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Christian S Haas

Publications and source records attributed to Christian S Haas.

18 recordsLinked to original sources
PubMed2006

Analysis of glomerular VEGF mRNA and protein expression in murine mesangioproliferative glomerulonephritis.

Capillary repair is crucial in the healing of glomerulonephritis (GN). The vascular endothelial growth factor (VEGF) has pro-angiogenic properties and plays an important role in glomerular capillary regeneration. Habu Snake Venom (HSV) GN, a murine model for mesangioproliferative GN, was induced in uninephrectomized C57/BL6 mice. Glomerular damage and capillary repair were assessed using morphometry, stereology, and confocal laser scanning microscopy. Mesangiolytic glomeruli were microdissected (days 1,3,7,14) using laser capture microdissection technique. VEGF mRNA expression was analyzed by real-time polymerase chain reaction and compared to intact glomeruli of healthy controls. Spatiotemporal VEGF gene and protein expression was determined using nonradioactive in situ hybridization and immunohistochemistry. On day 1, diseased animals developed focal mesangiolysis paralleled by a significant decrease in length density of glomerular capillaries that gradually returned to baseline levels thereafter, indicating capillary growth in response to initial injury. Glomerular VEGF mRNA expression increased on day 3 and returned back to baseline and beyond at day 14 when the glomerular recovery process was completed. Similarly, glomerular VEGF protein expression tended to be higher on day 3. The present study documents temporarily increased glomerular VEGF gene and protein expression during the healing of HSV GN, suggesting a potential role of VEGF in the repair of mesangiolytic glomerular damage.

Animals↗
PubMed2006

CXCL16-mediated cell recruitment to rheumatoid arthritis synovial tissue and murine lymph nodes is dependent upon the MAPK pathway.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor alpha (TNFalpha), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process. METHODS: Using a human RA ST-SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo. RESULTS: CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFalpha. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFalpha in vitro, suggesting that CXCL16 protein may function in large part independently of TNFalpha. CONCLUSION: Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.

Animals↗
PubMed2006

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

OBJECTIVE: To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. METHODS: The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. RESULTS: In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. CONCLUSION: Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2006

Inhibition of angiogenesis by interleukin-4 gene therapy in rat adjuvant-induced arthritis.

OBJECTIVE: Interleukin-4 (IL-4) can modulate neovascularization. In this study, we used a gene therapy approach to investigate the role of IL-4 in angiogenesis in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis. METHODS: Rats received an adenovirus producing IL-4 (AxCAIL-4), a control virus without insert, or control vehicle (phosphate buffered saline) intraarticularly before arthritis onset. At peak onset of arthritis, rats were killed. Vascularization was determined in the synovial tissue, and correlations with inflammation were assessed. Ankle homogenates were used in angiogenesis assays in vitro and in vivo, and protein levels of cytokines and growth factors were assessed by enzyme-linked immunosorbent assay. Synovial tissue expression of alphav integrins was determined by immunohistochemistry. RESULTS: IL-4 induced a reduction in synovial tissue vessel density, which was paralleled by a decrease in inflammation. AxCAIL-4 joint homogenates significantly (P < 0.05) inhibited both endothelial cell (EC) migration and tube formation in vitro. Similarly, AxCAIL-4 inhibited capillary sprouting in the rat aortic ring assay, and vessel growth in the in vivo Matrigel plug assay. The angiostatic effect occurred despite high levels of vascular endothelial growth factor (VEGF), and was associated with down-regulation of the proangiogenic cytokines IL-18, CXCL16, and CXCL5 and up-regulation of the angiogenesis inhibitor endostatin. Of interest, AxCAIL-4 also resulted in decreased EC expression of the alphav and beta3 integrin chains. CONCLUSION: In rat AIA, IL-4 reduces synovial tissue vascularization via angiostatic effects, mediates inhibition of angiogenesis via an association with altered pro- and antiangiogenic cytokines, and may inhibit VEGF-mediated angiogenesis and exert its angiostatic role in part via alphavbeta3 integrin. This knowledge of the specific angiostatic effects of IL-4 may help optimize target-oriented treatment of inflammatory arthritis.

