A primary culture of mouse proximal tubular cells, established on collagen-coated membranes

Abstract

A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures.

Impaired Lysosomal Function Underlies Monoclonal Light Chain–Associated Renal Fanconi Syndrome

“…These cells, which keep their differentiation and polarized transport processes, provide a particularly well suited model to investigate receptor-mediated endocytosis of apical ligands and their lysosomal processing. 13,22,23 We show that specific RFS-kLCs accumulate within lysosomes of PT cells, causing marked alterations in cell dynamics and lysosomal function. In turn, these alterations promote dedifferentiation of the cells and defective receptor-mediated endocytosis, sustaining the loss of reabsorptive capacity of the kidney tubule.…”

“…13,22 The mPTCs were exposed to low doses (25 mg/ml) of human kLCs associated with RFS (kCH or kDU), cast nephropathy (kRO), AL amyloidosis without RFS (kDE), or a mutant version of kCH (kCHm) (Supplemental Figure 2, A and B, Table 2). kCHm was obtained by directed mutagenesis of kCH, replacing the hydrophobic Ala at position 30 in V domain by the polar Ser that prevents crystal formation in PT cells.…”

Ultrastructural Aspects of Rat Renal Tubular Epithelium <i>in Vitro</i>: Scanning Electron Microscopy （SEM） Analyses at Various Stages of Culture

“…The major findings were : 1 a large number of small sphere-like structures and / or microvilli were apparent on the cell surface of the floating epithelium ; 2 cells with spherical structures, as well as cells with microvilli, were both capable of developing leaf-like structures after attaching to a cell culture vessel ; 3 the leaf-like structures resembling lamellipodia developed circumferentially at an early stage of culture ; and 4 long and branched filopodia developed which were much larger in diameter than the filopodia that developed at earlier stages. To our knowledge, small spherical structures on the cell surface of renal tubular epithelium have not been described previously, although SEM techniques have been used in cellular biological studies 20,21 . The coexistence of microvilli with spherical structures Fig.…”

Overproduction of phosphoprotein enriched in diabetes (PED) induces mesangial expansion and upregulates protein kinase C-β activity and TGF-β1 expression

“…Tissues were snap-frozen in liquid nitrogen and stored at −80°C for subsequent western blot analysis. Mouse tubular epithelial cells were isolated as previously described [36]. Cells were grown until confluent (8 to 12 days) in RPMI 1640 medium supplemented with 20% fetal calf serum, 2% (wt/vol.)…”