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

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 ß (TGF-ß) 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-ß applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release.Interestingly, TGF-ß activation induced different signaling pathways depending on the side of TGF-ß 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-ß-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-ß 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.

Synergistic induction of CTGF by cytochalasin D and TGFß-1 in primary human renal epithelial cells: Role of transcriptional regulators MKL1, YAP/TAZ and Smad2/3.

Changes in cell morphology that involve alterations of the actin cytoskeleton are a hallmark of diseased renal tubular epithelial cells. While the impact of actin remodeling on gene expression has been analyzed in many model systems based on cell lines, this study investigated human primary tubular epithelial cells isolated from healthy parts of tumor nephrectomies. Latrunculin B (LatB) and cytochalasin D (CytoD) were used to modulate G-actin levels in a receptor-independent manner. Both compounds (at 0.5µM) profoundly altered F-actin structures in a Rho kinase-dependent manner, but only CytoD strongly induced the pro-fibrotic factor CTGF (connective tissue growth factor). CTGF induction was dependent on YAP as shown by transient downregulation experiments. However, CytoD did not alter the nuclear localization of either YAP or TAZ, whereas LatB reduced nuclear levels particularly of TAZ. CytoD modified MKL1, a coactivator of serum response factor (SRF) regulating CTGF induction, and promoted its nuclear localization. TGFß-1 is one of the major factors involved in tubulointerstitial disease and an inducer of CTGF. Preincubation with CytoD but not LatB synergistically enhanced the TGFß-1-stimulated synthesis of CTGF, both in cells cultured on plastic dishes as well as in polarized epithelial cells. CytoD had no direct effect on the phosphorylation of Smad2/3, but facilitated their phosphorylation and thus activation by TGFß-1. Our present findings provide evidence that morphological alterations have a strong impact on cellular signaling of one of the major pro-fibrotic factors, TGFß-1. However, our data also indicate that changes in cell morphology per se cannot predict those interactions which are critically dependent on molecular fine tuning of actin reorganization.

Mitoxantrone-loaded superparamagnetic iron oxide nanoparticles as drug carriers for cancer therapy: Uptake and toxicity in primary human tubular epithelial cells.

Superparamagnetic iron oxide nanoparticles (SPIONs) are in use for many clinical diagnostic and experimental therapeutic applications, for example, for targeted drug delivery. To analyze the cellular responses to mitoxantrone-carrying SPIONs (SPION-MTO), and to the drug released from SPIONs, we used an in vitro system that allows comparison of primary human cells with different endocytotic capacities, namely, epithelial cells from proximal and distal parts of the nephron. SPIONs were selectively and rapidly internalized by proximal tubular cells with high endocytotic potential, but not by distal tubular cells. Uptake did not affect cell viability or morphology. In both cell types, free MTO (10-100 nM) induced double-strand DNA breaks and senescence, cell hypertrophy and reduced cell proliferation. However, cadherin-mediated cell-cell adhesion, cytoskeletal structures or polarity of the cells were not affected. Interestingly, a comparable response was also observed upon treatment with SPION-MTO and was independent of uptake of the particles. The effect of SPION-MTO on cells which did not internalize particles was primarily related to the release of MTO from drug-coated particles upon incubation in serum-containing cell growth medium. In conclusion, we show that whereas the uptake of SPIONs does not affect cellular functions or viability, the toxicity of drug-loaded SPIONs depends essentially on the type of drug bound to nanoparticles. Due to the relatively low systemic toxicity of MTO, the effects of MTO-SPIONs on human tubular cells were moderate, but they may become clinically relevant when more nephrotoxic drugs are bound to SPIONs.