Involvement of the tubular ClC-type exchanger ClC-5 in glomeruli of human proteinuric nephropathies

Abstract

Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.

Aim: To investigate whether ClC-5 is expressed in the glomerular compartment and whether it has a role in proteinuric nephropathies. ClC-5 expression was studied using Real-time PCR in manually- and laser-microdissected biopsies from patients with type 2 diabetes (n 37) and IgA nephropathy (n 10); in biopsies of membranous glomerulopathy (MG) (n 14) immunohistochemistry for ClC-5 (with morphometric analysis) and for WT1 was done.

Controls: cortical tissue (n 23) obtained from unaffected parts of tumor-related nephrectomy specimens.

Results: ClC-5 was expressed at glomerular level in all biopsies. Glomerular ClC-5 levels were significantly higher in diabetic nephropaty and MG at both mRNA and protein level (p<0.002; p<0.01). ClC-5 and WT1 double-staining analysis in MG showed that ClC-5 was localized in the podocytes. ClC-5 ultrastructural immunolocalization was demonstrated in podocytes foot processes. Our study is the first to demonstrate that ClC-5 is expressed in human podocytes. The ClC-5 overexpression found in biopsies of proteinuric patients suggests that proteinuria may play a part in its expression and that podocytes are likely to have a key role in albumin handling in proteinuric states.

Figure 1. Real-Time quantification of ClC-5 in manually-microdissected biopsies of NIDDM and IgA patients.

mRNA level of ClC-5, expressed as the ratio between the starting quantity means (SQm) of the target and housekeeping genes, in manually-microdissected biopsies from NIDDM and IgA patients at glomerular (glom) and tubulo-interstitial (ti) level.

Figure 2. Histological picture of a laser microdissected biopsy.

Example of laser-microdissection pre- (A) and post- (B) cut, showing that the glomerular tuft can be separated without any tubulo-interstitial contamination (Ematoxilin staining).

Figure 3. Real-Time quantification of ClC-5 in laser-microdissected biopsies of NIDDM and controls.

mRNA level of ClC-5 in laser-microdissected glomeruli from NIDDM cases and controls. ClC-5 mRNA levels were significantly higher in NIDDM glomeruli than in control glomeruli (p<0.002). The relative expression levels of the gene of interest was obtained by dividing the gene’s expression level by the mean level of the different housekeeping genes. The relative levels of the controls were set to one.

Figure 4. Immunoistochemical analysis in MG biopsies.

A. Glomerular ClC-5 immunostaining in MG patient (400×). B. ClC-5 staining in a control glomerulus (400×). Black arrows indicate cytoplasmatic ClC-5 staining (400×). C. Proximal tubular cells used for internal control purposes showed ClC-5 apical and sub-apical staining (200×). D. ClC-5 quantification by morphometric analysis on MG and control glomeruli. ClC-5 signal was significantly higher in MG than in control samples (p<0.01). The quantity was expressed as the mean of the area covered by pixels, as a percentage. The average of the morphometric data in each group of biopsies is reported. E. Absence of glomerular ClC-5 staining in a patient with Dent’s disease (400×). F. Negative control obtained by incubating the sample with no primary antibody or with nonimmune rabbit IgG (400×).

Figure 5. Double staining for ClC-5 and WT1 in MG and control biopsies.

Co-localization of ClC-5 (cytoplasmatic brown stain) and WT1 (nuclear blu-grey stain) in control (C) and in MG (A) glomeruli to identify podocytes (400×). B. Oil image immersion of a detail of panel A (1000×).

Figure 6. Immunolocalization of ClC-5 in ultrastucture of MG biopsies.

A. Transmission electron microscopy (TEM) of MG biopsy revealed small vesicles in the secondary foot processes of podocytes (direct magnification 9000×). B–C. Ultrastructural immunolocalization of ClC-5 in MG biopsy. Arrows indicate some of the gold particles located in podocytes (direct magnification 40000×).