ClC-3 is broadly expressed in many organs. Although ClC-3 was the first intracellular CLC to be cloned, it is subject to many controversies in respect to both its basic biophysical characteristics and its physiological functions. ClC-3, with ClC-4 and ClC-5 are located in proximal tubules residing predominantly on intracellular organelles, in particular in the endosomal pathway involved in albumin re-uptake.
In order to better understand ClC-3 role in kidney we analyzed its expression in kidney biopsies and renal cells.
Immunofluorescence for ClC-3 ((K-17) Santa Cruz) was performed on kidney biopsies of patients with glomerulonephritis. Real Time PCR for CLCN3 and housekeeping genes was performed on human mesangial (HMC) and proximal tubular (RPTC) cells RNA according to MIQE guidelines. HMC were grown with/without BSA (10 and 30 mg/ml) or D-glucose (final concentration 30 mM) for 12, 24, 48 and 72h. Statistical analysis was performed using Student’s t Test.
ClC-3 proteins was identified both at tubular and glomerular level of patients with glomerulonephritis (Figure 1). In the glomerular compartment, ClC-3 staining was located in the cytoplasm of cells that looked like mesangial cells judging from their morphology and localization.
RPTC are known to express CLCN3. Using Real Time PCR we confirmed CLCN3 presence in HMC, intriguingly at similar level of RPTC.
Cultivating HMC in proteinuria like conditions we observed, at concentrations of 10 and 30 mg/ml, a significant increase of CLCN3 at 48 and 72h (p<0.05 48h; p<0.01 72h). In high glucose environment, we disclosed a significant increase in CLCN3 at every time point evaluated (p<0.05).
For the first time we demonstrated the presence of ClC-3 in human glomeruli, in particular in HMC.
CLCN3 up-regulation due to albumin and glucose suggests the endosomal pathway involvement in mesangial cells and open new perspectives of its role in the glomerular compartment.
