Isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by CD10/CD13 double labeling

Abstract

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

Figure 1. Sorting proximal tubular cells using specific antibodies.

(A) Fluorescence plot showing cells labeled with antibodies against CD10 (APC: allophycocyanin) and CD13 (PE: phycoerythrin). FACS analysis revealed about 4% double-positive cells. (B) Fluorescence plot showing cells treated with isotypes to both antibodies to determine the upper threshold for non-specific fluorescence.

Figure 2. Representative morphology of primary CD10/CD13 double-negative cells, CD10/CD13 cells double-positive, CD13⁺ and CD10⁺cells.

(A) Primary cultures at passage 2 in serum-free medium. (B) Primary cultures at passage 2 in medium with 10% FBS. Magnification: ×100.

Figure 3. Expression of differentiation markers in different cell populations.

Representative western blots for (1) unsorted cells, (2) CD10/CD13 double-positive cells, (3) CD10⁺ cells, (4) CD13⁺ cells and (5) CD10/CD13 double-negative cells. Blots were incubated with antibodies against aquaporin-1, N-cadherin, MUC1. The β-actin protein was used as an internal control. Proteins were extracted from cells at passage 2.

Figure 4. Immunofluorescence detection of specific markers in (A) CD10/CD13 double-positive cells and (B) CD10/CD13 double-negative cells.

Actin was labeled by incubation with a phalloidin-FITC solution. Cells were labeled with antibodies to pan-cytokeratin, β-catenin, vimentin, aquaporin-1, E-cadherin and MUC1 (all Texas Red-conjugated). DAPI was used to counterstain nuclei. Magnification: ×200. The grey squares in the left panel images indicate the region of high magnification shown in the right panel. Experiments were performed with cells at different passages.

Figure 5. Immunofluorescence detection of specific markers in (A) CD13⁺ cells and (B) CD10⁺ cells.

Actin was labeled by incubation with a phalloidin-FITC solution. Cells were labeled with antibodies to pan-cytokeratin, β-catenin, vimentin, aquaporin-1, E-cadherin and MUC1 (all Texas Red-conjugated). DAPI was used to counterstain nuclei. Magnification: ×200. The grey squares in the left panel images indicate the region of high magnification shown in the right panel. Experiments were performed with cells at different passages.

Figure 6. Ultrastructural morphology of cells.

PT cells at passage 4 were seeded onto (A) uncoated transwell filters (×20 000), (B, C) collagen IV-coated filters (×20 000, ×140 000) and (D) Matrigel®-coated filters (×12 000). CD10/CD13 double-negative cells at passage 4 were seeded onto (E) uncoated transwell filters (×12 000) and (F) collagen IV-coated filters (×20 000). PT cells displayed a polarized morphology and exhibited tight junctions (TJ), long microvilli (M) and desmosomes (D). CD10/CD13 double-negative cells displayed a polarized morphology and exhibited tight junctions and short microvilli.

Figure 7. Functional characteristics of CD10/CD13 double-negative cells and PT cells.

(A) Transepithelial electrical resistance (TEER) measurements were performed in CD10/CD13 double-negative cells and PT cells. Cells at passage 3 were seeded onto uncoated (plastic) transwell filters (white bar), collagen IV-coated filters (black bar) or Matrigel®-coated filters (grey bar). (B) The MDCK cell line was used as positive control, and cells were seeded onto uncoated transwell filters. The TEER was recorded at the points indicated (d: day). Means ± SD of three experiments are reported. *: p<0.05 and **: p<0.001 compared with similar results for cells seeded on plastic. (C) Alkaline phosphatase activity measured in PT cells at passage 5 and in CD10/CD13 double-negative cells at passage 5. Means ± SD of four experiments are reported. *: p<0.05 compared with CD10/CD13 double-negative. (D) Amplification of SGLT2 (S1 segment marker), CA IV (S2 segment marker) and SGLT1 (S3 segment marker) fragments in 2 representative PT cells and in CD10/CD13 double-negative cells at passage 3. Cyclophilin A (PPiA) was used as a house-keeping gene.

Figure 8. Evaluation of PT cells and CD10/CD13 double-negative cells phenotypic stability.

(A) Fluorescence plot showing PT cells labeled with antibodies against CD10 (APC: allophycocyanin) and CD13 (PE: phycoerythrin) after four passages. Flow cytometry revealed about 94% double-positive cells. (B) Relative percentage of CD10/CD13 double-positive cells at passages 2, 3, 4 and 5 in the PT cells populations (n = 4). NS: non-significant (p>0.05). (C) Representative western blots for PT cells over 5 passages. Blots were incubated with antibodies against aquaporin-1, N-cadherin, MUC1. The β-actin protein was used as an internal control (D) Fluorescence plot showing the CD10/CD13 double-negative cell population labeled with antibodies against CD10 and CD13 after two passages. Flow cytometry revealed about 15% double-negative cells.