ClC-5, with Megalin (LRP2) and Cubilin, is part of the molecular complex involved at proximal tubular level in the endocytic re-uptake of low-molecular-weight (LMW) proteins and albumin too.
We previously demonstrated the presence of CLCN5 mRNA in microdissected glomeruli of patients with NIDDM.
Since human mesangial cells (HMC) are main actors of the histological alterations in DN, we performed an in vitro study mimicking diabetic environment in order to evaluate whether hyperglycemia was able to modulate the expression of the components of the macromolecular system involved in protein uptake at glomerular level.
HMC were cultivated with/without additional D-glucose (final concentration 30 mM) for 12, 24, 48 and 72h. Experiments were conducted in duplicate. Real Time PCR for CLCN5, LRP2, Cubilin and housekeeping genes was performed on HMC RNA according to MIQE guidelines. Data were analyzed with the ΔΔCt method. Statistical analysis was performed using Student’s t Test.
Using Real Time PCR we detected CLCN5, LRP2 and Cubilin mRNA expression in control HMC. HMC grown in high glucose media disclosed a significant increase in CLCN5 and Cubilin mRNA and a down-regulation in LRP2 at every time point analyzed (CLCN5 48, and 72h and Cubilin 12, 48 and 72h p<0.05; CLCN5 12 and 24h Cubilin 24h and LRP2 p<0.01) (Figure 1).
For the first time we identified CLCN5, LRP2 and Cubilin expression in human mesangial cells.
Interestingly, the diabetic environment modulates the expression of the components of the macromolecular system involved in protein uptake. In particular, glucose is able to modify in a different manner the expression of the two receptors LRP2 and Cubilin.
Since Cubilin is known to perform endocytosis without LRP2 cooperation in small intestine, the up-regulation of CLCN5 and Cubilin and the down-regulation of LRP2 may suggest a role for Cubilin without LRP2 in HMC too.
