Human Glomerular Diseases Research Articles

Inducible rodent models of acquired podocyte diseases

Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded.

Localization of 20-kD homologous restriction factor (HRF20) in diseased human glomeruli. An immunofluorescence study

The 20-kD homologous restriction factor (HRF20), which is identical to CD59, is a membrane-associated protein which inhibits the reaction of C9 to form membrane attack complex (MAC) of homologous complements. In various human glomerular diseases deposition of complement components is frequently seen and MAC is reported to associate with immune deposits. Using a specific monoclonal antibody, 1F5, against HRF20, we attempted to study the localization of HRF20 in human glomerulonephritides and to compare the localization of HRF20 with those of immune deposits and MAC. The frozen sections of kidney specimens were fixed in acetone at room temperature before staining. In normal kidneys and kidney specimens from the patients with minimal change nephrotic syndrome, membranous nephropathy, and IgA nephropathy, HRF20 was strongly localized in the peritubular capillaries and along Bowman's capsules. A weaker but well-defined staining was obtained in the mesangial area and faint staining was seen along the glomerular capillary walls. In contrast, glomerular capillary walls were rather strongly stained in the cases with diffuse lupus nephritis which had subendothelial dense deposits. These data suggest that HRF20 (CD59) is present in the human glomeruli and its expression is enhanced under certain conditions such as lupus nephritis.

Expression of α-Actinin-1 in Human Glomerular Mesangial Cells In Vivo and In Vitro

Recent studies have demonstrated important roles of alpha-actinins in glomerular disease, while little information is known about the expression profile of alpha-actinins in human glomerular mesangial cells. Here, immunofluorescence and confocal microscopy showed that alpha-actinin-1 exclusively distributed along mesangial cells in human glomeruli of IgA nephropathy. RT-PCR and Western blot further confirmed the expression of alpha-actinin-1 in primary cultured human mesangial cells. We also found that transforming growth factor-beta 1 (TGF-beta 1) stimulated ACTN1 gene transcription and that transiently transfected alpha-actinin-1 significantly increased TGF-beta 1-induced plasminogen activator inhibitor-1 (PAI-1) promoter activity in human mesangial cells. These findings suggest that alpha-actinin-1 may play a role in human glomerular disease.

RAGE Mediates Podocyte Injury in Adriamycin-induced Glomerulosclerosis

In the kidney, the receptor for advanced glycation end products (RAGE) is principally expressed in the podocyte at low levels, but is upregulated in both human and mouse glomerular diseases. Because podocyte injury is central to proteinuric states, such as the nephrotic syndrome, the murine adriamycin nephrosis model was used to explore the role of RAGE in podocyte damage. In this model, administration of the anthracycline antibiotic adriamycin provokes severe podocyte stress and glomerulosclerosis. In contrast to wild-type animals, adriamycin-treated RAGE-null mice were significantly protected from effacement of the podocyte foot processes, albuminuria, and glomerulosclerosis. Administration of adriamycin induced rapid generation of RAGE ligands, and treatment with soluble RAGE protected against podocyte injury and glomerulosclerosis. In vitro, incubation of RAGE-expressing murine podocytes with adriamycin stimulated AGE formation, and treatment with RAGE ligands rapidly activated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, via p44/p42 MAP kinase signaling, and upregulated pro-fibrotic growth factors. These data suggest that RAGE may contribute to the pathogenesis of podocyte injury in sclerosing glomerulopathies such as focal segmental glomerulosclerosis.

Basic science meets clinical medicine: identification of a CD2AP-deficient patient

Recent years have witnessed an explosion of research into the molecular basis of glomerular disease resulting in nephrotic- range urinary protein leak using both human genetics and animal models. Löwik et al. describe the first case report of an early-onset nephrotic syndrome presenting in conjunction with a homozygous CD2AP mutation. These data demonstrate the convergence between basic and clinical approaches and their potential to transform our understanding of the pathogenetic mechanisms underlying human glomerular disease.

Expression of filtrin in human glomerular diseases

Filtrin (NEPH3/KIRREL2) is a recently characterized member of the nephrin-like proteins of the immunoglobulin superfamily, and it has been suggested to participate in the maintenance of the glomerular filtration barrier in the kidney. In this study, the gene and protein expression of filtrin were examined in patients with acquired proteinuric diseases. Filtrin mRNA levels in renal biopsies were measured with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in two sets of patients with proteinuria. The mRNA levels were normalized to the housekeeping gene GAPDH and also related to the podocyte-specific genes nephrin and podocin. Immunofluorescence microscopy was employed to explore changes in the glomerular distribution of filtrin. Reduced glomerular expression of filtrin mRNA was observed in all studied diagnostic groups. In focal segmental glomerulosclerosis, the filtrin mRNA level was only one-tenth of the control samples (P approximately 5.0x10(-6)), and this finding was confirmed in a second set of samples. The ratios of filtrin to nephrin and podocin demonstrated a marked decrease in the expression of filtrin relative to the podocyte marker genes. However, no correlation between the expression of filtrin and the levels of serum creatinine and proteinuria was observed. Immunostaining showed changes in the expression pattern of filtrin in renal biopsies. Immunoelectron microscopic studies localized filtrin at the slit diaphragm of the podocyte foot processes. Down-regulation of the filtrin gene and protein expression in the renal biopsies together with the localization to the inter-podocyte filtration slit imply a potential role for this molecule in the pathogenesis of proteinuric diseases.

