Demonstration of 2 distinct glomerular populations by a sieving technic in the kidney cortex of the pig

Amelioration of sieving technics for glomeruli isolation aims at obtention of purer and more homogeneous preparations, presenting a high preservation degree for glomerular functional studies. We have recently demonstrated the necessity to use sieves adapted to the glomeruli size of different considered animals as well as to use kidneys having exactly the same weight in order to obtain very homogeneous glomeruli suspensions. This study presents a new amelioration in the homogeneity of the glomeruli diameter through a preliminary dissection of the renal cortex in order to isolate glomeruli situated in the same particular cortical zone. Pig renal cortex, because of its size, agrees well with a fine dissection in order to dissociate the superficial and the juxta-medullary zones. Glomeruli are isolated with 160/120 micro mesh sieves. Diameter mean value is 218.8 +/- 30.1 micro in superficial glomeruli and 270.4 +/- 30.1 micro in juxta-medullary ones, highly significant difference, (p less than 0.001). Moreover, repartition frequency histograms of the juxta-medullary glomerular populations diameter presents a large shifting to higher values. This renal dissection preceding the glomeruli isolation sieving technique contributes to better the homogeneity of the isolated glomeruli suspensions and opens the way to the original study of the comparative in vitro vasoreactivity of these two different glomerular populations after incubation with physiological or pharmacological reagents.     

Comparative study of the vasoreactivity of isolated human cortical and juxtamedullary glomeruli using verapamil and dopamine

The purpose of the present study was to compare the area variations of human cortical and juxtamedullary glomeruli after incubation with verapamil and dopamine. The glomeruli were isolated by sieving; areas were measured by a video analyser. The surface of the cortical glomeruli increases largely with verapamil (+14,3%) and dopamine (+11,5%). But, the area of the juxtamedullary glomeruli remains unchanged in the same experimental conditions. Evidence of such a vasoreactivity difference between cortical and juxtamedullary glomeruli will permit a better understanding in the regulation of renal haemodynamics.     

Demonstration of direct vasoconstrictive effect of cyclosporin on glomeruli isolated from human kidney

Cyclosporin (CsA), a potent immunosuppressive agent, can induce severe renal damage, characterized by lesions in the tubulo interstitial region and decreased renal function. Isolated glomeruli can be considered as a fruitful model to evidence the direct effect of drugs at the level of glomerular structures. The present study aims to evidence, in this model, vasoactive response of isolated human renal glomeruli induced by different CsA concentrations. Area changes of such isolated glomeruli can be assessed by a semi-automatic morphometric method, using a video-camera device coupled with a microprocessor (Biocom 2000). Experiments are done under double bind conditions. CsA-induced decrease in glomeruli area is dose- and time-dependent. CsA effect is rapid in that glomerular area is significantly decreased even after 5 min. incubation; vasoconstrictive effect is more important (and maximum) after 10 min. incubation; significant decrease can be noted with 10(-7) M (-11.41%) and 10(-6) M (-10.26%). In conclusion, the isolated glomerulus model allows us to demonstrate the direct vasoconstrictive effect of CsA, that can in part explain the renal functional changes and the acute renal failure often described in human clinic.     

An epithelial precursor is regulated by the ureteric bud and by the renal stroma

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.     

Renal tissue reconstitution by the implantation of renal segments on biodegradable polymer scaffolds

Renal units were created in vivo by transplanting isolated renal segments on three-dimensional, biodegradable polymer scaffolds. Renal segments, freshly isolated from rat kidneys, were seeded on polymer scaffolds and subcutaneously implanted in athymic mice for two and four weeks. Three-dimensional renal reconstructs were formed with glomeruli and tubules, showing a possibility of reconstituting renal structures by transplanting renal segments.     

The localisation of sorbitol pathway activity in the rat renal cortex and its relationship to the pathogenesis of the renal complications of diabetes mellitus.

