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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.

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.

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPRdn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPRdn 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 GIPRdn 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 GIPRdn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPRdn 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. GIPRdn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.

The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies. Rodent models are most widely used but have limitations in translational research. Porcine models have the potential to bridge the gap between basic studies and clinical trials in human patients. This article provides an overview of concepts for the development of porcine models for diabetes and obesity research, with a focus on genetically engineered models. Diabetes-associated ocular, cardiovascular and renal alterations observed in diabetic pig models are summarized and their similarities with complications in diabetic patients are discussed. Systematic multi-organ biobanking of porcine models of diabetes and obesity and molecular profiling of representative tissue samples on different levels, e.g., on the transcriptome, proteome, or metabolome level, is proposed as a strategy for discovering tissue-specific pathomechanisms and their molecular key drivers using systems biology tools. This is exemplified by a recent study providing multi-omics insights into functional changes of the liver in a transgenic pig model for insulin-deficient diabetes mellitus. Collectively, these approaches will provide a better understanding of organ crosstalk in diabetes mellitus and eventually reveal new molecular targets for the prevention, early diagnosis and treatment of diabetes mellitus and its associated complications.

The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. Using a commercially available optical reflectometer (F20, Filmetrics®, USA), the thicknesses of 0.5 μm thick semi-thin Epon (glycid ether)-sections and of 1-3 μm thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters.

In ecotoxicology, evaluation of toxicities and no observed effect concentrations (NOEC) of test compounds in experimental fish is commonly based on molecular-, biochemical- and analytical chemistry analyses of organ/tissue samples and the assessment of (histo-) pathological lesions. Standardization of organ/tissue sampling locations, sample numbers, and sample processing contributes to warrant the reproducibility and inter- and intra-study comparability of analysis results. The present article provides the first comprehensive tissue sampling guidelines specifically adapted to rainbow trout ( Oncorhynchus mykiss ) as a frequently used fish species in ecotoxicological studies. A broad spectrum of ~40 different organs and tissues is covered. Appropriate sampling locations, sample sizes and sample numbers for subsequent routine histopathological evaluation (all organs/tissue) and for molecular analyses (~30 organs/tissues) are described in detail and illustrated with schematic drawings and representative macroscopic and histological images. These field-proven sampling guidelines were developed based on the pertinent literature and practical experience in ecotoxicological fish studies. They are intended to serve as a standard reference for any routine ecotoxicological study using rainbow trout as a test system. A broad application of the featured tissue sampling procedures will help to improve the reproducibility of analyses and to reduce inter- and intra-study variability induced by sampling bias and (normal) inter-sample morphological variation, and will therefore provide a robust basis for reliable characterization of toxicity and NOEC identification of diverse test substances and aquatic pollutants.

Rainbow trout ( Oncorhynchus mykiss ) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium ( i . e ., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.

Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was crucial to maintaining glomerular podocyte function, while dysregulation of mTOR facilitated glomerular diseases. Genetic deletion of mTOR complex 1 (mTORC1) in mouse podocytes induced proteinuria and progressive glomerulosclerosis. Furthermore, simultaneous deletion of both mTORC1 and mTORC2 from mouse podocytes aggravated the glomerular lesions, revealing the importance of both mTOR complexes for podocyte homeostasis. In contrast, increased mTOR activity accompanied human diabetic nephropathy, characterized by early glomerular hypertrophy and hyperfiltration. Curtailing mTORC1 signaling in mice by genetically reducing mTORC1 copy number in podocytes prevented glomerulosclerosis and significantly ameliorated the progression of glomerular disease in diabetic nephropathy. These results demonstrate the requirement for tightly balanced mTOR activity in podocyte homeostasis and suggest that mTOR inhibition can protect podocytes and prevent progressive diabetic nephropathy.

‘Autosomal dominant tubulointerstitial kidney disease – UMOD ’ (ADTKD- UMOD ) is caused by impaired maturation and secretion of mutant uromodulin (UMOD) in thick ascending limb of Henle loop (TAL) cells, resulting in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). To gain insight into pathophysiology, we analysed proteome profiles of TAL-enriched outer renal medulla samples from ADTKD- UMOD and control mice by quantitative LC-MS/MS. In total, 212 differentially abundant proteins were identified. Numerous ER proteins, including BiP (HSPA5), phosphorylated eIF2α (EIF2S1), ATF4, ATF6 and CHOP (DDIT3), were increased abundant, consistent with UPR. The abundance of hypoxia-inducible proteins with stress survival functions, i.e. HYOU1, TXNDC5 and ERO1L, was also increased. TAL cells in ADTKD- UMOD showed a decreased proportion of mitochondria and reduced abundance of multiple mitochondrial proteins, associated with disturbed post-translational processing and activation of the mitochondrial transcription factor NRF1. Impaired fission of organelles, as suggested by reduced abundance of FIS1, may be another reason for disturbed biogenesis of mitochondria and peroxisomes. Reduced amounts of numerous proteins of the OXPHOS and citrate cycle pathways, and activation of the LKB1-AMPK-pathway, a sensor pathway of cellular energy deficits, suggest impaired energy homeostasis. In conclusion, our study revealed secondary mitochondrial dysfunction in ADTKD- UMOD.

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.