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Determinants of vascular permeability in the kidney glomerulus.

The human kidneys filter 70 liters of blood plasma every day. The hallmark of almost all kidney diseases, whether acquired or genetic, is the leakage of plasma proteins into the urine because of alterations in the glomerular filtration unit of the kidney. In this regard, the human mutations in nephrin, podocin, alpha-actinin-4, COL4A3, and COL4A5 genes expressed in the glomeruli have been implicated to cause alterations in glomerular filtration apparatus. Nevertheless, the expression of these proteins in relation to each other in mouse models for glomerular vascular leak is unknown. Additionally, within the glomerulus, the central question of whether the primary filtration barrier is the basement membrane or the epithelial slit diaphragm remains ambiguous. Therefore, in this study, we examined the localization and expression of glomerular epithelial slit diaphragm and glomerular basement membrane proteins implicated in glomerular vascular leak using mice deficient in either the alpha3 chain of type IV collagen, the major constituent of glomerular basement membrane, or LMX1B transcription factor, which regulates the expression of key glomerular type IV collagen genes COL4A3 and COL4A4 or nephrin, a glomerular epithelial slit diaphragm-associated protein. This study demonstrates that decreased expression of slit diaphragm protein, nephrin, correlates with a loss of glomerular filter integrity. Additionally, we demonstrate that defects induced by proteins of glomerular basement membrane lead to an insidious plasma protein leak, whereas the defects induced by proteins in the glomerular epithelial slit diaphragms lead to a precipitous plasma protein leak.

Adaptor Proteins, Signal Transducing↗

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

Chromatography, Liquid↗

Vascular anatomy of the pig kidney glomerulus: a qualitative study of corrosion casts.

Pig kidney glomerular vascular anatomy was studied by scanning electron microscopy of vascular corrosion casts. A generalized vascular architecture is presented to describe the pig kidney glomerulus based upon the observation of 3,800 vascular cast glomeruli. The relative simplicity of the pig glomerular vascular architecture has allowed the characterization of different vascular segments more completely than has been possible in other mammals. Based upon relationships to the afferent arteriole, a nomenclature and definition of primary, secondary, tertiary and anastomotic vessels is proposed for the distributing vessels comprising the glomerular tuft. The existence and formation of a large central hemispheric vessel deep within the confines of a glomerular hemisphere is micrographically documented. Micrographic evidence is presented supporting the formation of the single efferent arteriole by the merging of two central hemispheric vessels within the confines of the glomerular tuft. Failure of the merging of these two vessels may result in multiple efferent arterioles.

Animals↗

11beta-hydroxysteroid dehydrogenase type 2 is expressed in the human kidney glomerulus.

Our previous study demonstrated that the GR is expressed in the human kidney glomerulus. The function of the GR of glomerular cells might be affected by the concentration of intracellular glucocorticoids, which is modulated by 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2). Because the expression of 11betaHSD2 in the glomerular cells remains unclear, we used competitive RT-PCR and immunoblotting to detect the expression of 11betaHSD2 mRNA and protein in isolated human glomeruli, in whole kidney cortex as a positive control, and in a human glomerular visceral epithelial cell line. 11betaHSD2 mRNA was detected in all samples. Specific antihuman 11betaHSD2 antibody recognized a single band at 41 kDa, consistent with the molecular mass of human 11betaHSD2, in the samples of the isolated glomeruli and whole kidney cortex. Furthermore, definite 11betaHSD2 enzymatic activity was also determined with the sample of isolated glomeruli. We also performed immunohistochemistry by light and electron microscopy to determine the cellular and subcellular localization of 11betaHSD2 in the human glomeruli. Immunoreactivity of the enzyme was clearly observed in the glomerular visceral epithelial cells and endothelial cells as well as in the distal convoluted tubules and collecting ducts. The subcellular localization of 11betaHSD2 was shown to be endoplasmic reticulum. These results suggest that 11betaHSD2 might play a crucial role in modulating the intracellular concentration of glucocorticoids in human glomerular cells.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

[Electrophoretic analysis of rat kidney glomerulus basal membrane].