A series of in vivo and in vitro investigations was performed to examine the localisation of sorbitol pathway activity in the rat renal cortex and to investigate the possible relation that the acculumation of sorbitol pathway intermediates in renal cortical tissue may have to the pathogenesis of renal complications in diabetes mellitus. Neither of the sorbitol pathway intermediates, sorbitol or fructose, were detected either in intact glomeruli which had been isolated from rats rendered chronically diabetic with streptozotocin, or in metabolically active glomeruli which had been incubated in vitro in high glucose media. Such data agreed with previously published observations that the enzyme aldose reductase is not present in renal glomeruli, and suggested that changes in sorbitol pathway activity cannot be directly related to the pathogenesis of diabetic glomerulosclerosis. Sorbitol was detected in low concentrations (3.1 mu-mol/g protein) in cortical tubules which had been isolated from the renal cortex of rats rendered chronically diabetic with streptozotocin. This concentration of sorbitol was higher than that in the intact renal cortex of the diabetic animal (0.3 mu-mol/g protein) or in the cortical tubules of non-diabetic animals (0.5 mu-mol/g protein). It is apparent that the renal cortical tubule is a major site of sorbitol pathway activity in the renal cortex. However, there is presently no obvious causal relationship between the accumulation of such relatively low concentrations of sorbitol in the renal cortical tubule and the pathogenesis of glomerulosclerosis or cortical tubular lesions in diabetes.     

Presence of gamma-glutamyltransferase in the renal microvascular compartment

The association between the brush border enzyme alkaline phosphatase and gamma-glutamyltransferase was determined by sucrose density gradient analysis of crude kidney homogenates, isolated glomeruli, and isolated microvessels. As previously established there is an overlap of these enzyme activities in the crude homogenate corresponding to a density of 1.17 g.cm-3. In contrast, isolated glomeruli sedimented with a peak of 1.25 g. cm-3 and exhibited gamma-glutamyltransferase activity but little alkaline phosphatase activity; homogenizing isolated glomeruli shifted the fragments to a density coincident with that observed for the crude homogenate gamma-glutamyltransferase peak. A second population of capillaries, isolated microvessels, were homogenized and analysed on the sucrose density gradient. These fragments sedimented over the same range as crude homogenate gamma-glutamyltransferase peak but were devoid of alkaline phosphatase activity and yet exhibited remarkable gamma-glutamyltransferase activity. The results indicate homogenization of renal cortex results in a heterogeneous collection of particles from both tubular and microvascular locations exhibiting gamma-glutamyltransferase activity which overlap with the brush border alkaline phosphatase containing membranes. However, isolation of microvessels and glomeruli prior to homogenization allows separation of gamma-glutamyltransferase from alkaline phosphatase activity; between 10 and 20% of the total homogenate gamma-glutamyltransferase activity is estimated to be associated with the microvascular compartment.     

Masugi's nephritis: preparation of an active nephrotoxic serum

An active nephrotoxic serum (NTS) was prepared by immunization of rabbits with isolated renal glomeruli of rats and Freund's complete adjuvant. The glomeruli were isolated from the renal cortex by the sieving procedure. Intravenous injection of the NTS to rats induced the development of marked proteinuria and the nephrotic syndrome. Histological and electron microscopic studies of renal tissue performed by days 5 and 15 since the first injection of the NTS revealed extracapillary proliferative glomerulonephritis. IgG depositions on the basal membranes of the glomerular capillaries were linear on day 5 and granular on day 15 since the first injection of the NTS.     

Nitric oxide (NO) donor 3-morpholinosydnonimine antagonizes cyclosporin A-induced contraction in two in vitro glomerular models

Cyclosporin A induces in vivo a severe nephrotoxicity characterized by a large decrease in renal hemodynamics. The aim of this study is to establish the ability of the known NO donor 3-morpholinosydnomine (SIN-1) to prevent the cyclosporin A-induced contraction by using rat isolated glomeruli and cultured glomerular mesangial cells. Isolated rat glomeruli are obtained from the renal superficial cortex by a sieving method. Mesangial cells are cultured in RPMI 1640 with 15% fetal calf serum. The planar surface area (PSA) of either isolated glomeruli or mesangial cells is assessed using anage analyzer. Each glomerusus or mesangial cell serves as its own control through calculation of the area before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or in control solution with cyclosporin A alone or cyclosporin A and SIN-1. Cyclosporin A (10^–6 mol/L) induces an important time-dependent contraction of either glomerulus or mesangial cell. When pretreated with different concentrations of SIN-1 (10^–4 to 10^–9 mol/L), only a slight size decrease is noted. In conclusion, a direct constrictive effect of cyclosporin A in isolated glomeruli and mesangial cells can be prevented bythe NO donor SIN-1, suggesting an important involvement of the nitric oxide pathway in the cyclosporin A-induced nephrotoxicity.Abbreviations CyA cyclosporin A - SIN-1 3-morpholinosydnonimine - FCS fetal calf serum - PSA planar surface area     

