PV-1 is a novel endothelial protein shown by immunocytochemical tests to be specifically associated with the stomatal diaphragms of caveolae in lung endothelium. Although the highest expression levels of both mRNA and protein are in the lung, PV-1 also has been found to be expressed in other organs. Using a specific antibody to the extracellular domain of PV-1, we have extended the survey on the presence of this protein at light and electron microscope level in several rat organs. Here we show that by immunofluorescence the antibody recognizes with high specificity the endothelium of the fenestrated peritubular capillaries of the kidney and those of the intestinal villi, pancreas, and adrenals. By immunolocalization at electron microscope level, the antibody recognizes specifically the diaphragms of the fenestrae and the stomatal diaphragms of caveolae and transendothelial channels in the endothelia of these vascular beds. No signal was detected in the continuous endothelium of the heart, skeletal muscle, intestinal muscularis, or brain capillaries or the nondiaphragmed fenestrated endothelium of kidney glomeruli. Taken together, our findings define the only antigen to be localized thus far in fenestral diaphragms. They also show that the stomatal diaphragms of caveolae and transendothelial channels and the fenestral diaphragms might be biochemically related, in addition to being morphologically similar structures.

Malignant brain tumors exhibit distinct metabolic characteristics. Despite high levels of lactate, the intracellular pH of brain tumors is more alkaline than normal brain. Additionally, with increasing malignancy, brain tumors display intratumoral hypoxia. Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes that are induced by tissue hypoxia. They participate in regulation of pH homeostasis by catalyzing the reversible hydration of carbon dioxide. The aim of our study was to investigate whether brain tumors of different histology and grade of malignancy express elevated levels of CA IX and XII as compared to normal brain. We analyzed 120 tissue specimens from brain tumors (primary and metastatic) and normal brain for CA IX and XII expression by immunohistochemistry, Western blot, and in situ hybridization. Whereas normal brain tissue showed minimal levels of CA IX and XII expression, expression in tumors was found to be upregulated with increased level of malignancy. Hemangioblastomas, from patients with von Hippel-Lindau disease, also displayed high levels of CA IX and XII expression. Comparison of CA IX and XII staining with HIF-1alpha staining revealed a similar microanatomical distribution, indicating hypoxia as a major, but not the only, induction factor. The extent of CA IX and XII staining correlated with cell proliferation, as indicated by Ki67 labeling. The results demonstrate that CA IX and XII are upregulated in intrinsic and metastatic brain tumors as compared to normal brain tissue. This may contribute to the management of tumor-specific acid load and provide a therapeutic target.

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development.

The peripheral nervous system and the immune system were shown to have neurohumoral interactions. This study extends observations that demonstrated neuronal modulation of spontaneous interleukin-6 (IL-6) secretion in the spleen by norepinephrine (NE) and β-endorphin. Spontaneous IL-6 secretion in vivo was markedly reduced by removal of macrophages with the clodronate technique. Furthermore, spontaneous IL-6 secretion was significantly inhibited at physiological concentrations of cortisol (10−7 M). In the presence of 10−7 M cortisol, addition of norepinephrine (NE; 10−5 M) and isoproterenol (10−6 and 10−5 M) significantly increased spontaneous IL-6 secretion (+20%; P = 0.0280, P = 0.0005, and P = 0.0050, respectively). In contrast, addition of β-endorphin significantly inhibited spontaneous IL-6 secretion in the presence of 10−7 M cortisol (−40%; 10−11 M, P = 0.0410; 10−10 M, P = 0.0005). To study the effect of endogenously released transmitters on spontaneous IL-6 secretion, spleen slices were electrically stimulated with 1, 5, 10, 50, and 100 Hz. Spontaneous IL-6 secretion was markedly reduced at a frequency of 10 Hz with 10−7 M cortisol present ( P < 0.0001). This indicates that the combination of nerve firing at 5–10 Hz and physiological cortisol conditions inhibits spontaneous IL-6 secretion. Inhibition of spontaneous IL-6 secretion from spleen macrophages is most probably due to a net inhibitory effect of opioidergic transmission under these conditions.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Background: The pathophysiology of ischemic cerebral lesions following aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. There is growing evidence that inflammatory reactions could be involved in the pathogenesis of such delayed occurring ischemic lesions. The aim of this study was to evaluate adhesion molecules with regard to these lesions following SAH. Methods: Serum and cerebrospinal fluid (CSF) samples were taken daily from 15 patients up to day 9 after SAH and evaluated for intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). Results: CSF and serum samples correlated well during nearly the whole time course (p < 0.0001). A secondary increase in ICAM-1 and VCAM-1 in the serum and CSF correlated with an increase in flow velocity in the transcranial Doppler (p > 0.0001 and p < 0.007) but not to a delayed lesion in the CT scan. Conclusion: We believe that inflammatory processes are involved in the pathogenesis of cerebral vasospasm but they might only be a part of a multifactorial pathogenesis.

The development of the renal vascular system requires the coordinated action of soluble morphogenic factors and specific extracellular matrix components. Despite intensive research it remains unknown whether the humoral or the environmental component is more important in the development of renal microvessels. The prolonged serum-free culture of embryonic kidney cortex explants was achieved by means of a newly developed perfusion culture system. This system made the investigation of renal vascular development under defined organotypic conditions possible. Thus, growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hormones (aldosterone, vitamin D3) could be applied without the interference with serum components. Medium supplementation with VEGF or aldosterone in combination with vitamin D3 resulted in the coordinated proliferation of endothelial cells in the explant. A well-developed collecting duct epithelium and numerous tubular structures were always observed. In contrast, only a uniform cell layer was found between fibrous organ capsule and the collecting duct epithelium after bFGF application, but neither tubular structures nor endothelial cells. Thus, the experiments indicate that bFGF alone has no stimulating effect on the growth of the renal microvasculature under perfusion culture conditions.