Chronic high glucose inhibits albumin reabsorption by lysosomal alkalinization in cultured porcine proximal tubular epithelial cells (LLC-PK₁)

Abstract 

Lysosomal acidification is a key step of albumin reabsorption in proximal tubular epithelial cells (PTECs). This study was performed to examine the influence of chronic high glucose on lysosomal acidification in cultured PTECs. Porcine PTECs (LLC-PK₁ cells) were cultured in 16.7mM (300mg/dl) glucose (HG) alone or with 0.5mM phlorizin for 24 weeks and subsequently for 12 weeks in 5.5mM (100mg/dl) glucose (NG). Chronic HG inhibited the fluorescein isothiocyanate (FITC)-albumin (A) uptake progressively, while phlorizin reversed the inhibition. NG for 12 weeks after HG normalized the uptake. The time-dependent uptake of FITC-A was inhibited by HG and bafilomycin A₁ (BafA₁) after 15min and by 4,4′-diisothiocyanato-2,2′-disulfonic acid (DIDS) and N-ethyl-N-isopropyl-amiloride (EIPA) after 3min. Cellular ATP was depleted by HG and restored by NG. Lysosomal pH, assessed by an acidotropic fluorescent probe, was alkalinized (pH 4.5–7.8) with 5.5–27.8mM glucose and normalized by subsequent NG. BafA₁ alkalinized lysosomes, and the concentration required to 50% change for the pH and 50% inhibition of FITC-A uptake was similar. EIPA inhibited FITC-A uptake, but did not influence lysosomal pH. DIDS inhibited FITC-A uptake, and unexpectedly lowered lysosomal pH. Real time PCR showed that HG reduced the mRNA level for vacuolar H⁺-ATPase, but did not alter those of chloride channel-5 and Na⁺–H⁺-exchanger-3. In conclusion, the chronic HG inhibits albumin reabsorption by lysosomal alkalinization in PTECs, probably due to ATP depletion and down-regulation of vacuolar H⁺-ATPase.

Keywords: Lysosomal alkalinization, High glucose, Proximal tubular epithelial cells, Acidification machinery, Albumin reabsorption