Human Glomeruli Research Articles

Application of Scanning Electron Microscopy for observing glomerulus maturation in human kidney

The ultrastructurg of the mammalian kidney has been explored by many authors, both in adult and fetal animals. As one of the most feasible means to elucidate the complicated three dimensional structure, scanning electron microscopy (SEM) has played an important role in the study of the kidney especially on the morphology of renal glomeruli. So far none of the authors has applied SEM to the human fetus for investigating the development of the glomerulus. In the present report SEM has been used to demonstrate the structure of human fetal metanephric glomeruli with special reference to the morphological differentiation of the podocyte.Materials and Methods: The metanephric kidneys were collected from 4 human fetuses ranging from 11 to 14 weeks of gestation, 2-12 h after death following therapeutic or spontaneous abortions. All specimens were fixed with 2.5% glutaraldehyde in phosphate buffer and cut into small pieces with a razor blade.

Localization and quantification of [125I]-endothelin binding sites in human fetal and adult kidneys — Relevance to renal ontogeny and pathophysiology

Endothelins, 21 amino acid peptides, produced by endothelial cells are potent vasoconstrictors and mitogens. According to experimental studies in animals, endothelins seem to be involved in the regulation of renal hemodynamics. In order to gain insight into its potential effects in man, a quantitative analysis of its binding sites was performed in human kidneys. Because of the proliferative action of endothelin in cell culture we also compared binding sites in fetal and adult kidneys. Binding sites for [125I]-endothelin-1,2,3 were visualized by in-vitro autoradiography and quantified by densitometry. In both adult and fetal tissue, specific binding sites occurred in the cortex, medulla, and renal vessels. Unlabeled endothelins and sarafotoxin, a peptide with a high sequence homology to endothelins, inhibited [125I]-endothelin-1 binding with IC50 in the 9.8 to 0.023 nM range, whereas unrelated peptides (angiotensin II, atrial natriuretic peptide) and the calcium antagonist nitrendipine failed to compete for [125I]-endothelin-1 binding sites. Linear Scatchard analysis revealed that the number of binding sites (expressed per tissue equivalent: TE) were consistently higher in fetal than in adult kidneys, while affinities did not differ significantly in cortex, medulla, and vessels (fetal/adult: cortex KD 43.4 +/- 19.6/55.9 +/- 16.7 nM; BMax 13.5 +/- 7.8/2.7 +/- 1.3 fmol/mg TE; medulla KD 26.3 +/- 10.9/34.6 +/- 7.4 nM; BMax 10.1 +/- 0.9/3.7 +/- 1.1 fmol/mg TE; vessels KD 41.1 +/- 22.9/23.7 +/- 8.1 nM; BMax 12.9 +/- 3.9/4.1 +/- 1.2 fmol/mg TE). Medullary capillaries and veins showed strong binding in human and rat kidneys which may be important for the pathophysiology of acute renal failure. Human adult and fetal glomeruli had only a few binding sites. This contrasts to findings in the rat kidney in which glomeruli have a high concentration of endothelin binding sites; although this does not role out an influence per se, it does point out the need to subject the assumption of a relevant glomerular effect of endothelin in man to closer scrutiny. The diffuse and strong binding in fetal kidney may indicate a role for endothelin in the process of renal maturation.

12-Lipoxygenase Products Are Potent Inhibitors of Prostacyclin-Induced Renin Release

In isolated human or rat glomeruli, arachidonic acid can be metabolized by the cyclooxygenase pathway to prostaglandins or by the lipoxygenase pathway to hydroxyeicosatetraenoic acids (HETES). We have recently shown that 12-lipoxygenase products are potent inhibitors of renin release. Since prostacyclin (PGI2) is a potential renin secretagogue, we studied the direct effects of 12-lipoxygenase products on prostacyclin-induced renin secretion. Treatment of rat renal cortical slices with picomolar concentrations of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-HETE blocked the prostacyclin- or iloprost (an analog of PGI2)-induced renin secretion. The inhibitory effects of 12-lipoxygenase products were not exhibited by the 5-lipoxygenase-derived products, leukotriene B4 and 5-HPETE. These results suggest that HETES are not only potent modulators of prostacyclin actions on renin, but that the concerted actions of these compounds in cells may be critical determinants of the juxtaglomerular cell secretion of renin.

