In this study we show that CTGF is secreted from different types of polarized epithelial cells derived from the human renal tubular system. We provide evidence that CTGF secretion is specifically regulated depending on the cell type, the stimulus and the side of application. Proximal tubular cells were the main target cells of LPA stimulation and Rho kinase signaling was activated in these cells. By contrast, TGF-β primarily stimulated different types of distal tubular epithelial cells. Basolateral TGF-β activated Smad signaling whereas MAP kinases were essential for apical activation of CTGF secretion by TGF-β.
Epithelial cells derived from human kidneys are functionally and structurally heterogeneous depending on their origin along the renal tubular system. This is also reflected in terms of epithelial barrier function and, at the cellular level, tight junctions. Renal proximal tubules are characterized as ‘leaky’ nephron segments with paracellular transport of NaCl, which may in part relate to the expression of claudin-2 [31, 32]. Claudin-2 has also been shown to be involved in transepithelial resistance determined in different types of MDCK cells . Primary cells obtained from healthy sections of human tumor nephrectomies differ in their composition as cells are obtained from various parts of the nephron. This was reflected in our measurements of transcellular resistance of the polarized cells which varied depending on the cellular composition. Preparations which consisted almost exclusively of cells of distal origin showed higher resistance than preparations which contained a higher proportion of proximal cells.
By immunocytochemistry, human cells of proximal and distal origin can be distinguished by their expression of different cell-cell adhesion proteins . Morphologically, E-cadherin positive cells could be further subdivided in cobble-stone like cells which showed a strong E-cadherin immunoreactivity, and more elongated cells with less intense staining for E-cadherin. Studies are ongoing to further characterize the origin of these cells, because they markedly differed in their response to TGF-β as outlined in detail below. Proximal tubular cells uniquely express the mesenchymal cell-cell adhesion molecule N-cadherin. These cells appeared to be less polarized than distal cells, and their adherence to membranes is less tight. In the coculture system of primary cells proximal cells were surrounded by distal cells and thus exposed to mechanical stress. Proximal cells tended to form multilayered structures, which often stained positive for CTGF in line with the known sensitivity of CTGF expression to mechanical stimulation [34, 35].
Proximal tubular cells thus seem to be more susceptible to stimuli which modulate the actin cytoskeleton, be it by mechanical stress or by stimuli such as LPA, which activates Rho-Rho kinase signaling and modulates actin stress fibers . We have shown earlier that induction of CTGF is sensitive to Rho-Rho kinase signaling in mesenchymal cells and that alterations in the actin cytoskeleton may induce CTGF expression by modulating G-actin and MAL/SRF dependent transcription in renal fibroblasts and endothelial cells, respectively [28, 36, 37]. By contrast, regulation of MAL/SRF-mediated gene regulation was mediated by Rac1 signaling rather than Rho-Rho kinase signaling in MDCK cells activated by calcium deprivation . In polarized hPTEC, inhibition of Rho kinase signaling affected LPA-induced CTGF up-regulation but not TGF-β-mediated induction. This may be due to different signaling pathways activated by both stimuli and by the cell types activated. LPA activated almost exclusively N-cadherin positive proximal tubular cells.
Secretion of CTGF was clearly favored to the apical side of the cells. Thus far, rather little is known about the molecular mechanisms regulating CTGF secretion. Comparison of CTGF secretion with the secretion of fibronectin indicated that the apical secretion of CTGF was a regulated vectorial process which differed from the secretion of fibronectin, the basolateral secretion of which was much more pronounced. Other secreted proteins such as e.g. metalloproteinases were detected comparably in the apical and basolateral compartment of the transwell cultures (data not shown) further endorsing the specificity of CTGF secretion.
Based on the data obtained with LPA, it was clear that proximal tubular epithelial cells secreted CTGF to the apical side. However, as long as the molecular mechanisms of CTGF secretion have not been deciphered, we cannot rule out that other stimuli may activate also basolateral secretion of CTGF from proximal tubular cells.