A technique was developed for analysis of basal membrane proteins from rat kidney glomerulus using two-dimensional electrophoresis by O'Farrell. Basal membranes were isolated from the glomerulus by means of detergent treatment. Various procedures of basal membrane solubilization were studied. About 25 protein fractions with molecular mass from 25 kDa to 330 kDa were detected in the basal membrane after single-dimensional electrophoresis in presence of sodium dodecyl-sulfate. Two-dimensional electrophoresis of the basal membrane proteins and staining of gels with silver nitrate enabled to detect approximately 50 polypeptide fractions with molecular mass from 25 kDa up to 250 kDa at pH 5-7.

Animals↗

Large-scale identification of genes implicated in kidney glomerulus development and function.

To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large-scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non-glomerular kidney tissue, FACS-sorted podocytes and brain capillaries, which identified over 300 glomerular cell-enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus-enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

Animals↗

SPARC and the kidney glomerulus: matricellular proteins exhibit diverse functions under normal and pathological conditions.

In the last decade, numerous studies have emphasized the important functions that matricellular proteins subserve during angiogenesis, wound healing, and the maintenance of organ and tissue integrity. Matricellular proteins are defined as a group of secreted regulatory macromolecules that are not structural components of the extracellular matrix (ECM) but rather mediate interactions between the ECM and cells. One of these matricellular proteins, termed SPARC (secreted protein acidic and rich in cysteine), is produced during the process of wound healing and is prominent in several types of injury. An excessive deposition of glomerular matrix and an elevated proliferation of certain glomerular cells characterize a variety of kidney diseases. The proliferation of these cells is associated typically with the remodeling process that occurs after kidney injury, and is, at least in part, modulated by the altered expression of ECM, various growth factors, and the elevated production of matricellular proteins (e.g., SPARC). The secretion of one or more of the matricellular proteins can lead to expansion of the glomerular basement membrane, infiltration of immunocompetent cells, and, in some cases, to a reversal of the pathological condition. However, these proteins can also contribute collectively to renal fibrosis, glomerulosclerosis, glomerulonephritis, and the eventual loss of renal function. The purpose of this review is to evaluate the multiple functions of SPARC in the kidney glomerulus under normal and pathological conditions.

Animals↗

Effects of hypophysectomy on age-related changes in the rat kidney glomerulus: observations by scanning and transmission electron microscopy.

The kidney glomeruli of 9-month-old intact, 23-month-old intact, and 23-month-old hypophysectomized female rats were examined by scanning and transmission electron microscopy. With increasing age, the glomeruli increased in diameter and more podocyte microvilli were found. Hypophysectomy (4 months before sacrifice) reduced these values to levels approaching values seen in the 9-month intact controls. Transmission electron microscopy revealed that increased numbers of podocytes and endothelial cells had cytoplasmic dense bodies with advancing age. Granular cytoplasmic material, seen in podocytes, did not alter in frequency with age. Hypophysectomy reduced the number of cells containing the dense bodies as well as the number of podocytes containing granular material. Basal laminar thickness, while increasing with age, was unaffected by hypophysectomy. The results show that hypophysectomy can return some structural age-related changes to values seen in younger subjects. The effects are similar in some respects to the effects of dietary restriction.

Aging↗

[Functional morphology of the kidney. A review of the histophysiology of the kidney glomerulus, the nephrons and the collecting tubule system].

The kidney is a composite organ, the specific activities of which are attributable to the peculiar construction of the individual components of the glomerula, nephrons and collecting tubules. This comprehensive review will address the essential physiological mechanisms of urine formation, such as filtration, secretion, reabsorption and concentration, and correlate these functions to morphological structures where possible. The complex structure of the renal glomerulus as the basis for the formation of primary urine (blood-urine barrier) will be documented on the basis of electron micrographs. In addition to this, the ultrastructure of the epithelium in the various tubular segments and collecting tubules will be discussed from a histophysiological stand-point, including its significance in the excretion of waste substances and maintenance of a constant fluid environment in the body (Homeostasis).

Animals↗

[Interaction between guanylate cyclases in the kidney glomerulus].

Cyclic GMP mediates regulation of the basic functions in the kidney. The membrane- and cytosolic (soluble) guanylate cyclase systems in the kidney glomeruli modulate reciprocally their activities. In physiological conditions, this compensatory regulation results in maintaining the stable cGMP level.

Animals↗