Attenuation of cardiac hypertrophy by G-CSF is associated with enhanced migration of bone marrow-derived cells.

Granulocyte-colony stimulating factor (G-CSF) has been shown to promote mobilization of bone marrow-derived stem cells (BMCs) into the bloodstream associated with improved survival and cardiac function after myocardial infarction. Therefore, the aim of the present study was to investigate whether G-CSF is able to attenuate cardiac remodelling in a mouse model of pressure-induced LV hypertrophy focusing on mobilization and migration of BMCs. LV hypertrophy was induced by transverse aortic constriction (TAC) in C57BL/6J mice. Four weeks after TAC procedure. Mice were treated with G-CSF (100 µg/kg/day; Amgen Biologicals) for 2 weeks. The number of migrated BMCs in the heart was analysed by flow cytometry. mRNA expression and protein level of different growth factors in the myocardium were investigated by RT-PCR and ELISA. Functional analyses assessed by echocardiography and immunohistochemical analysis were performed 8 weeks after TAC procedure. G-CSF-treated animals revealed enhanced homing of VLA-4(+) and c-kit(+) BMCs associated with increased mRNA expression and protein level of the corresponding homing factors Vascular cell adhesion protein 1 and Stem cell factor in the hypertrophic myocardium. Functionally, G-CSF significantly preserved LV function after TAC procedure, which was associated with a significantly reduced area of fibrosis compared to control animals. Furthermore, G-CSF-treated animals revealed a significant improvement of survival after TAC procedure. In summary, G-CSF treatment preserves cardiac function and is able to diminish cardiac fibrosis after induction of LV hypertrophy associated with increased homing of VLA-4(+) and c-kit(+) BMCs and enhanced expression of their respective homing factors VCAM-1 and SCF.

No amelioration of uromodulin maturation and trafficking defect by sodium 4-phenylbutyrate in vivo: studies in mouse models of uromodulin-associated kidney disease.

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/ß, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.

Type of uromodulin mutation and allelic status influence onset and severity of uromodulin-associated kidney disease in mice.

Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin (UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line Umod(C93F), leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line Umod(A227T). In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and Cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Mutation of the Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in mice is associated with severe polyuria and a urea-selective concentrating defect without hyperreninemia.

The bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter NKCC2, located in the thick ascending limb of Henle's loop, plays a critical role in the kidney's ability to concentrate urine. In humans, loss-of-function mutations of the solute carrier family 12 member 1 gene (SLC12A1), coding for NKCC2, cause type I Bartter syndrome, which is characterized by prenatal onset of a severe polyuria, salt-wasting tubulopathy, and hyperreninemia. In this study, we describe a novel chemically induced, recessive mutant mouse line termed Slc12a1(I299F) exhibiting late-onset manifestation of type I Bartter syndrome. Homozygous mutant mice are viable and exhibit severe polyuria, metabolic alkalosis, marked increase in plasma urea but close to normal creatininemia, hypermagnesemia, hyperprostaglandinuria, hypotension,, and osteopenia. Fractional excretion of urea is markedly decreased. In addition, calcium and magnesium excretions are more than doubled compared with wild-type mice, while uric acid excretion is twofold lower. In contrast to hyperreninemia present in human disease, plasma renin concentration in homozygotes is not increased. The polyuria observed in homozygotes may be due to the combination of two additive factors, a decrease in activity of mutant NKCC2 and an increase in medullary blood flow, due to prostaglandin-induced vasodilation, that impairs countercurrent exchange of urea in the medulla. In conclusion, this novel viable mouse line with a missense Slc12a1 mutation exhibits most of the features of type I Bartter syndrome and may represent a new model for the study of this human disease.

Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis.

