Isolation, Culture and Characterization of Human Renal Proximal Tubule and Collecting Duct Cells

Abstract

The complexity and heterogeneity of the human nephron with regard to cell types make well-defined in vitro systems of renal cells valuable for studies of the pathogenetic mechanisms involved in nephrotoxicity. In our laboratory renal proximal tubule cells (PTC) and collecting duct cells (CDC) have been isolated, cultured and characterized from cadaver kidneys (postmortem time <24 h) for use in studies of renal cytotoxicity induced by therapeutics and bacteria. PTC seeded at 10⁶ cells/ml formed confluent monolayers within 7 days. Histochemical markers were used to determine the origin of the cell cultures. Cells were negative for factor VIII, positive for cytokeratin and γ-glutamyltransferase (GGT), and bound winged pea lectin. CDC, isolated from the renal papillae, formed monolayers within 14 days of seeding. CDC were negative for factor VIII and GGT, positive for cytokeratin and bound peanut lectin. PTC and CDC isolates and cultures exhibited typical epithelial cell ultrastructure: cell junctions, intermediate filaments, microvilli, and numerous mitochondria. The morphological and histochemical evidence confirms that PTC and CDC can be isolated and cultured for use in in vitro studies.

The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker

“…The F4 band of the Percoll gradient, composed of proximal tubules, was carefully removed, washed and centrifuged. The final pellet was resuspended in serum-free, hormonally defined culture media [18] and seeded at a density of 1.5 mg pellet/cm 2 in six-well plates coated with rat tail collagen I and human fibronectin (Sigma). The culture medium was changed every 48 h. Infection was performed when cells reached confluence, covering the wells with 1 ml of recombinant adenovirus stock for 1.5 h at 37°C.…”

Diclofenac toxicity in Gyps vulture is associated with decreased uric acid excretion and not renal portal vasoconstriction

“…The following morning cell homogenates were passed through a 250 and 38 μm filter to isolate the RTE cells (Trifillis, 1999). The isolated cells were washed three times by centrifugation at 200 ×g with PBS and re-suspended in DMEM supplemented with 10% FCS, streptomycin and penicillin G (Trifillis, 1999).…”

Genomic profiling identifies alterations in TGFβ signaling through loss of TGFβ receptor expression in human renal cell carcinogenesis and progression

“…The gene names and corresponding GenBank number and chromosome location are listed to these relevant laboratory models, we harvested normal renal epithelial (NRE) tissue from nephrectomy specimens and established them as primary cultures (Trifillis, 1999). As shown in Figure 4a and b, these primary cultures of NRE express TBR3, TBR2, and TBR1 mRNA and protein in vitro.…”

“…As shown in Figure 4a and b, these primary cultures of NRE express TBR3, TBR2, and TBR1 mRNA and protein in vitro. NRE cells can be grown in culture for 10 passages and are easily isolated and characterized (Sens et al, 1999;Trifillis, 1999). We characterized NRE cells for cytokeratin expression and tubule-specific gene expression, for example, megalin [(data not shown; (Dedir et al, 1996;Trifillis, 1999;Terzi et al, 2000)].…”

“…NRE cells can be grown in culture for 10 passages and are easily isolated and characterized (Sens et al, 1999;Trifillis, 1999). We characterized NRE cells for cytokeratin expression and tubule-specific gene expression, for example, megalin [(data not shown; (Dedir et al, 1996;Trifillis, 1999;Terzi et al, 2000)]. Thus, we have relevant cell models in which TBR2 and TBR3 expression can be manipulated to examine the impact of TBR biology on human RCC carcinogenesis and progression, in vitro.…”