Glomeruli from ischemic rat kidneys produce increased amounts of platelet activating factor

The production of Platelet Activating Factor (PAF-acether) by glomeruli isolated from rats subjected to a 60 min renal ischemia has been studied. PAF-acether has been purified by Sep-Pak columns and measured by its ability to release serotonin from previously loaded rabbit platelets. Glomeruli from ischemic kidneys had higher amounts of PAF-acether activity than glomeruli from sham operated rats. These data suggest a role for PAF-acether in the renal functional alterations induced by renal ischemia.     

APOBEC3G expression is restricted to epithelial cells of the proximal convoluted tubules and is not expressed in the glomeruli of macaques.

The Vif protein of human immunodeficiency virus-1 (HIV-1) interacts with members of the APOBEC family of cytidine deaminases. In this study, we isolated RNA from renal cortex as well as from isolated glomeruli and tubulointerstitial fractions from two pigtailed macaques that were exsanguinated and perfused with saline. RT-PCR results indicate that APOBEC3G was detected in the tubule fractions but not in the glomerular fractions. Immunoblot analysis using lysates prepared from these same fractions and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on renal cortical sections. Our results clearly show that the glomeruli do not express APOBEC3G but that select tubules within the cortex express APOBEC3G at high levels. To further differentiate the distribution of APOBEC3G expression, serial sections were stained with the lectins Dolichos biflorus agglutinin (DBA) and Phaseolus vulgaris erythroagglutinin (PHA-E), which differentially bind to epithelial cells of the tubules and glomeruli. Our results indicate that APOBEC3G expression was restricted to PHA-E-staining tubules and not DBA-staining tubules, suggesting that APOBEC3G expression was restricted to proximal convoluted tubules. These findings suggest that infection of epithelial cells of proximal renal tubules could suppress Vif-defective HIV-1 replication, whereas infection of cells of the glomeruli, a major target of HIV-associated nephropathy, could act as a reservoir for the replication of Vif-defective HIV-1.     

Involvement of Rho-kinase in P2Y-receptor-mediated contraction of renal glomeruli

The involvement of Rho-kinase in P2Y-receptor induced contraction of isolated rat renal glomeruli was investigated. The contraction effects have been investigated based on changes in the intracapillary volume of isolated glomeruli. ATP was found to induce time- and concentration-dependent contraction of isolated glomeruli. Other tested nucleotides (ADP, UTP) and ATP analogues (beta,gamma-methylene-ATP, 2-methylothio-ATP) contracted glomeruli in similar magnitude whereas AMP had no effect. Furthermore, the contractive effect of ATP was prevented in the presence of an antagonist of P2Y-receptors, reactive blue 2. However, a selective antagonist of A1-receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), had no effect. Contraction induced by ATP, ADP, and UTP, in contrast to 2-methylothio-ATP and beta,gamma-methylene-ATP, was prevented in the presence of Rho-kinase's inhibitor, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632). These findings suggest the involvement of Rho-kinase pathways in P2Y-induced contraction of isolated glomeruli.     

Localization of an Ia-bearing glomerular cell in the mesangium

Using trypsin to render intact, isolated rat glomeruli permeable to antibody, and using an electron microscopic immunoperoxidase technique, we have localized a phagocytic immunologically-relevant cell bearing Ia determinants to the renal mesangium. Thus there are at least two functionally distinct cell types in the renal mesangium: one is a contractile smooth musclelike cell, and the other a phagocytic cell that bears immunologically-relevant surface determinants.     

Gene expression in the normal adult human kidney assessed by complementary DNA microarray

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.     

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.     

