Alterations of podocytes in a murine model of crescentic glomerulonephritis

Recent observations suggest a central role of podocytes in crescent formation. In experimental glomerulonephritis podocytes disrupt the parietal epithelial layer and attach on its basement membrane, thus forming bridges between the tuft and Bowman's capsule, and they are a major constituent of crescents. In order to explain these findings we hypothesize that inflammation triggers motility in podocytes. In the present study we asked whether podocytes display alterations which suggest a migratory behavior in glomerulonephritis. Glomerulonephritis was induced in mice by injection of a rabbit serum against the glomerular basement membrane. The kidneys were perfusion-fixed 6days later and examined by light and electron microscopy as well as by immunohistochemistry. In glomerulonephritis the apical cytoplasm of podocytes displayed numerous actin-containing microprotrusions. Cortactin, a protein involved in the regulation of actin polymerization, was predominantly expressed in foot processes of podocytes in control mice. It was redistributed to the cell body in glomerulonephritis. In untreated mice β1-integrin was restricted to the foot processes. In glomerulonephritis it was additionally found in the cytoplasm and in the apical cell membrane. Recycling of integrins is a crucial event in initiation of cell migration. ICAM-1 and CD44, the ligation of which induces migratory behaviors, were absent from healthy podocytes but expressed by some podocytes in glomerulonephritis. Thus, in glomerulonephritis podocytes display some characteristic features of migrating cells. This might explain their ability to break through the parietal epithelium and to become a constituent of early crescent

Novel Microscopic Techniques for Podocyte Research

Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement—a severe change of well-formed foot processes into flat and broadened processes—visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80–20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research

Visualization of serotonin effects on renal vessels of rats

Visualization of serotonin effects on renal vessels of rats. We studied the effects of serotonin (5-hydroxytryptamine, 5-HT) on glomerular blood flow (GBF) and on renal vessel diameters in the hydronephrotic kidney and in vascular casts of normal kidneys of rats. 5-HT (60 min after local application of 10-8 mol · liter-1) constricted the arcuate arteries (-10 ± 2% to -14 ± 2%, mean ± SEM), dilated the interlobular arteries (+13 ± 2%) and afferent arterioles (+17 ± 3%), and decreased GBF (-44 ± 5%). In contrast to normal autoregulation, reduction of renal perfusion pressure after local application of 5-HT from 118 ± 3 mm Hg by 10 and 20 mm Hg reduced GBF by 12 ± 2% and 23 ± 3%, respectively. The 5-HT2 antagonist, ritanserin (60 min after local application of 10-6 mol · liter-1), dilated all preglomerular vessels and increased GBF. In the presence of ritanserin, 5-HT lost nearly all vascular effects. During infusion of 5-HT (5 µg · min-1 i.v. for 20 min) vascular reactions were similar to those under local application. After cyclooxygenase inhibition with indomethacin, infusion of 5-HT failed to constrict the arcuate arteries whereas vasodilation of the small preglomerular vessels remained unaffected. Analyzing vascular casts of normal kidneys we observed considerable vascular spasms and an average vasoconstriction of the interlobar arteries of 19 ± 9% after i.v. infusion of 5-HT. We believe that 5-HT decreases GBF by 5-HT2 receptor-mediated constriction of the large renal vessels which are modulated by the prostaglandin system, whereas 5-HT dilates the small preglomerular vessels, most likely via 5-HT1-like receptors. Furthermore, our data indicate that 5-HT impairs the myogenic component of renal autoregulation in the low pressure range

Studying the role of fascin-1 in mechanically stressed podocytes

Glomerular hypertension causes glomerulosclerosis via the loss of podocytes, which are challenged by increased mechanical load. We have demonstrated that podocytes are mechanosensitive. However, the response of podocytes to mechanical stretching remains incompletely understood. Here we demonstrate that the actin-bundling protein fascin-1 plays an important role in podocytes that are exposed to mechanical stress. Immunofluorescence staining revealed colocalization of fascin-1 and nephrin in mouse kidney sections. In cultured mouse podocytes fascin-1 was localized along actin fibers and filopodia in stretched and unstretched podocytes. The mRNA and protein levels of fascin-1 were not affected by mechanical stress. By Western blot and 2D-gelelectrophoresis we observed that phospho-fascin-1 was significantly downregulated after mechanical stretching. It is known that phosphorylation at serine 39 (S39) regulates the bundling activity of fascin-1, e.g. required for filopodia formation. Podocytes expressing wild type GFP-fascin-1 and non-phosphorylatable GFP-fascin-1-S39A showed marked filopodia formation, being absent in podocytes expressing phosphomimetic GFP-fascin-1-S39D. Finally, the immunofluorescence signal of phosphorylated fascin-1 was strongly reduced in glomeruli of patients with diabetic nephropathy compared to healthy controls. In summary, mechanical stress dephosphorylates fascin-1 in podocytes in vitro and in vivo thereby fascin-1 may play an important role in the adaptation of podocytes to mechanical forces

