Deborah A. Cieslinski

Interaction between growth factors and retinoic acid in the induction of kidney tubulogenesis in tissue culture

Kidney tubulogenesis is the initial step in renal organogenesis. The precise molecular determinants of this pattern formation are presently unknown, although soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. To define the molecular determinants of renal proximal tubule morphogenesis, primary cultures of rabbit renal proximal tubule cells in hormonally defined, serum-free media were treated with transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), and the retinoid, all trans-retinoic acid (RA), singly or in combination. Utilizing phase contrast and light and transmission electron microscopy, the simultaneous administration of TGF-beta 1 (10 ng/ml), EGF (1 nM), and RA (0.1 nM) transformed a confluent monolayer of renal proximal tubule cells within 5 to 6 days into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border. All three factors were necessary and sufficient to induce this phenotypic transformation. Further studies demonstrated that RA promoted the deposition of the A and B1 chains of laminin, a cell attachment protein of the basement membrane, in a small subset of proximal tubule cells in culture, as deduced by indirect immunofluorescent microscopy. Additional studies demonstrated that soluble purified laminin fully substituted for RA in this system to promote renal tubulogenesis when combined with TGF-beta 1 and EGF. These results demonstrate that the growth factors, TGF-beta 1 and EGF, and the retinoid, RA, promote tubulogenesis in adult renal proximal tubule cells in tissue culture in a manner reminiscent of inductive embryonic kidney morphogenesis. These observations define a coordinated interplay between growth factors and retinoids to induce pattern formation and morphogenesis. Furthermore, the demonstration of RA-induced laminin deposition as a critical event in this morphogenic process identifies laminin as a possible target protein for RA to act as a morphogen.

Comparison of toxicity of radiocontrast agents to renal tubule cells in vitro.

We have previously reported that radiocontrast agents induce direct renal tubule cell toxicity in vitro. The observed toxic effects were markedly potentiated by concomitant hypoxia. In addition, we have reported that the ionic radiocontrast agent diatrizoic acid is more toxic than the nonionic radiocontrast agent iopamidol in this system. Using suspensions enriched in rabbit renal proximal tubule segments, we compared the direct toxicities of the ionic dimeric ioxaglic acid to the nonionic monomeric compound iopamidol. Toxicity was assessed by comparing tubule potassium and calcium content, ATP levels, and respiratory rates after exposure to clinically achievable concentrations of radiocontrast agents. Ioxaglate (25 mM) produced significant declines in tubule cation content and respiratory rate with 30 min of hypoxia followed by 60 min of reoxygenation compared to molar-equivalent concentrations of iopamidol under similar conditions. Meglumine, a cationic compound frequently present in ionic contrast agent solutions, and ioxaglate tubule toxicity was additive. Iopamidol and ioxaglate exhibited similar tubule cell toxicity when comparison was based on iodine content. These experimental results suggest that the intrinsic nephrotoxic potential of ioxaglic acid is greater than that of iopamidol on a molar basis, but that the nephrotoxic potential of the two radiocontrast agents is similar when comparison is based upon iodine content.

Adenosine Triphosphate Stimulates Thymidine Incorporation but Does Not Promote Cell Growth in Primary Cultures of Renal Proximal Tubule Cells

Acute addition of adenosine triphosphate (ATP) stimulated thymidine incorporation in confluent, quiescent primary cultures of rabbit renal proximal tubule cells in a dose-responsive manner. Similar increases in thymidine incorporation was observed with adenosine diphosphate and adenosine monophosphate but not with adenosine. The effect of chronic administration of ATP, however, suppressed cell growth. This suppression appears to be due to an effect of ATP to cause detachment of cells from culture plates, resulting in an increase in thymidine incorporation acutely but in suppression of cell growth chronically. ATP is, therefore, not a direct growth promoter of renal proximal tubule cells in primary culture.