Mobilization of bone marrow-derived stem cells (BMCs) was shown to have protective effects after myocardial infarction (MI). However, the classical mobilizing agent, granulocyte-colony stimulating factor (G-CSF) relapsed after revealing an impaired homing capacity. In the search for superior cytokines, erythropoietin (EPO) appears to be a promising agent. Therefore, we analyzed in a murine model of surgically induced MI the influence of EPO treatment on survival and functional parameters as well as BMC mobilization, homing, and effect on resident cardiac stem cells (CSCs). Human EPO was injected intraperitoneally after ligation of the left anterior descendens (LAD) for 3 days with a total dose of 5000 IU/kg 6 and 30 days after MI, and pressure volume relationships were investigated in vivo. Cardiac tissues were analyzed by histology. To show the effect on BMCs and CSCs, FACS analyses were performed. Homing factors were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA. EPO-treated animals showed a significant improvement of survival post-MI (62 vs. 36%). At days 6 and 30, all hemodynamic parameters associated with attenuated remodeling, enhanced neovascularization, and diminished apoptotic cells in the peri-infarct area were improved. BMC subpopulations (CD31(+), c-kit(+), and Sca-1(+) cells) were mobilized, and homing of Sca-1(+) and CXCR4(+) BMCs toward an SDF-1 gradient into the ischemic myocardium was enhanced. However, there was no beneficial effect on CSCs. We have shown that EPO application after MI shows cardioprotective effects. This may be explained by mobilization of BMCs, which are homing via the CXCR-4/SDF-1 axis. However, EPO has no beneficial effects on resident CSCs. Therefore, new treatment regimes using EPO together with other agents may combine complementary beneficial effects preventing ischemic cardiomyopathy.

Novel missense mutation of uromodulin in mice causes renal dysfunction with alterations in urea handling, energy, and bone metabolism.

Uromodulin-associated kidney disease is a heritable renal disease in humans caused by mutations in the uromodulin (UMOD) gene. The pathogenesis of the disease is mostly unknown. In this study, we describe a novel chemically induced mutant mouse line termed Umod(A227T) exhibiting impaired renal function. The A227T amino acid exchange may impair uromodulin trafficking, leading to dysfunction of thick ascending limb cells of Henle's loop of the kidney. As a consequence, homozygous mutant mice display azotemia, impaired urine concentration ability, reduced fractional excretion of uric acid, and a selective defect in concentrating urea. Osteopenia in mutant mice is presumably a result of chronic hypercalciuria. In addition, body composition, lipid, and energy metabolism are indirectly affected in heterozygous and homozygous mutant Umod(A227T) mice, manifesting in reduced body weight, fat mass, and metabolic rate as well as reduced blood cholesterol, triglycerides, and nonesterified fatty acids. In conclusion, Umod(A227T) might act as a gain-of-toxic-function mutation. Therefore, the Umod(A227T) mouse line provides novel insights into consequences of disturbed uromodulin excretion regarding renal dysfunction as well as bone, energy, and lipid metabolism.

Parathyroid hormone treatment after myocardial infarction promotes cardiac repair by enhanced neovascularization and cell survival.

AIMS: An ongoing concept is that stem cells have the potential to regenerate the injured myocardium. In addition to direct vasorelaxing effects on the vasculature, which are mediated by an increased cAMP production leading to a decreased calcium influx in smooth muscle cells, parathyroid hormone (PTH) was recently shown to facilitate stem cell mobilization. Therefore, we analysed in a murine model of experimental myocardial infarction (MI) the influence of PTH treatment on survival, functional parameters, stem cell migration, and expression of vascular endothelial growth factor A (VEGF-A). METHODS AND RESULTS: Mice (C57BL/6) were treated with PTH (80 microg/kg/d) for up to 14 days after coronary artery ligation. Functional and immunohistochemical analyses were performed at days 6 and 30 after MI. Stem cells and VEGF expression in the myocardium were analysed by FACS and qRT-PCR at day 2 after MI. PTH-treated animals revealed a significant improvement of post-MI survival and myocardial function that was related to a subsequent reduction of left ventricular wall thinning and scar extension. Infarcted hearts of PTH-treated mice revealed increased numbers of CD45(+)/CD34(+) progenitor cells as well as an upregulation of VEGF-A mRNA associated with increased neovascularization and cell survival. CONCLUSIONS: PTH application after MI increases migration of angiogenic CD45(+)/CD34(+) progenitor cells to the ischaemic heart, which may attenuate ischaemic cardiomyopathy. As PTH is already used in patients with osteoporosis, our findings may have a direct impact on the initiation of clinical studies in patients with ischaemic heart disease.

Diets influence the diabetic phenotype of transgenic mice expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn).

