Cultures Of Glomerular Cells Research Articles

Characterizing Glomerular Barrier Dysfunction with Patient-Derived Serum in Glomerulus-on-a-Chip Models: Unveiling New Insights into Glomerulonephritis.

Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.

Analysis of gene expression and use of connectivity mapping to identify drugs for treatment of human glomerulopathies.

Human glomerulonephritis (GN)-membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS) and IgA nephropathy (IgAN), as well as diabetic nephropathy (DN) are leading causes of chronic kidney disease. In these glomerulopathies, distinct stimuli disrupt metabolic pathways in glomerular cells. Other pathways, including the endoplasmic reticulum (ER) unfolded protein response (UPR) and autophagy, are activated in parallel to attenuate cell injury or promote repair. We used publicly available datasets to examine gene transcriptional pathways in glomeruli of human GN and DN and to identify drugs. We demonstrate that there are many common genes upregulated in MN, FSGS, IgAN, and DN. Furthermore, these glomerulopathies were associated with increased expression of ER/UPR and autophagy genes, a significant number of which were shared. Several candidate drugs for treatment of glomerulopathies were identified by relating gene expression signatures of distinct drugs in cell culture with the ER/UPR and autophagy genes upregulated in the glomerulopathies ("connectivity mapping"). Using a glomerular cell culture assay that correlates with glomerular damage in vivo, we showed that one candidate drug - neratinib (an epidermal growth factor receptor inhibitor) is cytoprotective. The UPR and autophagy are activated in multiple types of glomerular injury. Connectivity mapping identified candidate drugs that shared common signatures with ER/UPR and autophagy genes upregulated in glomerulopathies, and one of these drugs attenuated injury of glomerular cells. The present study opens the possibility for modulating the UPR or autophagy pharmacologically as therapy for GN.

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

Prostaglandin E2promotes cellular recovery from established nephrotoxic serum nephritis in mice, prosurvival, and regenerative effects on glomerular cells

We postulated that prostaglandin E2 (PGE2), which exhibits regulatory functions to control immune-mediated inflammation, fibrosis, oxidative stress, and tissue/cellular regeneration, has the potential to improve the course of nephritis. Therefore, the therapeutic potential of prostanoid on established nephritis in mice was evaluated focusing on its role on renal cellular recovery, with emphasis on its cytoprotecting and growth-promoting effects. Acute nephritis was induced in mice by single injection of nephrotoxic serum (NTS), followed by PGE2 administration with severity of nephritis evaluated over time. Mice injected with PGE2 recovered promptly with normalization of blood urea nitrogen and urine protein levels and histology. Recovery was observed with dosing of prostanoid at day 1, as well as day 4. With the use of selective EP1-4 receptor agonists, EP3 receptor has been identified as important in mediating beneficial effects of PGE2 in our system. PGE2 normalized glomerular cell losses during nephrotoxic serum-induced nephritis, restored synaptopodin distribution and F-actin filaments arrangement in glomeruli. In cell culture, PGE2 reduced nephrotoxim serum (NTS)-induced apoptosis of glomerular cells and promoted cell reproliferation after NTS-mediated injury. In conclusion, PGE2 treatment promotes resolution of glomerular inflammation. Consistent with this observation, the regenerative and cytoprotective effects of prostanoid on glomerular cells in culture were observed, suggesting that PGE2 may be beneficial in the treatment of glomerulonephritis.

Drosophila perlecan regulates BMP signaling essential for synaptic growth

Glomerular heparan sulfate alterations: Mechanisms and relevance for proteinuria. Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.