Adjuvants, Immunologic↗
PubMed2006

Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis. In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCdelta inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCdelta, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.

Animals↗
PubMed2005

Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFkappaB.

Cell adhesion molecules are critical in monocyte (MN) recruitment in immune-mediated and hematologic diseases. We investigated the novel role of recombinant human migration inhibitory factor (rhMIF) in up-regulating vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and their signaling pathways in human MNs. rhMIF-induced expression of VCAM-1 and ICAM-1 was significantly higher compared with nonstimulated MNs. rhMIF induced MN VCAM-1 and ICAM-1 expression in a concentration-dependent manner (P < .05). Antisense oligodeoxynucleotides (ODNs) and inhibitors of Src, PI3K, p38, and NFkappaB significantly reduced rhMIF-induced MN VCAM-1 and ICAM-1 expression (P < .05). However, Erk1/2 and Jak2 were not involved. Silencing RNA directed against MIF, and inhibitors of Src, PI3K, NFkappaB, anti-VCAM-1, and anti-ICAM-1 significantly inhibited rhMIF-induced adhesion of HL-60 cells to human dermal microvascular endothelial cells (HMVECs) or an endothelial cell line, HMEC-1, in cell adhesion assays, suggesting the functional significance of MIF-induced adhesion molecules (P < .05). rhMIF also activated MN phospho-Src, -Akt, and -NFkappaB in a time-dependent manner. rhMIF induced VCAM-1 and ICAM-1 up-regulation in 12 hours via Src, PI3K, and NFkappaB as shown by Western blotting and immunofluorescence. MIF and MIF-dependent signaling pathways may be a potential target for treating diseases characterized by up-regulation of cell adhesion molecules.

Cell Adhesion↗
PubMed2005

Accelerated development of arthritis in mice lacking endothelial selectins.

The selectins, along with very late antigen-4 and CD44, have been implicated in mediating leukocyte rolling interactions that lead to joint recruitment and inflammation during the pathogenesis of rheumatoid arthritis. Previously, we showed that P-selectin deficiency in mice resulted in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. Here, we report that mice deficient either in E-selectin or in E-selectin and P-selectin (E/P-selectin mutant) also exhibit accelerated development of arthritis compared with wild type mice in the CIA model, suggesting that these adhesion molecules perform overlapping functions in regulating joint disease. Analyses of cytokine and chemokine expression in joint tissue from E/P-selectin mutant mice before the onset of joint swelling revealed significantly higher joint levels of macrophage inflammatory protein-1alpha and IL-1beta compared to wild-type mice. IL-1beta remained significantly increased in E/P-selectin mutant joint tissue during the early and chronic phases of arthritis. Overall, these data illustrate the novel finding that E-selectin and P-selectin expression can significantly influence cytokine and chemokine production in joint tissue, and suggest that these adhesion molecules play important regulatory roles in the development of arthritis in E/P-selectin mutant mice.

Animals↗
PubMed2005

Amelioration of rat adjuvant-induced arthritis by Met-RANTES.

OBJECTIVE: CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal-modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1alpha (MIP-1alpha; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. METHODS: Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. RESULTS: Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1alpha and MIP-1beta at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor alpha, interleukin-1beta, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. CONCLUSION: The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.

Animals↗
PubMed2005

Chemokine receptor expression in rat adjuvant-induced arthritis.

OBJECTIVE: Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvant-induced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors. METHODS: ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro. RESULTS: CCR1, a receptor for macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 and RANTES/CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP-1alpha/CCL3 and RANTES/CCL5, was up-regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down-regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down-regulation during AIA. CXCR4, a receptor for stromal cell-derived factor 1/CXCL12), was prominently up-regulated on ECs, preceding the peak of inflammation. CONCLUSION: These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade-targeting treatment strategies in inflammatory arthritis.

Animals↗
PubMed2004

Characterization of the renal phenotype in a mouse model of Marfan syndrome.