Transgenic mice overexpressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn)) have recently been shown to develop diabetes mellitus due to disturbed postnatal development of the endocrine pancreas. In this study, the effects of feeding a high fibre/low calorie diet on the diabetic phenotype of GIPR(dn) transgenic mice were examined. Transgenic and control animals received either a conventional breeding diet (BD) or a high fibre diet (HF). Both fasting and postprandial blood glucose levels and HbA1C levels were largely elevated in transgenic mice vs. controls (p<0.05), irrespective of the diet fed. Food and water intake and the daily urine volume of GIPR(dn) transgenic mice were higher than that of control mice (p<0.05). Transgenic animals receiving the HF diet showed significantly lower blood glucose and HbA1C levels as well as less food and water intake than transgenic mice fed BD. The 365-day survival of transgenic mice was significantly lower than that of control mice. Transgenic animals fed the HF diet lived significantly longer than their counterparts receiving BD. GIPR(dn) transgenic mice develop a severe diabetic phenotype which can be ameliorated by a HF diet, thereby resembling some aspects of the pathophysiology of human type 2 diabetes mellitus.

Ribonuclease (RNase) Prolongs Survival of Grafts in Experimental Heart Transplantation.

BACKGROUND: Cell damage, tissue and vascular injury are associated with the exposure and release of intracellular components such as RNA, which promote inflammatory reactions and thrombosis. Based on the counteracting anti-inflammatory and cardioprotective functions of ribonuclease A (RNase A) in this context, its role in an experimental model of heart transplantation in rats was studied. METHODS AND RESULTS: Inbred BN/OrlRj rat cardiac allografts were heterotopically transplanted into inbred LEW/OrlRj rats. Recipients were intravenously treated every other day with saline or bovine pancreatic RNase A (50 µg/kg). Toxic side effects were not found (macroscopically and histologically). Heart tissue flow cytometry and quantitative morphological analyses of explanted hearts at postoperative day 1 or postoperative day 4 showed reduced leukocyte infiltration, edema, and thrombus formation in RNase A-treated rats. In allogeneic mixed lymphocyte reactions, RNase A decreased the proliferation of effector T cells. RNase A treatment of rats resulted in prolonged median graft survival up to 10.5 days (interquartile range 1.8) compared to 6.5 days (interquartile range 1.0) in saline treatment (P=0.001). Treatment of rats with a new generated (recombinant) human pancreatic RNase 1 prolonged median graft survival similarly, unlike treatment with (recombinant) inactive human RNase 1 (each 50 µg/kg IV every other day, 11.0 days, interquartile range 0.3, versus 8.0 days, interquartile range 0.5, P=0.007). CONCLUSIONS: Upon heart transplantation, RNase administration appears to present a promising and safe drug to counteract ischemia/reperfusion injury and graft rejection. Furthermore, RNase treatment may be considered in situations of critical reperfusion after percutaneous coronary interventions or in cardiac surgery using the heart-lung machine.

Overexpression of a dominant negative GIP receptor in transgenic mice results in disturbed postnatal pancreatic islet and beta-cell development.

The expression of a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) under the control of the rat pro-insulin gene promoter induces severe diabetes mellitus in transgenic mice. This study aims to gain further insight into the effect of the expression of a dominant negative GIPR on glucose homeostasis and postnatal development of the endocrine pancreas. The diabetic phenotype of GIPRdn transgenic animals was first observed between 14 and 21 days of age (urine glucose>1000 mg/dl). After onset of diabetes, serum glucose was significantly higher and insulin values were significantly lower in GIPRdn transgenic mice vs. non-transgenic littermate controls. Morphometric studies of pancreatic islets and their endocrine cell types were carried out at 10, 30 and 90 days of age. The total islet and total beta-cell volume of transgenic mice was severely reduced as compared to control mice, irrespective of the age at sampling (p<0.05). The total volume of isolated insulin positive cells that were not contained within established islets was significantly reduced in transgenic mice, indicating disturbed islet neogenesis. These findings demonstrate in vivo evidence that intact signaling of G-protein coupled receptors is involved in postnatal islet and beta-cell development and neogenesis of the pancreatic islets.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND: Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS: We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS: A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS: The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Permanent neonatal diabetes in INS(C94Y) transgenic pigs.