A renal glomerular cell culture model using a collagen vitrigel membrane and its application to the evaluation of serum filtration

A renal glomerular cell culture model using a collagen vitrigel membrane and its application to the evaluation of serum filtration

Inhibiting albumin glycation attenuates dysregulation of VEGFR-1 and collagen IV subchain production and the development of renal insufficiency

Glomerular cells in culture respond to albumin containing Amadori glucose adducts (the principal serum glycated protein), with activation of protein kinase C-beta(1), increased expression of transforming growth factor (TGF)-beta1, the TGF-beta type II signaling receptor, and the extracellular matrix proteins alpha(1)(IV) collagen and fibronectin and with decreased production of the podocyte protein nephrin. Decreasing the burden of glycated albumin in diabetic db/db mice significantly reduces glomerular overexpression of TGF-beta1 mRNA, restores glomerular nephrin immunofluorescence, and lessens proteinuria, mesangial expansion, renal extracellular matrix protein production, and increased glomerular vascular endothelial growth factor (VEGF) immunostaining. In the present study, db/db mice were treated with a small molecule, designated 23CPPA, that inhibits the nonenzymatic condensation of glucose with the albumin protein to evaluate whether increased glycated albumin influences the production of VEGF receptors (VEGFRs) and type IV collagen subchains and ameliorates the development of renal insufficiency. Renal levels of VEGF and VEGFR-1 proteins and serum creatinine concentrations were significantly higher and renal levels of alpha(3)(IV) collagen and nephrin proteins and endogenous creatinine clearance values were significantly lower in control diabetic than in age-matched nondiabetic (db/m) mice. These changes were significantly attenuated in db/db littermate mice treated from 9 to 18 wk of age with 23CPPA. The findings indicate that inhibiting excess nonenzymatic glycation of serum albumin improves renal molecular biology abnormalities and protects against the development of renal insufficiency in the db/db mouse.

Leucine-rich repeat-containing G-protein-coupled receptor 8 in mature glomeruli of developing and adult rat kidney and inhibition by insulin-like peptide-3 of glomerular cell proliferation

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3-LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3-LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3-LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.

Glial fibrillary acidic protein-positive cells of the kidney are capable of raising a protective biochemical barrier similar to astrocytes: expression of metallothionein in podocytes.

Blood-tissue exchange and homeostasis within the organs depend on various interactions between endothelial and perivascular cells (Buniatian, 2001). Podocytes possess anatomical and cellular features intermediate between those of astrocytes and hepatic stellate cells (HSCs). Podocytes, like HSCs, are associated with fenestrated capillaries and, similar to astrocytes, interact with the capillaries via the basement membrane and participate in permeability-limiting ultrafiltration. The fact that podocytes come in direct contact with xenobiotics prompted us to investigate whether they express metallothionein (MT), an anticytotoxic system characteristic of astrocytes. In comparative studies, cryosections of 1- and 3-month-old rat kidney and adult rat brain, as well as podocytes and astrocytes from early and prolonged primary cultures of glomerular explants and newborn rat brain, respectively, were investigated. The cells were double-labeled with antiserum against glial fibrillary acidic protein (GFAP) and monoclonal antibody (MAb) against the lysine-containing epitope of Cd/Zn-MT-I (MAb MT) or MAb against alpha-actin. In kidney sections, MT immunoreactivity was detected in GFAP-positive glomerular cells and in interstitial fibroblasts. The pattern of staining for MT and GFAP in glomerular cells was similar to that of astrocytes in vivo. In glomerular cell cultures, MT was expressed in cobblestone-like podocytes which contained Wilms' tumor protein and lacked desmin. MT was upregulated at later culture periods, during which podocytes acquired features typical of undifferentiated astrocytes. This study hints at the existence of common regulatory mechanisms of blood-tissue interactions by neural and non-neural perivascular cells. These mechanisms appear to be used in an organ-specific manner.

Polygonal epithelial cells in glomerular cell culture: Podocyte or parietal epithelial origin?

Podocytes are unique cells with regard to morphology, their inability to proliferate in situ, and their apparent essential function for glomerular filtration and permselectivity. Upon transfer into culture conditions, polygonal epithelial cells grow out from isolated glomeruli and these cells have been postulated to represent dedifferentiated podocytes, even though they are negative for podocyte-specific markers and undergo cell division. However, it is controversial whether they originate from podocytes or parietal epithelial cells (PECs) of Bowman's capsule, because isolated glomeruli contain more or less Bowman's capsule. In this review, we shall summarize recent progress in the identification of the origin of the polygonal cells and the characterization of podocytes in culture.

Glomerular heparan sulfate alterations: Mechanisms and relevance for proteinuria

Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.

Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury

The glomerulus has been implicated as a target of hydrocarbon injury in vitro and in vivo. In the present studies, the phenotypic profiles of cultured rat glomerular cells (GCs) following repeated cycles of hydrocarbon injury were evaluated. Cultured GCs were incubated for 24 hours with benzo[a]pyrene (BaP; 3 micromol/L), a prototypical polycyclic aromatic hydrocarbon, and were allowed to recover overnight before two additional cycles of chemical challenge during serial propagation in vitro. At the end of this regimen, control cultures were characterized by predominance of fusiform cells that grew in "hills and valleys," while GCs subjected to hydrocarbon injury displayed an epithelial morphology characterized by a rounded, polygonal shape clearly distinct from that normally exhibited by glomerular mesangial cells (GMCs) in culture. Indirect immunofluorescent detection of cell markers was conducted to identify cells of mesenchymal or epithelial origin. Measurements of DNA synthesis and cell number were performed to determine proliferative capacities of the different cell types in response to hydrocarbon challenge. Immunofluorescence studies revealed that control GC cultures contained mostly alpha-smooth muscle (SM) actin-positive cells, with a few (5.1% +/- 2.6) E-cadherin-positive cells occasionally identified. In contrast, BaP-treated cultures exhibited a mixed cell population in which E-cadherin-positive cells were predominant (66.6% +/- 4.1). Single-cell cloning of naive cultures of GCs yielded four clones, three of which exhibited a fusiform morphology and were alpha-SM actin positive (SCC 1 through SCC 3) and one (SCC 4E) that exhibited epithelial characteristics similar to those found in hydrocarbon-treated cultures. Immunofluorescence studies showed that epithelial cells in hydrocarbon-treated cultures, as well as SCC 4E-derived clones, were vimentin positive and cytokeratin negative, characteristics similar to glomerular visceral epithelial cells (GVECs). DNA synthesis and cell proliferation in clone SCC 1 were decreased following acute BaP challenge, while growth rates in SCC 4E-derived clones were unaffected by hydrocarbon injury. Repeated cycles of hydrocarbon challenge in clonal populations yielded different profiles of DNA synthesis, with significant decreases in SCC 1 and no changes in SCC 4E. These observations suggest that hydrocarbon injury induces differential responses in cells of the glomerulus, resulting in inhibition of GMCs and selective growth advantage of GVECs. These alterations are reminiscent of critical events described in the pathogenesis of focal segmental glomerulosclerosis and raise important questions about the pathogenesis of hydrocarbon-induced nephropathies.

Glycosaminoglycans: use in treatment of diabetic nephropathy.

Glycosaminoglycans: use in treatment of diabetic nephropathy.

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury

Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.

Biology of glomerular cells in culture.

The glomerulus is a complex structure including four cell types, namely mesangial, visceral epithelial, parietal epithelial and endothelial cells. Mesangial cells resemble smooth muscle cells and play a major role in the synthesis of the components of the glomerular basement membrane and in the vasoreactivity of the glomerular tuft. In particular, they express receptors for angiotensin II which mediate mesangial cell contraction, this effect resulting in the decrease of the filtration area. They are also the site of synthesis of a variety of inflammatory agents which are involved in the development of glomerular injury in glomerulonephritis. Visceral epithelial cells, also referred to a podocytes, also participate in the synthesis of the normal constituents of the glomerular basement membrane. They express receptors for atrial natriuretic factor and possess on their surface a number of ectoenzymes. They also, in concert with mesangial cells, release metalloproteases which contribute to the degradation of the extracellular matrix. Parietal epithelial cells have been little studied. They represent the main constituent of the crescents observed in extracapillary proliferative glomerulonephritis. Endothelial cells secrete vasodilatory agents such as nitric oxide and prostacyclin and vasoconstrictor agents such as endothelin which act on the adjacent mesangial cells. New methods of culture of glomerular cells are in progress. Their aim is to keep as long as possible the physiological phenotype of these cells. Another progress is the availability of stable transformed cell lines which represent an abundant source of material for biochemical studies.

