Human renal cells from the thick ascending limb and early distal tubule: Characterization of primary isolated and cultured cells by reverse transcription polymerase chain reaction

Abstract

Human renal tubular cells of well-defined nephron origin are an important basis in the research of various physiological and pathophysiological mechanisms in the kidney. Whereas an exceeding amount of data has been obtained on proximal tubular cells, only limited data of cells of the human thick ascending limb and the early distal tubule (TALDC) are available. TALDC have been isolated immunomagnetically according to their specific antigen expression of Tamm-Horsfall glycoprotein (THG). Cells were either directly processed for analysis or cultured under normal cell culture conditions. Differentiation of primary isolates and cultured cells was assessed by reverse transcription polymerase chain reaction using characteristic markers. As controls, we used RNA from whole human kidney and cultured HK-2 cells. Additional characterizations were made by morphological analysis and western blotting. Primary isolated TALDC express the characteristic markers epidermal growth factor receptor, Na-K-2Cl transporter 2, epithelial calcium canal, and THG but were negative for Pax-2, aquaporin-2 and -3. Cultured TALDC were positive for epidermal growth factor receptor and Na-K-2Cl transporter 2 but have lost their epithelial calcium canal and THG expression and started to express Pax-2. All probes were positive for the specific markers kidney-specific cadherin and cytokeratin-8. Furthermore, differentiation of cultured TALDC was shown by cell morphology and their characteristic protein expression pattern. Our results highlight the purity of primary isolates and the differentiation of cultured TALDC, and show that they can be used as an in vitro system studies of the human thick ascending limb of Henle's loop and early distal tubule.