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

Kv2.1 channels prevent vasomotion and safeguard myogenic reactivity in rat small superior cerebellar arteries

Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and ‘multiplex’ single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10–100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response

Leukocyte telomere length and mitochondrial DNA copy number associate with endothelial function in aging-related cardiovascular disease

BackgroundWe investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD).MethodsIn total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis.Resultscf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P < 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (β = 0.229, P = 0.002) and leu-mtDNA (β = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (β = −0.426, P < 0.0001).ConclusionTL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction

Epac-mediated relaxation in murine basilar arteries depends on membrane permeability of cyclic nucleotide analogues and endothelial aging

Cerebral blood supply is finely tuned by regulatory mechanisms depending on vessel caliber the disruption of which contributes to the development of diseases such as vascular dementia, Alzheimer's and Parkinson 's diseases. This study scopes whether cAMP-mimetic-ligands relax young and aged murine cerebral arteries, whether this relates to the activation of PKA or Epac signaling pathways and is changed with advanced age. The hormone Urocortin-1 relaxed submaximally contracted young and old basilar arteries with a similar pD(2) and DmAx (similar to -8.5 and similar to 90% in both groups). In permeabilized arteries, PKA activation by 6-Bnz-cAMP or Epac activation by 8-pCPT-2'-O-Me-cAMP also induced relaxation with pD(2) of -6.3 vs. -5.8 in old for PKA-ligands, and -4.4 and -4.0 in old for Epac-ligands. Furthermore, aging significantly increased submaximal Ca2+-induced force. The effect of 8-pCPT-2'-O-Me-cAMP on intact arteries was attenuated by aging or nitric oxide synthase inhibition. No relaxing effect in both age-groups was observed after treatment with PKA-activator, Sp-6-Phe-cAMPS. In conclusion, our results suggest that in intact basilar arteries relaxation induced by cAMP-mimetics refers only to the activation of Epac and is impaired by smooth muscle and endothelial aging. The study presents an interesting option allowing therapeutic discrimination between both pathways, possibly for the exclusive activation of Epac in brain circulatory system

Endothelin-1-induced activation of rat renal pelvic contractions depends on cyclooxygenase-1 and Rho kinase

Upper urinary tract peristalsis is generated in the proximal renal pelvis that connects to the renal parenchyma at the pelvis-kidney junction. It may be exposed to the high renal endothelin-1 (ET-1) concentrations. Dietary NaCl restriction increases renal pelvic ETA receptor expression. We investigated the contribution of ETA and ETB receptors to ET-1-stimulated rat renal pelvic contractions and whether the sensitivity of renal pelvic contractile activity to ET-1 stimulation increases with dietary NaCl restriction. We tested whether ET-1-induced contractile activity depends on cyclooxygenase (COX)-1 or -2 and to what extent spontaneous as well as agonist-induced peristalsis depends on Rho kinases (ROCK). Contractions of isolated renal pelvises were investigated by myography. ET-1 concentration-dependently increased pelvic contractile activity up to 400% of basal activity. ETA but not ETB receptor blockade inhibited ET-1-induced pelvic contractions. Basal and ET-1-stimulated contractions were similar in renal pelvises from rats on a high-NaCl diet or on a NaCl-deficient diet. COX-1 inhibition reduced spontaneous and almost completely blocked the ET-1-induced pelvic contractions. ROCK inhibition reduced spontaneous and ET-1 stimulated pelvic contractile activity by 90%. RT-PCR revealed that both ROCK isoenzymes are present in the renal pelvic wall. Western blot analyses did not show increased phosphorylation of ROCK substrates myosin phosphatase target subunit 1, ezrin, radixin, and moesin in ET-1-treated isolated renal pelvises. ET-1 is a powerful ETA receptor-dependent activator of renal pelvic contractions. COX-1 and ROCK activity are required for the ET-1 effects on pelvic contractions, which are not significantly affected by dietary NaCl intake