Receptor mediated uptake of apo B and apo E rich lipoproteins by human glomerular epithelial cells

Various pathological disorders are accompanied by the deposition of lipids into glomerular cells. To gain insight into these disorders, it is essential to know if glomerular cells possess lipoprotein receptors. We therefore characterized the activity of lipoprotein receptors in cultured epithelial cells of the human glomerulus. Podocytes were chosen as they are directly exposed to lipoproteins in pathological states like in glomerular proteinuria (such as, nephrotic syndrome). Isolated human glomeruli (purity greater than 95%) were incubated in buffered RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum at 37 degrees C and 5% CO2. Outgrowing cells were vimentin and keratin positive. Monolayer cultures of human glomerular epithelial cells upon incubation in lipoprotein deficient serum for 48 hours expressed a receptor-dependent uptake of lipoproteins. These cells showed about 10% of the maximal capacity for LDL uptake as compared to fibroblasts; however, the Km values for binding, internalization and degradation were similar in the cultures of glomerular epithelial cells and fibroblasts. The Km values for degradation of LDL, chylomicron remnants, beta-VLDL from cholesterol-fed rabbits and VLDL from familial LCAT-deficiency patients were 14.2, 4.9, 2.9, 4.5 micrograms protein/ml medium, respectively, for glomerular epithelial cells. The avid uptake of 125I-labeled apo E-containing lipoproteins was further substantiated by their poor displacement by a 25-fold excess of unlabeled LDL and their ability to down regulate the apo B,E receptor activity. LDL as well as beta-VLDL were able to suppress the incorporation of 14C acetate into sterols and to stimulate 3H-cholesterylester formation. These experiments show that cultured glomerular epithelial cells express lipoprotein receptor activity. Plasma concentrations of apo E-containing lipoproteins are increased in certain renal diseases (such as, nephrotic syndrome); these lipoproteins could be rapidly removed by glomerular epithelial cells and lead to lipid deposition in glomeruli.

Ultrastructural immunogold studies of heparan sulphate proteoglycan in normal human glomeruli and glomerulonephritis

The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis, mesangial IgA disease, and anti-glomerular basement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis, no HSPG was found in the electron-dense deposits. These findings contrast with our previous studies using the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM.

Glomerular CR1 Express in situ Cofactor Activity for Degradation of C3b

Adherence of sheep erythrocytes (E) sensitized with IgM antibodies (A) and C3b (EAC3b) to C3b/C4b receptors (CR1) in cryostat sections of human renal glomeruli was studied using the closed chamber technique. The adsorption was stable for at least 3 h at 37 degrees C. In the presence of purified factor I, the indicator cells, however, detached from the sections after 30 min at 37 degrees C. Factor H was not required. The release was not due to loss of CR1 activity in the tissue. The detached indicator cells were negative in the immune adherence test and were agglutinated by antibody to C3d, but not by antibody to C3c. Western blot of the detached indicator cells revealed the presence of C3d and C3c was found in the chamber fluid. Accordingly, detachment of the indicator cells was due to degradation of C3b to C3d with the release of C3c into the chamber fluid. Protease inhibitors did not prevent the detachment of the indicator cells. EAC3b incubated with sections of renal glomeruli preincubated with anti-CR1 antibody were not degraded. The results therefore indicate that CR1 in situ in renal glomeruli can provide the necessary cofactor activity for factor I-mediated degradation of C3b to C3d and C3c.

