Improved separation method for rat proximal and distal renal tubules

Abstract

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

“…PTH caused concentration-dependent cyclic AMP increases ( l0-9 M PTH: P < 0.01 compared with control), arginine vasopressin caused no change in cyclic AMP, and All elicited concentration-dependent cyclic AMP decreases (10-6 M All: P = 0.09 compared with control). These data were consistent with known proximal tubule cell responses to each hormone ( 12,(27)(28)(29). Further elucidation of proximal tubule cell function was provided by 22Na transport studies.…”

Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ

“…Our conclusion is that PT isolated by traditional methods indeed can carry with them endothelial cell contamination, in addition to being up to 5% contaminated with other types of epithelial cells (that may express caveolins) (17). It is, however, possible that some isolation procedures may produce more highly purified tubules (20). Indeed, neither caveolins nor the endothelial markers ICAM1 and CD31 were reported in a recent systematic transcriptome analysis (http://dir.nhlbi.nih.gov/papers/lkem/pttr/ Default.aspx) performed on isolated rat PTs (53).…”

Angiotensin II Type 2 Receptor Agonist Directly Inhibits Proximal Tubule Sodium Pump Activity in Obese But Not in Lean Zucker Rats

“…14,15 Rats were anesthetized with sodium pentobarbital (50 mg/kg, IP). After a midline incision, selective perfusion of the kidneys was performed with modified Krebs-Hensleit buffer containing collagenase type IV (230 U/mL) and hyaluronidase type III (250 U/mL).…”