Direct effects of dexamethasone on human podocytes

Glucocorticoids are widely used in the treatment of human glomerular diseases, but their mode of action is poorly understood particularly in steroid-sensitive nephrotic syndrome, which is most common in childhood and is characterized by a lack of inflammation in the kidney. The podocyte is a key cell in the glomerulus in health and disease: until recently, human podocytes have been difficult to study in vitro. We have developed a conditionally immortalized human podocyte cell line transfected with a temperature-sensitive simian virus 40 transgene: when the transgene is inactivated in vitro, these cells adopt the phenotype of differentiated podocytes. We have used these cells to evaluate, using immunocytochemistry, reverse transcriptase-polymerase chain reaction, and Western blotting, direct effects of the glucocorticoid dexamethasone at concentrations designed to mimic in vivo therapeutic corticosteroid levels. Dexamethasone upregulated expression of nephrin and tubulin-alpha, and downregulated vascular endothelial growth factor. Effects on cell cycle were complex with downregulation of cyclin kinase inhibitor p21 and augmentation of podocyte survival, without any effect on apoptosis. We report cytokine production by human podocytes, especially interleukin (IL)-6 and -8; IL-6 expression was suppressed by dexamethasone. These potent direct effects on podocytes illustrate a novel mode of action of glucocorticoids and suggest potential new therapeutic strategies for glomerular disease.

Cell-cycle regulatory proteins in the podocyte in collapsing glomerulopathy in children

Podocyte is a terminally committed cell in G1 arrest of cell cycle, and is unable to overcome G1/S transition phase in children with minimal change disease (MCD) and classic focal segmental glomerulosclerosis (FSGS), in contrast to dysregulated proliferative phenotype of idiopathic collapsing glomerulopathy (CGN) in adults. Forty-two kidney biopsies, MCD (14), FSGS (12), CGN (4), and normal (CON) (12), were evaluated by immunohistochemistry using dual staining for expression of p27, p21, and p57, and cyclins D and A, in podocytes of children with CGN. On light microscopy, all podocytes expressed p27, whereas p21 and p57 expression was seen in a portion of podocytes in normal kidney biopsies. Cyclin D was expressed in a small percentage of podocytes. Cyclin A expression was absent in normal biopsies. The staining for p27 decreased significantly, in order, from normal (100%) to MCD (45.8%) to CGN (24.2%) to FSGS (16.6%). p21 staining was significantly decreased from normal (69.8%) to CGN (15.5%) to MCD (2.2%) to FSGS (0.6%), and the difference between CGN and MCD and FSGS was also significant. There was no significant difference in staining of p57. Cyclin D staining was significantly increased in CGN (26.8%) compared to normal (7.2%), MCD (1.6%), and FSGS (0.0%), and the difference between CGN and MCD and FSGS was also significant. De novo cyclin A staining was only observed in children with CGN. Thus, p27 and p21 but not p57 was decreased in CGN, as in FSGS when compared to normal. Both cyclins D and A staining were increased in CGN. The staining pattern in CGN would suggest that podocyte is able to overcome G1/S transition phase, and has a proliferative phenotype. We propose, based on the significant contrast observed in podocytes injury response between CGN (proliferative) and classic FSGS (non-proliferative), that CGN not be considered as a morphological variant of FSGS.

Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling

Loss of renal function is often associated with the injury of kidney glomeruli. It is therefore necessary to understand the mechanisms leading to progressive glomerular diseases; this may be addressed using proteomics. Until now, however, analysis of the glomeruli proteome using 2-DE has been technically hampered by low protein yields from scarce samples. To circumvent this problem, we developed a procedure which allows the human and mouse glomeruli proteome to be analysed. In this study, two different approaches were used to isolate mouse and human glomerular protein from kidney cortex. Mouse glomeruli were extracted by embolisation magnetic beads into the glomerular capillaries. Laser capture microdissection (LCM) was utilised to harvest glomeruli from human biopsy material. Human and murine samples were analysed using a fluorescence saturation labelling technique. Using 3 microg mouse glomerular protein a total of 2900 spots were resolved for differential proteome analysis. Moreover, it was also demonstrated for the first time that only ten glomeruli (0.5 microg) picked by LCM from a slide of a human kidney biopsy material were sufficient to visualise 900 spots. This novel strategy paves the way for future experiments aimed at investigating functional proteomics of glomerular diseases in humans and in mice.