Cyclosporin nephrotoxicity assessed in isolated human glomeruli and cultured mesangial cells

Cyclosporin nephrotoxicity assessed in isolated human glomeruli and cultured mesangial cells

Classical pathway of complement activation in normal and diseased human glomeruli

Monoclonal antibodies (mAb) reactive against complement components involved in the classical activation pathway were applied in an indirect immunoperoxidase technique for the histological study of normal and diseased human renal tissues. Prominent staining with antibodies against the C4d fragment was seen in all glomeruli and some renal arteriolar walls. The C4d staining was mesangial with light microscopy, whereas the subendothelial site of the glomerular basement membrane (GBM) also appeared to be positive in immunoelectron microscopy. In similar localization, albeit with distinctly weaker intensity, IgM and C4 binding protein (C4bp) were detected. In kidney biopsies from patients with various types of glomerulonephritis, C4d reactive antibodies stained the glomerular structures in a strong, diffuse or granular pattern in contrast to the more segmental distribution and weaker staining intensity in normal kidney specimens. Increased amounts of C4d, occasionally also of C4b, were paralleled in diseased kidney tissues by distinct deposits of IgM and/or IgG in the presence of C4bp. This study suggests that the C4d fragment in normal human glomeruli is indicative of a continuous, local complement activation via the classical pathway induced by the physiological deposition of IgM-containing immune complexes.

Vimentin-cross-reactive epitope of type 12 streptococcal M protein

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.

Inhibitory Effect of Human Atrial Natriuretic Peptide on Cyclic AMP Levels in Microdissected Human Glomeruli

Summary: We studied whether α-human atrial natriuretic peptide (α-hANP) had the capacity to regulate cyclic AMP (cAMP) levels in human glomeruli, since decreased cAMP in the glomerulus may increase the glomerular filtration rate (GFR) through increasing kf (glomerular capillary ultrafiltration coefficient). Human kidneys were obtained at surgery for carcinoma. Normal cortical tissues from these kidneys were used for the study. After incubating the renal cortical slices with 0.1% collagenase, glomeruli were dissected manually under the stereomicroscope. Two glomeruli were incubated (37 °C, 2 min) with parathyroid hormone (PTH) and/or α-hANP. cAMP was determined by radioimmunoassay. α-hANP at a concentration of 5 × 10-6M had no effect on glomerular cAMP accumulation in the basal condition. PTH stimulated cAMP formation in a dose-dependent manner. α-hANP inhibited significantly the increase in cAMP formation induced by PTH (p < 0.01). This action of α-hANP was dosedependent, with a maximum of 50% inhibition. PTH is one of the endogenous substances that are known to increase cAMP formation and decrease kf. Thus, it seems likely that α-hANP increases GFR through modulating the production of cAMP in human kidney.

Characterization of a plasminogen activator and its inhibitor in human mesangial cells

In the course of some pathological and experimental nephropathies, intraglomerular fibrin deposits develop, possibly as a consequence of inefficient fibrinolysis. In vitro human glomeruli exhibit fibrinolytic activity due to the synthesis of plasminogen activators (PAs) such as, tissue-type PA (t-PA) and urokinase-type PA (u-PA). Immunofluorescence studies have previously shown that t-PA is localized in the capillary tufts and u-PA in the visceral epithelial cells. We have now investigated the fibrinolytic activity of cultured human mesangial cells. Inhibitory activity towards u-PA or t-PA but not plasmin was found in both conditioned medium and cellular extracts. Analysis of the conditioned medium by zymography revealed a single band of PA-activity (Mr: 110 to 120 kDa). Immunoneutralization with anti-t-PA and anti-plasminogen activator inhibitor (PAI-1) IgG but not anti-u-PA or anti-PAI-2 removed this band. Reverse fibrin autography demonstrated the presence of PAI-1 in both cellular extracts and in conditioned medium. Western Blot analysis showed that two bands (50 kD and 120 kD) were recognized by the anti-PAI-1 antibody. By ELISA t-PA and PAI-1 antigens were found to increase progressively with time in the culture medium but not in cellular extracts. Both t-PA and PAI-1, but not u-PA and PAI-2, were also detected by immunofluorescence studies. Thus human glomerular mesangial cells synthesize and secrete t-PA and PAI-1 in vitro. PAI-1 is produced in excess, therefore t-PA is only found in the form of a complex with PAI-1.

