mRNA Expression of Extracellular Matrix Proteins in Renal in vitro Models of Metabolic Acidosis and Hyperglycaemia

Abstract

Introduction: Extracellular matrix proteins (ECM) deposition has been associated with tubulointerstitial fibrosis, leading to end-stage renal failure (ESRF). Several factors cause the extreme synthesis of ECM in the kidneys, including diabetes and metabolic acidosis. However, the detailed mechanisms leading to abnormal accumulation of ECM remain to be elucidated. Aim: To determine the effect of acidosis and hyperglycaemia on mRNA expression of MMP-2, MMP-9, collagen 1a1, collagen 4a1, TIMP1, and PAI-1 in renal proximal tubular epithelial cell (RPTEC). Method: RPTEC (HK-2) cells were cultured in 10% FCS-supplemented growth medium in 6-well plates until 80% of confluency was reached. Following this, cells were growth arrested for 24h in a serum-free medium and then exposed to acidosis (pH 6.7) or hyperglycaemia (25mM D-glucose, 30mM D-glucose) with separate controls for 24h and 72h, respectively. The cells were then harvested, and total RNA was isolated. The mRNA was converted to cDNA, and the expression of the target genes was quantified using qRT-PCR. GAPDH was used for expression of gene expression analysis, and the ∆∆Ct method was used to present the gene expression in terms of fold change. Result: Acidosis increased the mRNA expression of all ECM proteins; however, only the PAI-1 mRNA expression significantly increased. In hyperglycaemia, variable mRNA expression of the ECM proteins was measured over 72 hours. Conclusion: This study has shown that acidosis affects RPTEC as it increases the accumulation of all markers, which may lead to tubulointerstitial fibrosis. Hyperglycaemia had a variable effect on the expression of the ECM genes, suggesting that hyperglycaemia may lead to an increase in the deposition of ECM and then renal failure.