Increased tumor necrosis factor and IL-1 beta gene expression in the kidneys of mice with lupus nephritis

Because TNF and IL-1 can initiate immunologic and inflammatory events alone or synergistically, a local increase in the levels of one or both of these cytokines in vivo may cause irreparable tissue damage. The purpose of this study was to evaluate local TNF and IL-1 beta gene expression in vivo in the kidneys of MRL-Ipr mice with autoimmune lupus nephritis. TNF mRNA was detected in the renal cortex of MRL-Ipr mice but was not present in the cortex of normal congenic MRL-++ or C3H/FeJ mice. MRL-Ipr mice with lupus nephritis expressed higher amounts of TNF mRNA compared with MRL-Ipr mice prior to disease. In addition, freshly isolated, unstimulated glomeruli from MRL-Ipr mice with nephritis were found to secrete detectable levels of TNF, whereas glomeruli from MRL-++ mice did not. IL-1 beta mRNA, present in the renal cortex of C3H/FeJ, MRL-++, and young MRL-Ipr mice with normal kidneys, was also more abundantly expressed in MRL-Ipr mice with nephritis. Cultured macrophages from glomeruli of mice with nephritis were found to express TNF and IL-1 beta mRNA and product. These macrophages are prominent only in MRL-Ipr mice with renal disease and are the likely source of increased gene expression for both cytokines.     

Increased glomerular atrial natriuretic peptide receptor affinity in experimental nephrotic syndrome.

Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and diuretic effects. To better define the ANP hormonal system in the nephrotic syndrome, a condition associated with renal sodium retention, we undertook a study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly decreased serum albumin and total protein and significantly increased serum cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level of atrial natriuretic peptide was similar in both groups of rats. Competition binding inhibition studies in isolated glomeruli demonstrated one binding site in both groups of rats. The density of ANP binding sites in isolated glomeruli was similar in nephrotic and pair-fed rats while the binding affinity was increased significantly in the nephrotic rats. This is the first study to demonstrate alterations in renal ANP receptors in the nephrotic syndrome. Further studies will be necessary to determine whether alterations in glomerular ANP receptors contribute to renal sodium retention in the nephrotic syndrome.     

Abrogation of mercuric chloride-induced nephritis in the Brown Norway rat by treatment with antibodies against TNFalpha

HgCl(2) induces an autoimmune disease in the Brown Norway rat characterized by synthesis of autoantibodies (mainly, anti-GBM Abs), severe proteinuria and interstitial nephritis. Also, HgCl(2)- injected rats develop glomerular cell infiltrates consisting of ED1(+) cells (monocyte/macrophage), starting on day 4 and reaching a maximum on day 8. Treatment with anti-TNF-alpha antiserum had preventative effects as it reduced the urinary protein levels to close to the normal range and also blocked the influx of inflammatory cells in the renal glomeruli and interstitium, but circulating anti-GBM and lineal glomerular IgG deposits were unmodified. In addition, whole isolated glomeruli from HgCl(2)-induced nephritis secreted TNF-alpha commencing on day 8, being maximally detected on day 11 and preceding, between 2 to 3 days, the development of proteinuria. The administration of anti-TNF-alpha antiserum or anti-alpha4 integrin mAb completely abrogated the synthesis of TNF-alpha in glomeruli isolated from the respective treated groups of animals, in addition to the proteinuria. Taken together our results confirm that TNF-alpha plays an important role in the induction and development of HgCl(2)-induced nephritis and highlights the pathogenic importance of the local release of TNF in those renal diseases in which prominent glomerular macrophage accumulation is a constant feature.     

Circadian Changes in the Surface Area of Renal Glomeruli from Normal Rats

In an attempt to further investigate circadian changes in kidney function, the planar surface area (PSA) (µm 2) of renal isolated glomeruli from normal rats was monitored using a computerized image analyzer method. Eight male Sprague-Dawley (SPRD) rats, aged 12û14 weeks, were entrained to a 12-h light/12-h dark cycle in controlled environmental conditions of temperature (22±2°C) two weeks before the experiments started. Isolated glomerular preparations were obtained, using a mechanical sieving technique, at four different circadian times: 07:00, 13:00, 19:00 and 01:00. It was the finding of the present study that the PSA of the glomeruli varied significantly over the 24-h period, but showed a weak amplitude. The size of the glomeruli reached the highest values at night (21358.59±456.72 µm 2 at 21:17) with an acrophase at 21:39, as do all the other renal parameters, but also blood pressure and many vasoactive compounds involved in the regulation of the mesangial cell physiology, contractile element of the glomerulus. Such chronobiological data not only provided a clear example of complementarity between in vivo and ex vivo experiments, but evidenced that temporal changes do exist in kidney structure and could be correlated with rhythms in renal physiology.     

Novel expression of claudin-5 in glomerular podocytes

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.