Isolation and Culture of Renal Cortical Tubules From Neonate Rabbit Kidneys

Abstract

SUMMARYA method is described for the preparation of an enriched population of proximal tubules from the cortices of neonate (21-28 d old) rabbits. The method uses collagenase-hyaluronidase digestion, followed by gentle shear to yield a suspension of tubules and glomeruli. Tubular enrichment is achieved by discontinuous density gradient centrifugation in a Percoll gradient. Two fractions were obtained by this method. The denser fraction contained predominantly proximal tubules, was depleted of glomeruli and was enriched in the proximal tubule marker enzyme alkaline phosphatase. Qualitatively similar results to those obtained with neonate animals were obtained with adult tissue. Neonate tubules from the denser gradient fraction grew readily in tissue culture. When examined by electron microscopy the cells exhibited a marked polarity of ultrastructural organization and retained apical tight junctions. Despite this, there was an obvious loss of structure in comparison with in vivo proximal tubule cells. The use of primary culture techniques to study in vitro renal epithelial function is discussed.

Restricted growth of rat kidney proximal tubule cells cultured in serum‐supplemented and defined media

“…On a TCA-insoluble protein basis, the specific activity of AP after 2 days culture in HDM medium was decreased 41% in the rat primary cultures and 44% in the rabbit cultures relative to the purified PT preparation (specific data not presented). These decreases in enzyme activity are similar to those observed in other cell culture studies for AP as well as for other PT markers such as y-glutamy1 transpeptidase and leucine aminopeptidase (Chung et al, 1982;Curthoys and Bellemann, 1979;Richardson et al, 1982).…”

“…The Ficoll gradient purification scheme developed in this study for rat PTs was simple to use, produced sufficient material for cell culture, and gave comparable purification results to other reported procedures using Ficoll and Percoll gradients (Eveloff et al, 1980;Richardson et al, 1982;Scholer and Edelman, 1979). The final tubule fraction, however, was less enriched for PT marker enzymes than that reported by Vinay et al (19811, who utilized a continuous Percoll gradient.…”

“…Initiation of primary cultures of cells from specific nephron regions has been attempted by culturing individual microdissected segments (Horster et al, 1985;Horster, 1979;Wilson et al, 1985;Wilson and Horster, 1983). A more classical approach which has the advantage of providing sufficient material for biochemical analysis is to use cell or tubule preparations purified from a particular nephron segment by differential centrifugation in gradients of Ficoll (Eveloff et al, 1980;Scholer and Edelman, 1979) or Percoll (Richardson et al, 1982;Vinay et al, 1981).…”

Stress Fibers in situ in Proximal Tubules of the Rat Kidney.

“…For immunoblotting the electrophoresed polypeptides were transferred onto Hybond-C (Amersham) mem- Contraction of isolated tubules. Renal tubular segments were prepared enzymatically from kidney cortices as described previously (22). Such isolated tubules were placed in a chamber madeof polylysin-coated glass slides, coverslips and spacers, treated with 1% Triton X-100, 0.1 M KC1 in 10mM sodium phosphate buffer, pH 7.2 (19) for 10min on ice and then washed with 0.1 M KC1, 10 mMsodium phosphate buffer at pH 7.2 (contraction buffer).…”

EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases

“…The isolation procedure for renal tubules was according to that of Richardson et al (Richardson et al, 1982) with some modifications. Kidneys were minced and digested with collagenase (0.02%, Type II, Sigma-Aldrich Japan K.K.)…”