Isolation of a pure suspension of rat proximal tubules

Abstract

A suspension of cortical tissue fragments prepared by collagenase digestion of renal cortex obtained from fed and chronically acidotic (NH4Cl) rats was separated into four bands on a Percoll density gradient. By microscopic examination, vital staining with trypan blue, and histologic staining technique (periodic acid-Schiff) the F4 band was shown to contain only (greater than 98%) proximal tubules, whereas the F1 band was significantly enriched (70%) with distal tubules contaminated by glomeruli and short segments of proximal tubules. Intra/extracellular ratios for PAH of 15 were measured in the F4 band and of 2 in F1 band. ATP was 1.4 and 2.8 mumol/g in the F4 and F1 bands, respectively, and was stable for at least 60 min. The proximal F4 band was shown to be gluconeogenic (L-glutamine or L-lactate 2.5 mM as substrate) and to adapt to metabolic acidosis. The distal F1 band was shown to be glycolytic (glucose 2.5 mM) with no changes with acid-base status. All fractions were shown to metabolize glutamine, but the metabolic fate of this amino acid was different in proximal and distal structures. A F4/F1 activity ratio for the proximal cytoplasmic phosphoenolpyruvate carboxykinase enzyme of 2.6 and 4.3 was observed in normal and acidotic rats, respectively. In contrast, a F4/F1 ratio of 0.13 and 0.22 was observed for the distal cytoplasmic hexokinase enzyme. This preparation, therefore, allows the metabolism of a homogeneous population of proximal tubular fragments to be studied and can be used to obtain information on enzyme location within the nephron.

NFATc1 Identifies a Population of Proximal Tubule Cell Progenitors

“…PTCs were isolated using modification of the methods of Vinay et al 29 and as modified by Xu et al 30 Briefly, cortices from HgCl 2 -treated WT or Nfatc1 ϩ/Ϫ mice were collected and minced in Krebs-Hensleit saline (KHS) buffer containing 115 mM NaCl, 24 mM NaHCO 3 , 10 mM HEPES, 5 mM glucose, 5 mM KCl, 2 mM NaH 2 PO 4 , 1.5 mM MgSO 4 , and 1 mM alanine. This solution was then enriched with 0.15% (wt/ vol) collagenase type I, 0.5% (wt/vol) BSA, and 0.01% soybean trypsin inhibitor and incubated at 37°C for 1 h to digest the cortices.…”

Renal Ischemia/Reperfusion Injury

“…Proximal tubular epithelial cells (PTEC) were obtained from rat kidney cortices that were subjected to 45 min of ischemia and 3 h of reperfusion or sham operation, following the process described by Vinay et al (25) with some modifications. Briefly, renal cortices were incubated in gassed Krebs-Henseleit saline (pH 7.4) that contained 0.1 mg/ml collagenase type I (Sigma, St. Louis, MO) at 37°C for 1 h and sieved throughout 0.250-to 0.035-mm mesh.…”

Developmental Changes in Proximal Tubule Tight Junction Proteins

“…Rat proximal tubules were prepared using the same protocol previously used by our laboratory (15,16), according to the method described by others (17)(18)(19). Briefly, neonatal and adult rats were killed, and their kidneys were removed and the cortex was dissected, minced, and placed in Kreb's solution (4°C) that contained (in mM) 105 NaCl, 24 NaHCO 3 , 5 KCl, 1.5 CaCl 2 , 1.0 MgSO 4 , 2.0 Na 2 PO 4 , 5.0 glucose, 1.0 alanine, 4.0 lactate, 10.0 HEPES, and 0.2% BSA adjusted to 300 mOsmol/kg H 2 0 and that was gassed with 95% CO 2 /5% O 2 (pH 7.4).…”