Decay accelerating factor regulates complement activation on glomerular epithelial cells.

Epithelial cells of the glomerular capillary are the site of C5b-9 mediated injury in rat membranous nephropathy. We investigated the regulation of C activation by cultured glomerular epithelial cells (GEC). Rat and human GEC were more resistant to C injury by homologous C than heterologous C. In human GEC homologous C cytotoxicity was enhanced by antiserum to decay accelerating factor (DAF) indicating that homologous C activation was, at least in part, restricted by membrane DAF. Anti-DAF immunoprecipitated a 67-kDa protein from human glomeruli. In rat GEC, pronase and phosphatidylinositol-specific phospholipase C (which are known to inactivate human DAF) enhanced cytotoxicity by homologous C. Thus, DAF is present on human GEC in culture and in human kidney glomeruli, and a DAF-like protein is present on cultured rat GEC. These proteins regulate C activation in vitro and may play a role in controlling C activation on GEC in vivo.

Alpha 2-Adrenoceptor Stimulation Inhibits Cellular Cyclic AMP Production in Microdissected Human Glomeruli

The physiological role of numerically predominant alpha 2-adrenoceptor in the kidney is still unknown. This study examined the effect of alpha 2-adrenoceptor stimulation on the production of cAMP from isolated human glomeruli. Unaffected portions of human kidneys which had been removed because of renal cell carcinoma were used for the study. Glomeruli were dissected manually under a stereo microscope. In these glomeruli, alpha 2-adrenoceptor stimulation with epinephrine in the presence of propranolol inhibited significantly parathyroid hormone-dependent increases in cAMP production. This inhibitory effect of epinephrine was removed by adding a specific alpha 2-adrenoceptor antagonist, yohimbine, indicating that the inhibitory effect of epinephrine on cAMP formation was due to alpha 2-adrenoceptor stimulation. Thus, alpha 2-adrenoceptors are involved in the inhibition of cAMP production in human glomeruli.

Prostaglandins, thromboxane and leukotrienes in the control of mesangial function.

Mesangial cells are intercapillary cells of the kidney glomerulus, serving both smooth muscle and immune effector functions (1–3). Since the first report of contraction of cultured rat mesangial cells by the vasoactive peptides, angiotensin II (ANG II) and arginine vasopressin (AVP) (2,4), considerable interest focused on the hypothesis that mesangial cells may regulate glomerular blood flow and filtration in synergism with afferent and efferent resistances, by altering capillary surface area and hence the ultrafiltration coefficient, Kf (5,6). Changes of capillary surface area might result from mechanical stretching and constriction of vessel walls by surrounding mesangial cells or intraglomerular shunting of blood flow as a result of segmental occlusion of certain loops. Although no definitive evidence of such function has been provided thus far, a number of observations link the mesangial cell to a regulatory role on glomerular hemodynamics, including reports of selective changes of Kf upon induction of glomerular immune injury (7) or infusion of vasoactive agents (8–10). Further support to the concept of mechanical properties of the mesangium comes from studies in freshly isolated rat and human glomeruli, which are rapidly contracted by ANG II, as evaluated by different techniques (11,12).