TGF-β1 in glomerulosclerosis and interstitial fibrosis of adriamycin nephropathy

The role of transforming growth factor-beta 1 (TGF-beta 1) for renal injury was investigated in the chronic model of progressive renal disease in rats induced by the injection of adriamycin. The renal cortical tissues were sampled at weeks 4, 8 and 16 for histological examination, either cortical or glomerular cell culture, and RNA extraction. A progressive increase in fibronectin synthesis was found in metabolically labeled cortical or glomerular culture at week 8 or 16, correlating with the degree of glomerulosclerosis and interstitial fibrosis. TGF-beta bioassay (mink lung epithelial cell assay) showed a progressive increase in latent TGF-beta secretion from cortex and glomeruli, while the amount of active TGF-beta was small. The peak of latent TGF-beta levels at week 16 coincided with the intense TGF-beta 1 staining of inflammatory cells dispersed in the interstitium and glomeruli. Northern blotting demonstrated the difference in the mRNA expression patterns of TGF-beta 1 and latent TGF-beta 1 binding protein (LTBP) in the cortex. TGF-beta 1 mRNA was constantly high throughout the experiment, while LTBP mRNA increased progressively and reached a peak at week 16. Furthermore, mRNA levels of fibronectin, procollagen alpha 2(I), and TGF-beta type II and type III receptors increased progressively in a similar pattern to the renal histological changes. These temporal and spacial relationships between the renal histological changes and the increased expression of TGF-beta 1 and TGF-beta receptors may thus suggest that TGF-beta 1 plays an important role in the process of the renal fibrosis and sclerosis.

Glomerular cells in vitro versus the glomerulus in vivo

Early data on glomerular physiology and pathophysiology were largely based on morphological studies and on functional studies of either glomeruli in vivo or isolated glomeruli. The introduction of glomerular cell culture in the late seventies and early eighties has allowed a more specific approach to the role of individual glomerular cell types in the mediation of renal pathology [1–3]. Over the last five years there has been a tremendous increase in studies dealing with cultured glomerular cells. For example, the number of abstracts on mesangial cells in culture that have been presented at the annual meeting of the American Society of Nephrology has increased from about 10 in 1988 to about 150 in 1992. In contrast, only about 25 studies dealt with glomerular epithelial cells last year, and very few with glomerular endothelial cells, which is likely a reflection of the ease at which these cells can be grown out of isolated glomeruli. As discussed in several recent papers, these in vitro studies have shown that glomerular cells have a wide range of synthetic activities and response patterns [this issue of Kidney International, 1–8]. Based on the in vitro characterization of the capabilities of glomerular cells, physiological or pathological pathways have been suggested that may be operative in vivo . However, when working with cells isolated from glomeruli and when attempting to extrapolate to the in vivo situation, several issues need to be addressed. First, what type of cells has been obtained and how do their morphological characteristics compare to those observed in vivo ? Second, is there evidence to suggest that cultured cells exhibit the same synthetic profile as they do in vivo or do they synthesize products that can not be detected in vivo under normal or pathological circumstances? Third, are the responses of cultured glomerular cells comparable to those observed in vivo or do they show response patterns that are restricted to the petri dish? In the following review we will discuss these three issues by way of example rather than by attempting to give a complete overview. Finally, we will restrict our discussion to glomerular mesangial and epithelial cells, since too little information is available on glomerular endothelial cells either in culture or in vivo .

Localisation of α, μ and π class glutatione S-transferases in kidney: comparison with CuZn superoxide dismutase

We describe studies in whole kidney, cortical and medullary homogenates and, glomerular cells in culture to determine the relative levels of expression of α (Ya, Yc, Yk), μ (Yb1/Yb2), π (Yf) glutathione S-transferases (GST) and CuZn superoxide dismutase (CuZn SOD) in different regions of the nephron. Immunoblotting and immunohistochemistry were used to demonstrate relatively weak expression of α, μ GST and, CuZn SOD in the glomerulus compared to that in particularly distal tubules. Whilst expression of Ya was found within glomerular cells, Yc, Yk and Yf were not detected. Immunofluorescence showed that Ya and Yb1/Yb2 but not Yf were expressed in cultured epithelial and mesangial cells studied between passages 1 and 3. While Ya was distibuted in cytosol, Yb1/Yb2 was primarily located in nuclei.