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INS(C94Y) transgenic pigs. A line expressing high levels of INS(C94Y) mRNA (70-86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered ß-cell mass at the age of 8 days. At 4.5 months, INS(C94Y) transgenic pigs exhibited 41% reduced body weight, 72% decreased ß-cell mass (-53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. ß-cells of INS(C94Y) transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INS(C94Y) transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INS(C94Y) transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.

The cardioprotective effects of parathyroid hormone are independent of endogenous granulocyte-colony stimulating factor release.

AIMS: Parathyroid hormone (PTH) administration after myocardial infarction (MI) is known to attenuate ischaemic cardiomyopathy. This effect mainly resulted from an increase in mobilization and homing of CD34+/CD45+ cells into the ischaemic myocardium. PTH-related stem cell mobilization was shown to be related to endogenous granulocyte-colony stimulating factor (G-CSF) release. The aim of our study is to determine the role of G-CSF on the cardioprotective effects of PTH. METHODS AND RESULTS: G-CSF +/+ (C57BL/6) and G-CSF -/- mice were treated with PTH for 6 days after inducing a MI. The myocardial homing factor stromal cell-derived factor-1 (SDF-1) was analysed on day 2 with enzyme-linked immunosorbent assay. Stem cell populations in peripheral blood and hearts were examined by FACS on days 6 and 2, respectively. Cardiac function and immunohistochemistry were investigated on day 6 and day 30. PTH treatment resulted in a significant increase in CD45+/CD34+ cells in peripheral blood in G-CSF +/+ but not in G-CSF -/- mice. However, a significant increase in SDF-1 and enhanced migration of CD45+/CD34+ cells into the ischaemic myocardium was revealed after PTH administration in both G-CSF +/+ and G-CSF -/- mice. Enhanced stem cell homing was associated with improved cardiac function and post-MI survival after PTH treatment. Furthermore, infarct size, wall thickness, and neovascularization showed a significant improvement in both groups 30 days after MI. CONCLUSION: The cardioprotective effects of PTH were shown to be independent of endogenous G-CSF release and therefore from stem cell mobilization. This puts more emphasis on the role of stem cell homing into ischaemic myocardium.

Postnatal development of numbers and mean sizes of pancreatic islets and beta-cells in healthy mice and GIPR(dn) transgenic diabetic mice.

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR(dn) transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR(dn) transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR(dn) transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR(dn) transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR(dn) transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR(dn) transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.

Differential glomerular proteome analysis of two murine nephropathy models at onset of albuminuria.

PURPOSE: Early stages of various entities of progressive kidney diseases are commonly characterized by development of glomerular hypertrophy and albuminuria. The purpose of the present study was to identify protein biomarker candidates for these glomerular alterations. EXPERIMENTAL DESIGN: Quantitative differences in the glomerular proteomes of two unrelated murine nephropathy models in the defined stage of glomerular hypertrophy at onset of albuminuria were identified by 2-D DIGE and MALDI-TOF/TOF analysis. Investigated mouse models were (I): transgenic (tg) mice expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR(dn) ), a model of diabetes mellitus associated nephropathy and (II): growth hormone (GH)-tg mice, an established model of progressive glomerulosclerosis. RESULTS: In GIPR(dn) -tg mice, nine differentially abundant glomerular proteins were unambiguously identified, and eight in GH-tg mice (each versus controls). Four proteins (Annexin A4, Dihydropyrimidinase-related protein 2, Myosin regulatory light chain 2, Tropomyosin 1) displayed a congeneric differential glomerular abundance in both models, thus representing a common differential protein expression profile of glomerular hypertrophy at onset of albuminuria. The glomerular presence of these proteins was also detected in specimen of human focal and segmental glomerulosclerosis and diabetic nephropathy. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest a pathogenetic relevance of the identified proteins in early stages of chronic kidney diseases and their potential use as diagnostic markers.

Diabetic kidney lesions of GIPRdn transgenic mice: podocyte hypertrophy and thickening of the GBM precede glomerular hypertrophy and glomerulosclerosis.