Comparative Analysis of Podocyte Foot Process Morphology in Three Species by 3D Super-Resolution Microscopy

Since the size selectivity of the filtration barrier and kidney function are highly dependent on podocyte foot process morphology, visualization of foot processes is important. However, the size of foot processes is below the optical resolution of light microscopy. Therefore, electron microcopy has been indispensable to detect changes in foot process morphology so far, but it is a sophisticated and time-consuming technique. Recently, our group has shown that 3D structured illumination microscopy (3D-SIM), a super-resolution microscopy (SRM) technique, can visualize individual foot processes in human biopsies. Moreover, we have developed a software-based approach to directly quantify the structure of podocyte foot processes named Podocyte Exact Morphology Measurement Procedure (PEMP). As shown in patients suffering from minimal change disease (MCD), PEMP allows the quantification of changes of the foot process morphology by measuring the filtration slit density (FSD). Since rodents are frequently used in basic research, we have applied PEMP to quantify foot processes of mice and rats. Comparative analysis of nephrin-stained kidneys from humans, rats, and mice showed significant differences of the FSD. The highest FSD was measured in mice (3.83 ± 0.37 μm−1; mean ± SD) followed by rats (3.36 ± 0.42 μm−1) and humans (3.11 ± 0.26 μm−1). To demonstrate that PEMP can be used to determine foot process morphology also in affected animals, we measured the FSD in palladin-knockout mice on a 129S1 genetic background compared to wild-type littermates. Taken together, we established a method for the quick and exact quantification of podocyte foot process morphology which can be applied to diagnosis and basic research

ExprEssence - Revealing the essence of differential experimental data in the context of an interaction/regulation net-work

<p>Abstract</p> <p>Background</p> <p>Experimentalists are overwhelmed by high-throughput data and there is an urgent need to condense information into simple hypotheses. For example, large amounts of microarray and deep sequencing data are becoming available, describing a variety of experimental conditions such as gene knockout and knockdown, the effect of interventions, and the differences between tissues and cell lines.</p> <p>Results</p> <p>To address this challenge, we developed a method, implemented as a Cytoscape plugin called <it>ExprEssence</it>. As input we take a network of interaction, stimulation and/or inhibition links between genes/proteins, and differential data, such as gene expression data, tracking an intervention or development in time. We condense the network, highlighting those links across which the largest changes can be observed. Highlighting is based on a simple formula inspired by the law of mass action. We can interactively modify the threshold for highlighting and instantaneously visualize results. We applied <it>ExprEssence </it>to three scenarios describing kidney podocyte biology, pluripotency and ageing: 1) We identify putative processes involved in podocyte (de-)differentiation and validate one prediction experimentally. 2) We predict and validate the expression level of a transcription factor involved in pluripotency. 3) Finally, we generate plausible hypotheses on the role of apoptosis, cell cycle deregulation and DNA repair in ageing data obtained from the hippocampus.</p> <p>Conclusion</p> <p>Reducing the size of gene/protein networks to the few links affected by large changes allows to screen for putative mechanistic relationships among the genes/proteins that are involved in adaptation to different experimental conditions, yielding important hypotheses, insights and suggestions for new experiments. We note that we do not focus on the identification of 'active subnetworks'. Instead we focus on the identification of single links (which may or may not form subnetworks), and these single links are much easier to validate experimentally than submodules. <it>ExprEssence </it>is available at <url>http://sourceforge.net/projects/expressence/</url>.</p

Low Serum Testosterone Is Associated with Increased Mortality in Men with Stage 3 or Greater Nephropathy

