Vectorial secretion of CTGF as a cell-type specific response to LPA and TGF-β in human tubular epithelial cells

Abstract

Background: Increased expression of the pro-fibrotic protein connective tissue growth factor (CTGF) has been detected in injured kidneys and elevated urinary levels of CTGF are discussed as prognostic marker of chronic kidney disease. There is evidence that epithelial cells lining the renal tubular system contribute to uptake and secretion of CTGF. However, the role of different types of tubular epithelial cells in these processes so far has not been addressed in primary cultures of human cells.

Results: Tubular epithelial cells of proximal and distal origin were isolated from human kidneys and cultured as polarized cells in insert wells. The pro-fibrotic stimuli lysophosphatidic acid (LPA) and transforming growth factor β (TGF-β) were used to induce CTGF secretion.LPA activated CTGF secretion in proximal tubular cells when applied from either the apical or the basolateral side as shown by immunocytochemistry. CTGF was secreted exclusively to the apical side. Signaling pathways activated by LPA included MAP kinase and Rho kinase signaling. TGF-β applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release.Interestingly, TGF-β activation induced different signaling pathways depending on the side of TGF-β application. Smad signaling was almost exclusively activated from the basolateral side most prominently in cells of distal origin. Only part of these cells also synthesized CTGF indicating that Smad activation alone was not sufficient for CTGF induction. MAP kinases were involved in apical TGF-β-mediated activation of CTGF synthesis in proximal cells and a subset of epithelial cells of distal origin. This subpopulation of distal tubular cells was also able to internalize recombinant apical CTGF, in addition to proximal cells which were the main cells to take up exogenous CTGF.

Conclusions: Analysis of polarized human primary renal epithelial cells in a transwell system shows that vectorial secretion of the pro-fibrotic protein CTGF depends on the cell type, the stimulus and the signaling pathway activated. In all conditions, CTGF was secreted mainly to the apical side upon TGF-β and LPA treatment and therefore, likely contributes to increased urinary CTGF levels in vivo. Moreover, CTGF secreted basolaterally may be active as paracrine pro-fibrotic mediator.

Figure 1

Polarized culture of primary human tubular epithelial cells (hPTEC).A: Freshly isolated hPTEC were cultured for 8 days in transwell inserts. Cilia were stained with acetylated tubulin and nuclei were visualized by Hoechst. For comparison, non polarized cells are shown in the right panel. Scale bar: 10 μm. B: hPTECs were cultured for 8 to 13 days in transwell inserts. Transepithelial electrical resistance (TEER) was measured in three cultures of one preparation. Data are means +/− SD.

Figure 2

Incubation with apical or basolateral LPA stimulated apical secretion of CTGF.A: Polarized hPTEC were stimulated with LPA (10 μM) from the basolateral side (L-b) or from the apical side (L-a) for 2 and 4 h. CTGF was detected in the apical compartment. The graph summarizes data (means +/− SD) of 5 (2 h) and 3 (4 h) independent experiments. Values of secretion of control cells were set to 1 at each time point. * p < 0.05; ** p < 0.01, Dunnet’s multiple comparison test. B/C: Polarized hPTEC were stimulated with LPA (10 μM) from the apical or basolateral side for 1 h. CTGF and E-cadherin were visualized by immunocytochemistry. Nuclei were stained with Hoechst. Cells negative for E-cadherin represent proximal tubular cells. Newly synthesized CTGF was detectable in a perinuclear localization (C; nuclei colored in red; CTGF in green, scale bar: 10 μm). Arrows indicate examples of CTGF positive proximal cells. Scale bar: 50 μm. D: Polarized hPTEC were pre-incubated with the inhibitors (U0: U0106, 1 μM; SB203: SB203580 1 μM; Y27: Y27632, 10 μM) for 30 min and then stimulated with LPA (10 μM) from the apical and basolateral side for 4 h. Secreted CTGF was detected in the apical (a) and basolateral (b) compartment. The graph summarizes data obtained with two isolations. Apical secretion of LPA-stimulated cells was set to 1 in each experiment.