The microfibrillar protein fibrillin-1 is expressed abundantly in the vasculature and the glomerulus of the kidney. Mutations in the fibrillin-1 gene lead to Marfan syndrome. The most common complication of this disease is aortic dilatation due to elastic deficiencies of the vascular wall. Several case reports describe glomerular disease in patients with Marfan syndrome, and fibrillin-1 has been implicated in nephrogenesis. To study the role of fibrillin-1 in renal development and function, we characterized the renal phenotype of fibrillin-1-underexpressing mice. Kidney histology was evaluated by means of morphometry and stereology. Relative kidney weights, daily urine excretion, urinary albumin excretion, serum and urinary creatinine, as well as serum urea were not different than wild-type mice. Glomerular number and renal capillarization were normal. The size of the renal filtration surface was comparable in wild-type and fibrillin-1-underexpressing mice. There was no indication for glomerular, renal vascular, or tubulointerstitial injury. However, glomerular volume and mesangial area were reduced. No changes in glomerular cell numbers were detected, but the cellular volume of mesangial cells was significantly lower in glomeruli of fibrillin-1-underexpressing mice. Thus, despite the high abundance of fibrillin-1 in glomeruli of wild-type animals, underexpression of fibrillin-1 did not lead to functional deficiencies of the glomerulus. Alterations in renal histology were only subtle with a reduced glomerular volume and mesangial area likely due to a reduced mesangial cell volume.

Animals↗
PubMed2004

Matrix metalloproteinases in renal development.

Matrix metalloproteinases (MMPs) are enzymes with metal ion-dependent activity that degrade extracellular matrix (ECM) glycoproteins. MMPs play a vital role in various biological processes, such as embryogenesis, tissue remodeling, angiogenesis, and wound healing, and in certain disease processes, for example, metastasis of cancer cells. Following their activation, MMPs are believed to modulate both cell-cell and cell-matrix interactions, which in turn regulate cellular differentiation, migration, proliferation, and cell survival. Being involved in pericellular proteolysis, they maintain a gradient of ECM proteins by balancing ECM synthesis and degradation. Such a balance is critical for various mammalian developmental processes during embryonic life and also for the homeostasis of various organs and reparative processes in later life. During the past two decades the role of MMPs in the morphogenesis of various organs, including that of the metanephros, has been investigated extensively. Mammalian nephrogenesis comprises a series of intricate events characterized by a sustained remodeling and turnover of ECM, suggesting a potential role of MMPs in renal development. Conceivably, reciprocal inductive epithelial-mesenchymal interactions that take place at the very commencement of nephrogenesis are modulated by a number of ECM proteins. Their expression, especially at the epithelial-mesenchymal interface, are critical for metanephric development, and such a strategic expression is likely to be modified by a number of different macromolecules that exhibit spatiotemporal and stage-specific expression. Among them the most suitable candidate that could exert such a control would be MMPs. This review addresses the current status of our understanding of the functions and the role of MMPs in renal development.

Animals↗
PubMed2003

1,25-Dihydroxyvitamin D3 decreases podocyte loss and podocyte hypertrophy in the subtotally nephrectomized rat.

