Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. I. An improved method for preparation of proximal tubular cells and their functional characterization by alpha-methylglucose uptake

Toxicol Appl Pharmacol. 1989 Oct;101(1):135-43. doi: 10.1016/0041-008x(89)90219-6.

Abstract

Rat renal proximal tubular cells were isolated by successive EGTA and collagenase perfusions and purified by filtration and isopycnic centrifugation. The method is rapid and provides a much higher fraction of proximal tubular cells (90-95%) than comparable methods. The yield of viable (97 +/- 3%) cells is high (30 X 10(6) cells/g kidney). The intracellular ATP was 16 +/- 2 nmol/mg protein and remained essentially constant for at least 3 hr. The cells were characterized for transport of organic ions and glucose. Glucose transport was studied by alpha-[14C]methylglucose uptake; apparent Km and Vmax values were 3.4 +/- 0.5 mM and 4.1 +/- 0.9 nmol/min.mg protein, respectively. This transport could almost be completely inhibited by phloridzin, indicating that the uptake is mediated by the brush border glucose carrier.

MeSH terms

  • Adenosine Triphosphate / analysis
  • Animals
  • Biological Transport, Active
  • Cell Separation / methods*
  • In Vitro Techniques
  • Kidney Tubules, Proximal / cytology*
  • Kidney Tubules, Proximal / metabolism
  • Male
  • Methylglucosides / pharmacokinetics*
  • Methylglycosides / pharmacokinetics*
  • Microbial Collagenase
  • Niacinamide / analogs & derivatives
  • Niacinamide / pharmacokinetics
  • Perfusion
  • Rats
  • Rats, Inbred Strains
  • Time Factors
  • p-Aminohippuric Acid / pharmacokinetics

Substances

  • Methylglucosides
  • Methylglycosides
  • Niacinamide
  • methylglucoside
  • Adenosine Triphosphate
  • Microbial Collagenase
  • N-methylnicotinamide
  • p-Aminohippuric Acid