Figure 3

Secretion of CTGF from polarized hPTEC stimulated with TGF-β. Polarized hPTEC were stimulated with TGF-β for 6 and 24 h from the basolateral side (T-b) or from the apical side (T-a). CTGF was detected in the apical (A) or basolateral (B) compartment. The blots shown are from a representative experiment performed with duplicate inserts. The graphs summarize means +/− SD of 6 (A) and 3 (B) experiments with different hPTEC isolations. Expression of control cells in each experiment was set to 1. * p < 0.05, ** p < 0.01, Dunnet’s multiple comparison test. C: RNA was isolated from polarized cells treated with TGF-β for 3 h. CTGF mRNA expression was determined by real time quantitative RT-PCR and corrected for 18 S RNA. Data are means +/− SD of duplicate determinations of duplicate inserts. RNA in control cells was set to 1. * p < 0.05, ** p < 0.01, Dunnet’s multiple comparison test.

Figure 4

Secretion of fibronectin. Polarized hPTEC were stimulated with TGF-β from the basolateral and from the apical side for 24 to 72 h. Secreted CTGF and fibronectin were detected in the basolateral (b) or apical (a) compartment. The blot is representative of 3 experiments with different hPTEC isolations. In the experiment shown CTGF was below the detection limit in the basolateral compartment.

Figure 5

Preferential activation of Smad2/3 in polarized hPTEC of distal origin. Polarized hPTEC were treated with TGF-β for 1 h from the apical or basolateral side. E-cadherin (red) and Smad2/3 (green) were detected by immunocytochemistry. Cells negative for E-cadherin represent proximal cells. Upon activation with TGF-β, Smad2/3 was concentrated in the nuclei of proximal and distal cells, respectively. Arrows indicate nuclear Smad2/3 in proximal E-cadherin negative cells and arrow heads indicate nuclear Smad2/3 in distal weakly E-cadherin positive cells. Scale bar: 50 μm.

Figure 6

Accumulation of intracellular CTGF upon stimulation with basolateral TGF-β.A: hPTEC were polarized and then stimulated with TGF-β for 1 h from the basolateral side. N-cadherin (green) and CTGF (red) were detected by immunocytochemistry. Control cells showed CTGF expression only in clustered proximal tubular cells (arrow, upper panels). Treatment with TGF-β from the basolateral side activated CTGF primarily in distal tubular cells (lower panels). Scale bar: 20 μm. B: Higher magnification of an experiment performed as in A with cells stimulated with TGF-β from the basolateral side. E-cadherin (red) and CTGF (green) were detected by immunocytochemistry. Arrows indicate examples of positive cells. Scale bar: 20 μm. C: Polarized hPTEC were treated with brefeldin A (10 μM) for 30 min and then were incubated with TGF-β from the apical side for 2 h. E-cadherin (red) and CTGF (green) were detected by immunocytochemistry. Arrows indicate examples of CTGF positive proximal cells, which were negative for E-cadherin; arrow heads indicate examples of CTGF positive distal cells, which weakly express E-cadherin. Scale bar: 20 μm.

Figure 7

Role for MAP kinases in apical TGF-β-mediated secretion of CTGF.A: Polarized hPTEC were incubated with inhibitors (U0: U0106, 1 μM; SB203: SB203580, 1 μM; Y27: Y27632, 10 μM; SB43: SB431542: 10 μM) for 30 min and then stimulated with TGF-β from the apical side for 6 h. CTGF secretion was detected in the apical compartment. The graph summarizes means +/− SD of 3 independent experiments. Secretion of CTGF in TGF-β-stimulated cells was set to 1 in each experiment. ** p < 0.01, * p < 0.05 compared to TGF-β-stimulated cells, Dunnet’s multiple comparison test. B: Polarized hPTECs were stimulated in duplicate with TGF-β for 15 and 60 min from the apical side. Protein was isolated from polarized cells. ERK1/2 and phospho-ERK1/2 were detected by Western blotting.

Figure 8

Uptake of extracellular CTGF from the apical side.A: Polarized hPTEC were treated with recombinant CTGF (10 ng/ml) for 15 min from the apical side. Cells were stained for E-cadherin (red) and CTGF (green). CTGF immunoreactivity was detected primarily in E-cadherin negative, proximal cells. Scale bar: 20 μm. B: Cells were treated as in A. N-cadherin positive cells (red) are shown at higher magnification. CTGF (green) is detectable in vesicular structures. Scale bar: 20 μm. C: Cells were treated as in A. Cells weakly positive for E-cadherin (red) showed CTGF (green) immunoreactivity, whereas distal cells with strong E-cadherin cell-cell contacts were negative. Arrows indicate cells positive for CTGF. Scale bar: 20 μm.