1,25(OH)(2)D(3) has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH)(2)D(3) interfered with glomerulosclerosis. The cellular mechanisms involved have remained obscure, however. It was the purpose of the present study to assess glomerular structure and cellular composition in subtotally nephrectomized (SNX) rats treated with nonpharmacological doses of 1,25(OH)(2)D(3). Male Sprague-Dawley rats were sham operated (sham) or underwent SNX under general anesthesia and received either solvent or 1,25(OH)(2)D(3) (3 ng.100 g body wt(-1).day(-1) sc). Blood pressure (BP) and albuminuria were measured. After 16 wk, the remnant renal tissue was perfusion fixed and morphometric and stereological measurements were carried out. The expression of proliferating cellular antigen (PCNA), cyclin-dependent kinase inhibitor p27, Wilms tumor gene (WT1), and desmin, a marker of early podocyte damage, was investigated by immunohistology. BP, serum creatinine, and urinary albumin excretion were significantly higher in SNX than in sham rats. Albuminuria was significantly lower in SNX+1,25(OH)(2)D(3) compared with SNX+solvent rats. Mean glomerular tuft volume was significantly higher in SNX+solvent (2.69 +/- 0.21 gx 10(6) microm(3)) than in sham rats (1.44 +/- 0.17 and 1.28 +/- 0.14 x 10(6) microm(3)); it was significantly (P < 0.05) lower in SNX+1,25(OH)(2)D(3) rats (1.81 +/- 0.16 x 10(6) microm(3)). The main finding was a significantly higher number of podocytes in SNX+1,25(OH)(2)D(3) (88 +/- 9) and sham (98 +/- 17) compared with SNX+solvent rats (81 +/- 8.7). In parallel, the increase in podocyte volume in SNX+solvent rats was abrogated by treatment with 1,25(OH)(2)D(3), and immunohistochemistry revealed less expression of desmin, PCNA, and p27, suggesting less podocyte injury and activation of the cyclin cascade. This study identifies the podocyte as an important target cell for the renoprotective action of 1,25(OH)(2)D(3). This notion is suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocyte hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.

Animals↗
PubMed2003

Tacrolimus and cerivastatin pharmacokinetics and adverse effects after single and multiple dosing with cerivastatin in renal transplant recipients.

AIMS: In contrast to cyclosporin, only limited information exists on the interaction potential between the immunosuppressive agent tacrolimus and HMG-CoA reductase inhibitors, which are metabolized via the cytochrome P450 system. The aim of this study was to investigate the pharmacokinetics, and adverse effects of cerivastatin combined with tacrolimus in renal transplant patients. METHODS: Ten patients with stable kidney graft functions and LDL-cholesterol serum concentrations > 110 mg dl-1 were included in the study. After an observation period of 3 months, cerivastatin (0.2 mg daily) was administered for an additional 3 months. Tacrolimus steady-state pharmacokinetics and cerivastatin single- and multiple-dose pharmacokinetics were determined. Lipid concentrations, routine laboratory parameters and adverse events were obtained and analysed throughout the study period of 6 months. RESULTS: Blood tacrolimus trough concentrations were not affected by cerivastatin (mean +/- SD 8.6 +/- 2.1 ng ml(-1) before, and 8.7 +/- 2.4 ng ml(-1) at day 90 of cerivastatin dosing, with a 95% confidence interval on the difference = 0.97, 1.08). The mean area under the blood concentration-time curve to 24 h (AUC(0,24 h)) for cerivastatin was 14.5 +/- 2.53 micro g l(-1) h(-1) at day 1 after starting treatment and 19.02 +/- 3.55 micro g l(-1) h(-1) (3 months later), resulting in a 35% higher (AUC(0,24 h)) compared with the first dose. Total cholesterol, LDL-cholesterol and triglyceride concentrations were significantly lowered by cerivastatin whereas no significant effect of cerivastatin on serum creatininkinase concentrations was observed and no adverse effects were documented. CONCLUSIONS: Tacrolimus increased the AUC(0, 24 h) of cerivastatin by a mean of 35% in renal transplant patients. Cerivastatin had no detectable effect on the pharmacokinetics of tacrolimus.

Adult↗
PubMed2003

Imprinted mesodermal specific transcript (MEST) and H19 genes in renal development and diabetes.

