Matrix-independent stimulation of human tubular epithelial cell migration by Rho kinase inhibitors.

Proximal tubular epithelial cells differ from other epithelial cells in the expression of N-cadherin as major adherens junction protein instead of E-cadherin. Migration of proximal epithelial cells (HKC-8) was analyzed by scratch wounding and by a barrier assay, which allowed determination of migration velocity on different extracellular matrices. Migration velocity was about threefold higher on fibronectin compared to collagen IV. The differential migration velocity was reflected by the orientation of F-actin stress fibers. TGF-beta activated secretion of fibronectin and thus increased migration on collagen IV, but did not further promote migration on fibronectin. Pharmacological inhibition of Rho kinases (ROCKs) by Y-27632, hydroxyfasudil and H-1152, or siRNA against ROCKs significantly increased migration velocity independently of the extracellular matrix. Cells at the migration front showed long filopodia, which could not be mimicked by overexpression of consitutively active Cdc42, indicative of a more complex regulation of F-actin structures. N-cadherin was reorganized from tight zipper-like structures into loosened cell-cell contacts upon incubation with Y-27632, but HKC-8 cells still migrated as cohort. Migration through single cell pores in a modified Boyden chamber assay was also stimulated by ROCK inhibitors. ROCK inhibitors enhanced migration of primary cultures of renal tubular cells which consisted of proximal and distal tubular cells expressing N-cadherin and E-cadherin, respectively. There was no indication of a switch in cadherin expression in these cells or a preferential migration of N-cadherin expressing cells. Pharmacologic inhibition of ROCKs may thus favor repair processes in renal tubules by increasing the migratory capacity of tubular epithelial cells.

Organoaxial gastric volvulus: a rare cause of an acute abdomen.

We report the case of a 65-year-old male patient with massive epigastric pain of sudden onset and vomiting due to an organoaxial volvulus of the stomach. We comment on the surgical management in our case and discuss etiology and therapeutic options of this rare entity.

Recurrent gastric ulcer and cholangitis caused by ectopic drainage of the bile duct into the stomach.

We report a case of recurrent gastritis with pyloric stenosis and cholangitis due to a rare variation in bile duct anatomy. A 72-year-old female patient showed recurrent gastral ulcers and biliary colic with cholangitis caused by gallstones in the main bile duct with an ectopic orifice in the prepyloric region and concurrent inflammatory pyloric stenosis. After temporarily successful endoscopic treatment with stenting and pyloric dilatation, the patient suffered from recurrent cholangitis. Finally, the abnormal biliary anatomy required surgical treatment with biliodigestive anastomosis.

Actin-mediated gene expression depends on RhoA and Rac1 signaling in proximal tubular epithelial cells.

Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses.

Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.

BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-ß for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-ß, even though TGF-ß signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-ß. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-ß-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-ß-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability which included constant expression of E-cadherin. Our cell culture system of primary epithelial cells is thus suitable to understand and modulate cellular remodeling processes of distinct tubular cells relevant for human renal disease.