Ines Marek

Glomerular Regeneration Is Delayed in Nephritic α8-Integrin-Deficient Mice: Contribution of α8-Integrin to the Regulation of Mesangial Cell Apoptosis

Background/Aims: α8β1-Integrin is expressed in mesangial cells. In vitro studies suggest a role for α8-integrin in the regulation of cell proliferation and apoptosis. We tested the hypothesis that α8-integrin is essential for the healing process after mesangioproliferative glomerulonephritis. Methods: Mice homozygous for a deletion of the α8-integrin chain were compared with wild-type mice. To study glomerular healing, we used the habu toxin model of reversible mesangioproliferative glomerulonephritis. Animals received 6 mg/kg habu toxin intravenously; controls received saline only. Results: Early mesangiolysis occurred in wild-type and α8-integrin-deficient mice. However, mesangiolysis was no longer detectable after 7 days in wild types but persisted after 14 days in α8-integrin-deficient animals. Mesangial activation marker α-smooth muscle actin was detectable only at day 7 in wild-type mice but persisted until day 14 in α8-integrin-deficient mice. In wild types, glomerular cell proliferation and apoptosis peaked at day 7 and decreased thereafter but remained elevated in α8-integrin-deficient mice until day 28. In cultivated mesangial cells, α8-integrin expression was associated with increased cell survival. Conclusion: Interactions between α8-integrin and the mesangial matrix may contribute to healing of glomerular injury by influencing cell proliferation and apoptosis.

Tubulointerstitial De Novo Expression of the α8 Integrin Chain in a Rodent Model of Renal Fibrosis – A Potential Target for Anti-Fibrotic Therapy?

In the normal kidney, the α8 integrin chain is expressed only on mesangial cells and vascular smooth muscle cells. α8 integrin ligates several matrix molecules including fibronectin, osteopontin and fibrillin-1. Recently, we detected de novo expression of α8 integrin on epithelial cells in renal cysts. We hypothesized that the α8 integrin chain is induced in tubular epithelia undergoing dedifferentiation and contributes to the fibrotic response in the tubulointerstitium (TI) after unilateral ureteral obstruction (UUO). After induction of UUO in rats by ligation of the right ureter, increased expression of the α8 integrin chain and its ligands was observed. In the TI, α8 integrin was localized to cytokeratin-positive epithelial cells and to interstitial fibroblasts; and colocalized with its ligands. In mice underexpressing α8 integrin UUO led to collagen deposition and fibroblast activation comparable to wild types. Mice lacking α8 integrin showed even more TI damage, fibroblast activation and collagen deposition after UUO compared to wild type mice. We conclude that the expression of the α8 integrin chain and its ligands is strongly induced in the TI after UUO, but underexpression of α8 integrin does not attenuate TI fibrosis. Mice lacking the α8 integrin chain are even more susceptible to TI damage than wild type mice. Thus, interactions of α8 integrin with its ligands do not seem to contribute to the development or progression of TI fibrosis in UUO. Targeting α8 integrin might not be a useful approach for anti-fibrotic therapy.

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells

BackgroundExtracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin.ResultsWild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells.ConclusionsOur findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.

Alpha8 Integrin (Itga8) Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover

The α8 integrin (Itga8) chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration.

Under-expression of α8 integrin aggravates experimental atherosclerosis.

Integrins play an important role in vascular biology. The α8 integrin chain attenuates smooth muscle cell migration but its functional role in the development of atherosclerosis is unclear. Therefore, we studied the contribution of α8 integrin to atherosclerosis and vascular remodelling. We hypothesized that α8 integrin expression is reduced in atherosclerotic lesions, and that its under‐expression leads to a more severe course of atherosclerosis. α8 Integrin was detected by immunohistochemistry and qPCR and α8 integrin‐deficient mice were used to induce two models of atherosclerotic lesions. First, ligation of the carotid artery led to medial thickening and neointima formation, which was quantified in carotid cross‐sections. Second, after crossing into ApoE‐deficient mice, the formation of advanced vascular lesions with atherosclerotic plaques was quantified in aortic en face preparations stained with Sudan IV. Parameters of renal physiology and histopathology were assessed: α8 integrin was detected in the media of human and murine vascular tissue and was down‐regulated in arteries with advanced atherosclerotic lesions. In α8 integrin‐deficient mice (α8−/−) as well as α8+/− and α8+/+ littermates, carotid artery ligation increased media:lumen ratios in all genotypes, with higher values in ligated α8−/− and α8+/− compared to ligated α8+/+ animals. Carotid artery ligation increased smooth muscle cell number in the media of α8+/+ mice and, more prominently, of α8−/− or α8+/− mice. On an ApoE−/− background, α8+/− and α8−/− mice developed more atherosclerotic plaques than α8+/+ mice. α8 Integrin expression was reduced in α8+/− animals. Renal damage with increased serum creatinine and glomerulosclerosis was detected in α8−/− mice only. Thus, under‐expression of α8 integrin aggravates vascular lesions, while a complete loss of α8 integrin results in reduced renal mass and additional renal disease in the presence of generalized atherosclerosis. Our data support the hypothesis that integrin α8β1 has a protective role in arterial remodelling and atherosclerosis. Copyright

Trophoblast expression dynamics of the tumor suppressor gene gastrokine 2

Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.

The placental mTOR-pathway: correlation with early growth trajectories following intrauterine growth restriction?