BACKGROUND: Imprinted genes, mesodermal specific cDNA or transcript (MEST) and H19, are implicated in peri-implantation embryogenesis, and their expression was assessed in embryonic kidneys undergoing glucose-induced dysmorphogenesis. METHODS: MEST and H19 mRNA expression was assessed by Northern blot analysis in embryonic kidneys of mice harvested at day 15 to day 19 of gestation and of 1-week-old mice obtained from hyperglycemic mothers. A full-length mouse MEST cDNA was isolated, subcloned into an expression vector, a recombinant protein prepared and an antibody raised; the latter was used to assess protein expression by immunoprecipitation and immunofluorescence microscopy in day 13 metanephric explants subjected to high glucose ambience. Also, MEST mRNA expression was assessed in high d glucose-treated explants by competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses and by in situ tissue autoradiography. RESULTS: A high expression of MEST and H19 with respective transcript size of approximately 2.7 and approximately 2.4 kb was observed in fetal kidneys, and their expression decreased during the successive stages of gestation and was undetectable in the postnatal period. At day 13, the MEST mRNA was expressed in the mesenchyme, while H19 was expressed in the ureteric bud branches and epithelial elements of the metanephros. Their expression decreased with progression of gestation. By competitive RT-PCR and Northern blot and in situ autoradiographic analyses, both MEST and H19 expressions decreased in day 13 explants treated with high glucose and in the kidneys of fetuses obtained from diabetic mothers. The MEST protein expression was observed in the metanephric epithelial elements and ureteric bud branches instead of in the mesenchyme, and its expression decreased in glucose-treated dysmorphogenetic explants, as assessed by immunofluorescence and immunoprecipitation methods. CONCLUSION: MEST and H19 imprinted genes are strategically located in the mammalian embryonic metanephros. They are developmentally regulated and their concomitant decreased expression in high glucose ambience or diabetic state did not follow the prevailing dogma of reciprocal inactivation/activation of imprinted genes, and such a decrease may be responsible for the perturbed epithelial:mesenchymal interactions leading to dysmorphogenesis of the mammalian metanephros.

Animals↗
PubMed2003

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice.

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

Animals↗
PubMed2002

Isolation and functional analysis of mouse UbA52 gene and its relevance to diabetic nephropathy.

In delineating the mechanism(s) of diabetic nephropathy various novel genes have been isolated, whereas others remain to be discovered. We identified several up-regulated genes in the kidneys of diabetic newborn mice. Among them was UbA52, a ubiquitin ribosomal fusion protein. Its mRNA expression in the kidney was proportional to blood glucose levels. By in situ hybridization and immunohistochemistry, UbA52 was exclusively localized to renal tubules, and its expression was markedly increased in diabetic mice. The up-regulated UbA52 mRNA and protein expression were also observed in Madin-Darby canine kidney cells, a tubular cell line, treated with 30 mm glucose in both cell lysates and ribosomal fractions. To explore the mechanism(s) of its increased expression, UbA52 genomic DNA was isolated. A transcription start site at -22 bp from the initiation codon was identified and confirmed by primer extension analysis. The UbA52 promoter region included glucose response-related E-box sequences and stress response elements (STRE). Unlike in humans, mouse UbA52 gene had no introns in the coding or 5'-ATG-flanking regions. To identify the DNA segment with maximal promoter activity, deletion constructs were prepared using a pSEAP vector system and transfected into COS7 kidney cells. Maximal activity was confined to -198 to +68 bp, which included E-boxes and STRE motifs. A dose-dependent increase in the promoter activity was observed in cells exposed to high glucose. Mutations in the first E-box (CAGCTG --> TGGCTG) or STRE (CCCCT --> CATCT) resulted in a decrease in the SEAP activity under high glucose ambience. Given the presence of glucose-responsive motifs in the promoter region and decrease in the SEAP activity in E-box mutants in the presence of glucose, these data suggest that UbA52, a ribosomal fusion protein, may be relevant in the pathogenesis of diabetic nephropathy.

Animals↗
PubMed2002

Restricted expression of mouse GCMa/Gcm1 in kidney and thymus.

Mouse Glial Cells Missing a (mGCMa) belongs to a small family of transcription factors named after the prototypical GCM from Drosophila. mGCMa was expected to regulate gliogenesis, but instead found to be primarily expressed during development in trophoblasts of chorion and labyrinth. Its deletion causes abnormal placental labyrinth formation and results in embryonic lethality. Here we identify kidney and thymus as sites of mGCMa expression. In thymus, mGCMa is restricted to few small clusters of cells, in kidney to the S3 segment of proximal tubules. mGCMa expression is primarily postnatal, arguing for a role in organ function rather than organ development.

Animals↗