LPA stimulated CTGF expression applied from both sides. In line with these data CTGF receptors have been characterized in both, apical and basolateral membranes of polarized gastrointestinal Caco-2 cells . By contrast, TGF-β was reported to activate Caco-2 cells and MDCK cells only when applied to the basolateral side [40–42]. However, localization of TGF-β receptors seems to vary between different epithelia. Even with certain MDCK cells (subtype MDCKII) apical activation by TGF-β has been reported . Apical localization of TGF-β receptor I was also reported in porcine vas deferens epithelium . In the kidney, Wang et al. had provided evidence for apical expression of TGF-β receptors in a subset of rat cortical collecting duct cells, and showed in vitro reactivity of mouse proximal tubular cells and renal inner medullary cell lines (mIMCD-3 cells) upon apical stimulation with TGF-β . In our experiments, TGF-β was clearly active when applied from the apical side in a TGF-β receptor I-dependent manner. Immunocytochemical analyses revealed that in addition to proximal tubular cells only a subset of distal cells reacted to apical TGF-β, characterized by low expression of E-cadherin. Further studies are necessary to define the origin of these cells which obviously differ from MDCK cells.
An additional level of complexity arises from the diversity of TGF-β receptor signaling, which implies Smad dependent and independent pathways (summarized in ). Furthermore, TGF-β signaling depends on interactions of its recptors with other membrane-bound proteins including growth factor receptors and integrins [47, 48]. In our studies, apical TGF-β hardly activated the canonical Smad pathway which was detected upon basolateral application and may be essential for TGF-β secretion as detected in MDCK cells. In MDCKII cells, apical treatment with TGF-β increased transepithelial resistance, which was prevented by inhibition of p38 and ERK1/2 signaling . In line with those studies, we showed phosphorylation of ERK1/2 upon apical application of TGF-β related to increased CTGF synthesis. A role for active ERK1/2 in CTGF synthesis in non-polarized epithelial cells was also described in epithelial cells of the eye  and in proximal tubular cell lines HKC-8 [21, 22]. Activation N-Ras GTPase has been implicated in ERK-mediated CTGF induction in HKC-8 cells . Furthermore, evidence was provided for direct phosphorylation of ShcA proteins by TGF-β receptor I . Whether these or other signaling pathways are also involved in the apical induction of CTGF in primary human tubular cells remains to be investigated.
Basolateral stimulation of tubular epithelial cells by TGF-β rapidly activated Smad signaling in all distal tubular cells and also part of the proximal cells. By contrast, only distinct cell populations stained positive for intracellular CTGF at any given time point after TGF-β stimulation. This may in part be due to the fact that intracellular CTGF can only be detected when it accumulates in the secretory pathway. However, more likely it reflects the fact that activation of the Smad pathway is not sufficient for CTGF synthesis and secretion. For instance, induction of CTGF by TGF-β in epithelial cells cultured in dishes was strongly dependent on cell density, whereas Smad translocation was density-independent . The signaling pathways which regulate density-dependent CTGF synthesis are not yet known but may also modulate CTGF expression in polarized cells.
Cellular uptake of CTGF has been observed in different cell types, mesangial cells , fibroblasts , chondrocytes  or endothelial cells (Muehlich, Goppelt-Struebe, unpublished), and was also shown in vivo in proximal tubular cells in mice upon CTGF injection . Our experiments confirmed uptake of exogenous CTGF in proximal cells. In addition, we observed CTGF uptake in the subset of E-cadherin positive cells, which also secreted CTGF upon stimulation with apical TGF-β as discussed above.
It was interesting to note that upon stimulation with TGF-β, CTGF was not exclusively secreted to the apical side but also detectable at the basolateral side. Extrapolating to the in vivo situation, this may imply that the secreted CTGF is not lost in the urine but may be potentially active as a paracrine mediator. This aspect is supported by in vivo studies showing a role for tubular CTGF in the model system of remnant kidney disease in TGF-β1 transgenic mice . Activated epithelial cells may thus contribute to the increased CTGF plasma levels detected in chronic kidney disease also in humans [1–3]. Furthermore, the strong apical secretion of CTGF suggests that also in vivo, activated tubular cells may have a share in urinary CTGF levels not only by uptake of filtered CTGF but also by secretion. Whether or not urinary CTGF has a functional role by acting on further distal tubular cells, remains to be established. Thus far, the biological effects of CTGF on distal tubular cells have not yet been investigated.