Expression of the chemokine receptor CXCR1 in human glomerular diseases

Leukocyte infiltration, a hallmark of renal diseases, is orchestrated in part by the actions of chemokines. The chemokine CXCL8/interleukin (IL)-8 is expressed during renal diseases and allograft rejection, whereas the corresponding receptor CXCR1 has not been described previously. Expression of CXCR1 was characterized in peripheral blood using multicolor fluorescence-activated cell sorter analysis (FACS). CXCR1 was localized in 81 formalin-fixed, paraffin-embedded renal specimens by immunohistochemistry using a monoclonal antibody against human CXCR1. Included were biopsies with crescentic glomerulonephritis (CGN, n = 22), immunoglobulin (Ig) A nephropathy (n = 15), membranoproliferative glomerulonephritis (MPGN, n = 17), lupus nephritis (n = 12), membranous nephropathy (n = 11), and non-involved parts of tumor nephrectomies (n = 4). Consecutive tissue sections of human tonsils, allograft explants, and renal biopsies were stained for CD15- and CD68-positive cells. Expression of CXCR1 and CXCL8/IL-8 mRNA was quantified by real-time reverse transcriptase-polymerse chain reaction of microdissected renal biopsies (n = 35) of the same disease entities. By FACS CXCR1 expression was found on polymorphonuclear CXCR1 expression by polymorphonuclear leukocytes (PMNs), natural killer cells, and a subpopulation of monocytes. By immunohistochemistry, CXCR1 expression was found on infiltrating inflammatory cells (predominantly PMNs), as well as on intrinsic renal cells (arterial smooth muscle cells, endothelial cells of peritubular capillaries). The distribution pattern of CXCR1 differed between disease entities. The highest numbers of glomerular CXCR1-positive cells were present in biopsies with MPGN, followed by lupus nephritis, and CGN. CXCR1 might be involved in the recruitment of PMNs to the glomerular tuft, which could be targeted by CXCR1-blocking agents.

Comparative promoter analysis allows de novo identification of specialized cell junction-associated proteins

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.

Endothelin Receptors in the Kidney of Patients with Proteinuric and Non-Proteinuric Nephropathies

Background. Endothelin-1 (ET-1), which acts via the specific receptors ET-A and ET-B, has been implicated in the development of renal scarring. The activation of the endothelin system was observed in experimental models of glomerular diseases and was attributed to the toxic action of proteinuria on the tubular epithelial cells. The aim of this study was to investigate whether the endothelin system in the kidney is altered in glomerular diseases and possibly related to proteinuria. Methods. Thirty-seven patients with different types of glomerulonephritis and 14 controls were included. Patients presented either nephrotic syndrome (n=25) or mild proteinuria (<1g/24h, n=12). The expression of ET-A and ET-B receptors in the renal tissue was examined immunohistochemically. At the time of biopsy, urinary ET-1 was determined. Results. Both receptors were mainly localized within tubular epithelial cells, and their expression was significantly higher in patients with glomerulonephritis compared to controls. The expression of ET-B was higher in nephrotic compared to non-nephrotic patients, while no difference was observed in the expression of ET-A receptors. A significant positive correlation of the degree of proteinuria with the excreted ET-1 (r= 0.487, p<0.05) and the extent of immunostaining for ET-B receptors (r=0.420, p<0.05) was observed. The expression of ET-B receptors and the excretion of ET-1 decreased significantly in patients with remission of nephrotic syndrome after therapy. Conclusion. This study provides evidence that the endothelin system is activated in human glomerular disease, confirming data from experimental studies. Proteinuria seems to be related to the activation of endothelin system, though further investigation is necessary to clarify this issue.

Glomerular Disease Workshop

Recent observations regarding intrinsic glomerular cell biology, particularly in the podocyte, have provided exciting new insights into potential pathogenic mechanisms of human glomerular disease. Although both immune and nonimmune mechanisms of glomerular injury have been studied previously, experimental models of disease and recent techniques that provide tools for molecular profiling show great promise for identifying glomerular disease biomarkers. Despite these recent advances, additional work in both basic and clinical studies of glomerular disease is needed to advance the field. Standardization of animal models of distinct forms of glomerular disease would likely facilitate the search for biomarkers. Several factors limit current efforts to implement clinical trials of glomerular disease. Identification of disease biomarkers, development of disease-specific end points, and organization of collaborative clinical groups are critical for ultimately designing and implementing appropriately powered trials of glomerular disease.