Diabetic nephropathy is the leading cause of end-stage renal disease and the largest contributor to the total cost of diabetes care. Rodent models are excellent tools to gain more insight into the pathogenesis of diabetic nephropathy. In the present study, we characterize the age-related sequence of diabetes-associated kidney lesions in GIPR(dn) transgenic mice, a novel mouse model of early-onset diabetes mellitus. Clinical-chemical analyses as well as qualitative and quantitative morphological analyses of the kidneys of GIPR(dn) transgenic animals and nontransgenic littermate controls were performed at 3, 8, 20, and 28 wk of age. Early renal changes of transgenic mice consisted of podocyte hypertrophy, reduced numerical volume density of podocytes in glomeruli, and homogenous thickening of the glomerular basement membrane, followed by renal and glomerular hypertrophy as well as mesangial expansion and matrix accumulation. At 28 wk of age, glomerular damage was most prominent, including advanced glomerulosclerosis, tubulointerstitial lesions, and proteinuria. Real-time PCR demonstrated increased glomerular expression of Col4a1, Fn1, and Tgfb1. Immunohistochemistry revealed increased mesangial deposition of collagen type IV, fibronectin, and laminin. The present study shows that GIPR(dn) transgenic mice exhibit renal changes that closely resemble diabetes-associated kidney alterations in humans. Data particularly from male transgenic mice indicate that podocyte hypertrophy is directly linked to hyperglycemia, without the influence of mechanical stress. GIPR(dn) transgenic mice are considered an excellent new tool to study the mechanisms involved in onset and progression of diabetic nephropathy.

Synergy between CD26/DPP-IV inhibition and G-CSF improves cardiac function after acute myocardial infarction.

Ischemic cardiomyopathy is one of the main causes of death, which may be prevented by stem cell-based therapies. SDF-1alpha is the major chemokine attracting stem cells to the heart. Since SDF-1alpha is cleaved and inactivated by CD26/dipeptidylpeptidase IV (DPP-IV), we established a therapeutic concept--applicable to ischemic disorders in general--by combining genetic and pharmacologic inhibition of DPP-IV with G-CSF-mediated stem cell mobilization after myocardial infarction in mice. This approach leads to (1) decreased myocardial DPP-IV activity, (2) increased myocardial homing of circulating CXCR-4+ stem cells, (3) reduced cardiac remodeling, and (4) improved heart function and survival. Indeed, CD26 depletion promoted posttranslational stabilization of active SDF-1alpha in heart lysates and preserved the cardiac SDF-1-CXCR4 homing axis. Therefore, we propose pharmacological DPP-IV inhibition and G-CSF-based stem cell mobilization as a therapeutic concept for future stem cell trials after myocardial infarction.

Dominant-negative effects of a novel mutated Ins2 allele causes early-onset diabetes and severe beta-cell loss in Munich Ins2C95S mutant mice.

The novel diabetic mouse model Munich Ins2(C95S) was discovered within the Munich N-ethyl-N-nitrosourea mouse mutagenesis screen. These mice exhibit a T-->A transversion in the insulin 2 (Ins2) gene at nucleotide position 1903 in exon 3, which leads to the amino acid exchange C95S and loss of the A6-A11 intrachain disulfide bond. From 1 month of age onwards, blood glucose levels of heterozygous Munich Ins2(C95S) mutant mice were significantly increased compared with controls. The fasted and postprandial serum insulin levels of the heterozygous mutants were indistinguishable from those of wild-type littermates. However, serum insulin levels after glucose challenge, pancreatic insulin content, and homeostasis model assessment (HOMA) beta-cell indices of heterozygous mutants were significantly lower than those of wild-type littermates. The initial blood glucose decrease during an insulin tolerance test was lower and HOMA insulin resistance indices were significantly higher in mutant mice, indicating the development of insulin resistance in mutant mice. The total islet volume, the volume density of beta-cells in the islets, and the total beta-cell volume of heterozygous male mutants was significantly reduced compared with wild-type mice. Electron microscopy of the beta-cells of male mutants showed virtually no secretory insulin granules, the endoplasmic reticulum was severely enlarged, and mitochondria appeared swollen. Thus, Munich Ins2(C95S) mutant mice are considered a valuable model to study the mechanisms of beta-cell dysfunction and death during the development of diabetes.

Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis.

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.