High-Affinity Binding of the Converting Enzyme Inhibitor, Ramiprilat, to Isolated Human Glomeruli

Evidence for effects of angiotensin converting enzyme (ACE) on isolated human glomeruli was provided using specific binding of tritium-labeled ramiprilat, a potent inhibitor of ACE. [3H]ramiprilat bound to isolated glomeruli, depending on time and temperature, displaying a KD of 3.8 nmol/L and a Bmax of 853 fmol/mg protein. Specific binding represented more than 90% of total binding. Dissociation occurred rapidly after dilution of the sample with incubation buffer or after addition of an excess of unlabeled inhibitor. Binding of [3H]ramiprilat was also inhibited by increasing concentrations of enalaprilat, another ACE inhibitor. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely prevented binding. This was reversed by divalent Zn2+ and Ca2+ ions, but not by magnesium. Binding of [3H]ramiprilat to isolated glomeruli was maximal at pH 8, which also is optimal for ACE activity. The binding of [3H]ramiprilat to isolated human glomeruli is specific, and resembles the characteristics which have been found earlier for enzyme activity of ACE. Thus, binding of [3H]ramiprilat to isolated glomeruli can be assumed to be directed to ACE.

High affinity binding of ramiprilat on isolated human glomeruli

Evidence for angiotensin-converting enzyme (ACE) on isolated human glomeruli was furnished by specific binding of tritium-labeled ramiprilat, a potent inhibitor of ACE. 3H-ramprilat bound to isolated glomeruli, depending on time and temperature displaying a K D of 3.8 nmol/l and a B max of 853 fmol/mg protein. Specific binding represented more than 90% of total binding. Dissociation occurred rapidly after dilution of the sample with incubation buffer or after addition of an excess of unlabeled inhibitor. Binding of 3H-ramiprilat was also inhibited by increasing concentrations of enalaprilat, another ACE-inhibitor or by preincubation of the glomeruli with polyclonal antibodies against ACE. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely inhibited binding. This inhibitory effect of EGTA was reversed by divalent Zn 2+ and Ca 2+ ions but not by magnesium. Binding of 3H-ramiprilat to isolated glomeruli was maximal when the pH of the assay medium was brought to pH 8. In conclusion, the binding of 3H-ramiprilat to isolated human glomeruli is specific and resembles the characteristics which have been found earlier for enzyme activity of ACE. Thus, binding of 3H-ramiprilat to isolated glomeruli can be assumed to be directed to ACE.

88 High-affinity binding of an angiotensin converting enzyme inhibitor, ramiprilat, to isolated human glomeruli

H.A. Becker, Richard; Albus, U.; Kress, I.; Linz, Wolfgang; Vasmant, D.*; Delarue, F.; Sraer, J. D. Author Information

Profibrinolytic and procoagulant activities of human glomeruli from normal kidneys and rejected renal allografts

We studied the profibrinolytic activity (PFA) aw' the procoagulant activity (PCA) of glomeruli isolated from 17 human irreversibly rejected renal allografts and compared them to glomeruli prepared from 6 normal human kidneys. When studied by SDS-PAGE and zymography, glomeruli of both rejected and control kidneys were found to contain a 70 Kd form of tissue-type plasminogen activator (t-PA) but also a 120 kd form corresponding to t-PA complexed to an endothelial-type plasminogen activator inhibitor (PAI-1), as demonstrated by specific antibodies. In glomeruli from 5 out of 17 rejected kidneys no PA activity could be detected by zymography. Mean t-PA level in glomerular supernatants, measured by ELISA, was only slightly decreased and glomerular PFA measured by a radioenzymatic assay was not significantly different in rejected and in control kidneys. Conversely glomerular PCA was significantly decreased in all rejected kidneys when compared to controls. Thus these results suggest that during rejection the glomerular profibrinolytic activity is similar and that the glomerular procoagulant activity is decreased when compared to normal kidneys. This may explain, at least in part, that glomeruli are usually relatively spared by thrombosis during the rejection process.

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age

Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Effect of diabetes and age

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas Chromatograph-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF 1α and PGF 2α, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B 2 (TxB 2) and PGE 2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF 1α. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.