Individually microdissected nephron segments of defined epithelial origin from human kidneys were cultured in vitro in the present studies. Nephron segments of proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb of Henle, and cortical collecting tubule were grown in defined media. Each cell type retained differentiated characteristics as assessed by ultrastructural morphology, marker enzyme profiles, and adenylate cyclase response to selected hormones. These studies demonstrate the feasibility of using primary cultures of well-defined segments of the human nephron to study human renal tubular epithelia in vitro.

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Human polycystic kidney disease (PKD) epithelia were successfully grown in culture and expressed abnormal characteristics. Cysts lining epithelia of superficial and deep cysts were microdissected and compared to individual normal human proximal straight tubules (PST) and cortical collecting tubules (CCT) grown in defined media. PKD cyst epithelia differed from normal renal tubular epithelia in growth patterns and structural and functional properties. PKD epithelia grew more rapidly and showed cyst-like areas in otherwise confluent monolayers. Polygonal and elongate cells contained an epithelial-specific cytokeratin antigen and had polarized morphology. An extremely abnormal basement membrane morphology was seen and consisted of some banded collagen and numerous unique blebs or spheroids. These blebs were apparently extruded from intracellular vacuoles and stained with ruthenium red, suggesting a proteoglycan component. Cytochemistry of marker enzymes demonstrated the presence of NaK-ATPase and alkaline phosphatase, but a lack of gamma-glutamyl transpeptidase. The response of adenylate cyclase activity to vasopressin, parathyroid hormone, and forskolin was significantly diminished in PKD cells as compared to PST and CCT. These studies suggest a defect in cell growth and basement membrane synthesis in human PKD. Cultured PKD epithelia provide a new tool for the study of the pathogenesis of this disease.

The mechanism(s) whereby a denuded renal tubular epithelial cell surface becomes reestablished remains unknown. We therefore measured the rate of renewal of mechanical wounds made in confluent monolayers of two established renal tubular epithelial cell lines. We found that wounds of MDCK cells heal at a faster rate than wounds of LLC-PK1 cells. The magnitude of wound healing did not differ when cells grown on plastic were compared with cells grown on fibronectin, laminin, or collagen. Irradiation (4,000 rads) of MDCK and LLC-PK1 cells significantly reduced indexes of proliferation (5-bromo-2'-deoxyuridine and thymidine uptake) without affecting wound healing. Serum and epidermal growth factor (EGF) enhance whereas transforming growth factor-beta 1 (TGF-beta 1) impairs wound healing. Hepatocyte growth factor (HGF) stimulates wound healing at low concentrations and inhibits healing at high concentrations in MDCK cells while not affecting healing of LLC-PK1 cell wounds at any concentration. Several interleukins (IL-1, IL-2, IL-3, and IL-6) did not affect wound healing in either cell type. Healing of LLC-PK1 but not MDCK cells was impaired by exposure to a peptide containing a RGD sequence. Conversely, healing of MDCK but not LLC-PK1 cells was impaired by the REDV tetrapeptide. Healing of both LLC-PK1 and MDCK was impaired by heparin but not by the LDVPS peptide. These results demonstrate that mechanical wounds of LLC-PK1 and MDCK cells heal, at least in part, by migration. Healing is regulated by serum and growth factors including EGF, HGF, and TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)