Background: Chronic kidney disease (CKD) and low serum total testosterone (TT) concentrations are independent predictors of mortality risk in the general population, but their combined potential for improved mortality risk stratification is unknown. Methods: We used data of 1,822 men from the population-based Study of Health in Pomerania followed- up for 9.9 years (median). The direct effects of kidney dysfunction (estimated glomerular filtration rate 2), albuminuria (urinary albumin-creatinine ratio ≧2.5 mg/mmol) and their combination (CKD) on all-cause and cardiovascular mortality were analyzed using multivariable Cox regression models. Serum TT concentrations below the age-specific 10th percentile (by decades) were considered low and were used for further risk stratification. Results: Kidney dysfunction (hazard ratio, HR, 1.40; 95% confidence interval, CI, 1.02–1.92), albuminuria (HR, 1.38; 95% CI, 1.06–1.79), and CKD (HR, 1.42; 95% CI, 1.09–1.84) were associated with increased all-cause mortality risk, while only kidney dysfunction (HR, 2.01; 95% CI, 1.21–3.34) was associated with increased cardiovascular mortality risk after multivariable adjustment. Men with kidney dysfunction and low TT concentrations were identified as high-risk individuals showing a more than 2-fold increased all-cause mortality risk (HR, 2.52; 95% CI, 1.08–5.85). Added to multivariable models, nonsignificant interaction terms suggest that kidney dysfunction and low TT are primarily additive rather than synergistic mortality risk factors. Conclusion: In the case of early loss of kidney function, measured TT concentrations might help to detect high-risk individuals for potential therapeutic interventions and to improve mortality risk assessment and outcome

BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function

Podocyte loss and changes to the complex morphology are major causes of chronic kidney disease (CKD). As the incidence is continuously increasing over the last decades without sufficient treatment, it is important to find predicting biomarkers. Therefore, we measured urinary mRNA levels of podocyte genes NPHS1, NPHS2, PODXL and BDNF, KIM-1, CTSL by qRT-PCR of 120 CKD patients. We showed a strong correlation between BDNF and the kidney injury marker KIM-1, which were also correlated with NPHS1, suggesting podocytes as a contributing source. In human biopsies, BDNF was localized in the cell body and major processes of podocytes. In glomeruli of diabetic nephropathy patients, we found a strong BDNF signal in the remaining podocytes. An inhibition of the BDNF receptor TrkB resulted in enhanced podocyte dedifferentiation. The knockdown of the orthologue resulted in pericardial oedema formation and lowered viability of zebrafish larvae. We found an enlarged Bowman's space, dilated glomerular capillaries, podocyte loss and an impaired glomerular filtration. We demonstrated that BDNF is essential for glomerular development, morphology and function and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential CKD biomarker

Extensive alterations of the whole-blood transcriptome are associated with body mass index: results of an mRNA profiling study involving two large population-based cohorts

Background: Obesity, defined as pathologically increased body mass index (BMI),is strongly related to an increased risk for numerous common cardiovascular and metabolic diseases. It is particularly associated with insulin resistance, hyperglycemia, and systemic oxidative stress and represents the most important risk factor for type 2 diabetes (T2D). However, the pathophysiological mechanisms underlying these associations are still not completely understood. Therefore, in order to identify potentially disease-relevant BMI-associated gene expression signatures, a transcriptome-wide association study (TWAS) on BMI was performed. Methods: Whole-blood mRNA levels determined by array-based transcriptional profiling were correlated with BMI in two large independent population-based cohort studies (KORA F4 and SHIP-TREND) comprising a total of 1977 individuals. Results: Extensive alterations of the whole-blood transcriptome were associated with BMI: More than 3500 transcripts exhibited significant positive or negative BMI-correlation. Three major whole-blood gene expression signatures associated with increased BMI were identified. The three signatures suggested: i) a ratio shift from mature erythrocytes towards reticulocytes, ii) decreased expression of several genes essentially involved in the transmission and amplification of the insulin signal, and iii) reduced expression of several key genes involved in the defence against reactive oxygen species (ROS). Conclusions: Whereas the first signature confirms published results, the other two provide possible mechanistic explanations for well-known epidemiological findings under conditions of increased BMI, namely attenuated insulin signaling and increased oxidative stress. The putatively causative BMI-dependent down-regulation of the expression of numerous genes on the mRNA level represents a novel finding. BMI-associated negative transcriptional regulation of insulin signaling and oxidative stress management provide new insights into the pathogenesis of metabolic syndrome and T2D