Idiopathic intrauterine growth restriction (IUGR) is a result of impaired placental nutrient supply. Newborns with IUGR exhibiting postnatal catch-up growth are of higher risk for cardiovascular and metabolic co-morbidities in adult life. Mammalian target of rapamycin (mTOR) was recently shown to function as a placental nutrient sensor. Thus, we determined possible correlations of members of the placental mTOR signaling cascade with auxologic parameters of postnatal growth. The protein expression and activity of mTOR-pathway signaling components, Akt, AMP-activated protein kinase α, mTOR, p70S6kinase1 and insulin receptor substrate-1 were analysed via western blotting in IUGR v. matched appropriate-for-gestational age (AGA) placentas. Moreover, mTOR was immunohistochemically stained in placental sections. Data from western blot analyses were correlated with retrospective auxological follow-up data at 1 year of age. We found significant catch-up growth in the 1st year of life in the IUGR group. MTOR and its activated form are immunohistochemically detected in multiple placental compartments. We identified correlations of placental mTOR-pathway signaling components to auxological data at birth and at 1 year of life in IUGR. Analysis of the protein expression and phosphorylation level of mTOR-pathway components in IUGR and AGA placentas postpartum, however, did not reveal pathognomonic changes. Our findings suggest that the level of activated mTOR correlates with early catch-up growth following IUGR. However, the complexity of signals converging at the mTOR nexus and its cellular distribution pattern seem to limit its potential as biomarker in this setting.

Fibrillin-1 and alpha8 integrin are co-expressed in the glomerulus and interact to convey adhesion of mesangial cells

Fibrillin-1 is a microfibrillar extracellular matrix protein that was described to be a ligand for α8 integrin. α8 integrin is a matrix receptor specifically expressed in mesangial and smooth muscle cells of the kidney. In previous studies we detected glomerular expression of fibrillin-1. Moreover, fibrillin-1 promoted adhesion, migration, and proliferation of mesangial cells. We hypothesized that fibrillin-1 and α8 integrin might interact in the glomerulus, and thus, regulate mesangial cell properties. Our studies showed that fibrillin-1 and α8 integrin colocalize in the glomerular mesangium. Induction of experimental glomerulonephritis led to an increase of both fibrillin-1 and α8 integrin expression. In vitro studies revealed that mesangial cells deficient for α8 integrin adhere weaker to fibrillin-1 and migrate more easily on fibrillin-1 than wild-type mesangial cells. Baseline proliferation on fibrillin-1 is higher in α8 integrin-deficient mesangial cells, but the induction of proliferation is not different in α8 integrin-deficient and wild-type mesangial cells. We conclude that fibrillin-1 and α8 integrin interact, and thus, regulate mesangial cell adhesion and migration. The concomitant induction of both fibrillin-1 and α8 integrin in a self-limited model of glomerular injury points to a protective role of the interaction of fibrillin-1 with α8 integrin in the glomerulus resulting in reduced damage of the glomerular tuft as a consequence of firm adhesion of mesangial cells.

Expression of the Alpha8 Integrin Chain Facilitates Phagocytosis by Renal Mesangial Cells

Background/Aims: Healing of mesangioproliferative glomerulonephritis involves degradation of excess extracellular matrix, resolution of hypercellularity by apoptosis and phagocytosis of apoptotic cells. Integrin receptors participate in the regulation of phagocytosis. In mice deficient for alpha8 integrin (Itga8-/-) healing of glomerulonephritis is delayed. As Itga8 is abundant in mesangial cells (MC) which are non-professional phagocytes, we hypothesized that Itga8 facilitates phagocytosis of apoptotic cells and matrix components by MC. Methods: MC were isolated from wild type (WT) and Itga8-/- mice. Latex beads were coated with matrix components. Apoptosis was induced by cisplatin in macrophages and in DiI-stained MC. After coincubation of latex beads or apoptotic cells with MC, the phagocytosis rate was detected in WT and Itga8-/- MC via fluorescence microscopy and FACS analysis. Results: Itga8-/- MC showed reduced phagocytosis of matrix-coated beads and apoptotic cells compared to WT MC. Reduction of stress fibers was observed in Itga8-/- compared to WT MC. Inhibition of cytoskeletal reorganization by inhibition of Rac1 or ROCK during phagocytosis significantly decreased the rate of phagocytosis by WT MC but not by Itga8-/- MC. Conclusion: The expression of Itga8 facilitates phagocytosis in MC, likely mediated by Itga8-cytoskeleton interactions. An impairment of MC phagocytosis might thus contribute to a delayed glomerular regeneration in Itga8-/- mice.

Contribution of the α8 Integrin Chain to the Expression of Extracellular Matrix Components

Abstract In the kidney, the α8 integrin chain (itga8) is expressed in mesenchymal cells and is upregulated in fibrotic disease. We hypothesized that itga8 mediates a profibrotic phenotype of renal cells by promoting extracellular matrix and cytokine expression. Genetic itga8 deficiency caused complex changes in matrix expression patterns in mesangial and smooth-muscle cells, with the only concordant effect in both cell types being a reduction of collagen III expression. Silencing of itga8 with siRNA led to a decline of matrix turnover with repression of matrix metalloproteinases and reduction of matrix production. In contrast, de novo expression of itga8 in tubular epithelial cells resulted in reduced collagen synthesis. Overexpression of itga8 in fibroblasts did not change the expression of matrix molecules or regulators of matrix turnover. Thus, the influence of itga8 on the expression of matrix components was not uniform and celltype dependent. Itga8 seems unlikely to exert overall profibrotic effects in renal cells.