protein expression except in SLE with nephrotic syndrome where it correlated with glomerular ClC-5 expression (r=0,68 p=0,009).

Conclusions: Our study clearly confirms that glomeruli express ClC-5, but also reveals that ClC-5 expression is differently modulated at mRNA and protein level depending on the proteinuric nephropathies. The absence of a significant correlation between proteinuria/die and ClCN5 expression could be due to the low number of biopsies collected.