Podocyte Depletion Causes Glomerulosclerosis

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

Pathogenic mechanisms in membranoproliferative glomerulonephritis

This review considers new information on the pathogenesis of a long recognized and poorly understood form of glomerular injury, membranoproliferative glomerulonephritis. This disease has received growing attention as it is the principal renal manifestation of hepatitis C virus infection, which has become pandemic worldwide. This review briefly describes three murine models of membranoproliferative glomerulonephritis suitable for pathogenesis studies. We consider recent evidence implicating innate immune mechanisms in immune and autoimmune-mediated glomerulonephritis, and recent data pointing to the alternative pathway of complement activation in the amplification of glomerulonephritic injury. Understanding the contribution of complement activation and innate immunity to the evolution of membranoproliferative glomerulonephritis promises to provide new therapeutic targets for this disease. Inhibitors of the complement cascade are already being tested in clinical trials as therapeutic interventions for some human glomerular diseases. Successful tests of this approach in membranoproliferative glomerulonephritis are still awaited. Our understanding of how the innate immune system modulates glomerulonephritis is still in an early stage, and future studies should be directed at identifying targets and specific interventions that may also benefit patients with this disease.

WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment.

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 microg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

ACE inhibitors improve nephrin expression in Zucker rats with glomerulosclerosis

Podocyte injury is associated with many forms of human and experimental proteinuric glomerular disease. The aim of this study was to investigate the level of nephrin expression in a model of obesity and type II diabetes mellitus, the obese Zucker rat, as well as to investigate whether nephrin expression is influenced by treatment with quinapril or diltiazem, 2 drugs frequently used in type II diabetes mellitus. Obese Zucker rats were treated with either quinapril or diltiazem at a dose of 10 mg/kg body weight per day and 100 mg/kg body weight per day, respectively, for 6 months. Real time reverse transcription-polymerase chain reaction (RT-PCR) and immunoperoxidase assays were used to assess and quantify nephrin gene expression and other markers of podocyte damage, such as desmin and synaptopodin protein. Quinapril treatment prevented the reduction of nephrin levels compared with the control group, while diltiazem treatment did not prevent the reduction. Similar results were obtained when other phenotypic markers, such as desmin, were assessed. Similarly, synaptodin showed this tendency, although it did not achieve statistically significant differences. The podocyte phenotypic changes assessed in a model of obesity and type II diabetes mellitus were corrected by an angiotensin-converting enzyme (ACE) inhibitor. These results could be associated with an improvement in the slit diaphragm, and therefore, in the maintenance of the filtration barrier. Diltiazem did not achieve similar results.

CXCR3 Is Involved in Tubulointerstitial Injury in Human Glomerulonephritis

Chemokines play pivotal roles in the recruitment of inflammatory cells into the kidney. The chemokine receptors CXCR3 and CCR5 are expressed on activated T lymphocytes, and expression of CXCR3 by mesangial cells has been suggested. Detailed description of CXCR3 expression might form a rational basis for use as a diagnostic marker and for therapeutic CXCR3 targeting in human glomerulonephritis. We studied the expression of CXCR3 in renal biopsies by immunohistochemistry (n = 45), and real time RT-PCR (n = 78). Biopsies were from patients with IgA nephropathy, lupus nephritis, and membranoproliferative glomerulonephritis. Furthermore, cultured human mesangial cells (HMC) were studied for CXCR3 expression, and for functional responses to the ligands CXCL10/IP-10 and CXCL9/Mig. CXCR3-positive cells were rarely found in glomerular tufts, but formed a major part of the tubulointerstitial infiltrates. Consistently, CXCR3 mRNA expression was too low to be quantified in glomerular compartments, and was not detectable in HMC. The published staining for CXCR3 of mesangial cells could be traced to cross-reactivity of an antibody for CXCR3 with a potentially related chemokine receptor as revealed by FACS analysis. Despite an absence of CXCR3 expression, mesangial cells reacted to CXCR3 ligands by proliferation and migration, which was blocked by pertussis toxin but not by an anti-CXCR3 antibody. These results indicate that HMC do not express the classical CXCR3, but may potentially express a related receptor with shared ligand specificity. By immunohistochemistry the number of CXCR3-positive cells, mainly interstitial T cells, correlated with renal function, proteinuria, and percentage of globally sclerosed glomeruli. A significant morphological and numerical correlation between CD3, CXCR3, and CCR5-positive cells indicated a CXCR3/CCR5 double-positive T cell population. No apparent difference in the CXCR3 expression pattern was found between disease entities. CXCR3 expression was localized to interstitial T cells, and these cells correlated strongly with important prognostic markers. Therefore interstitial CXCR3, as well as CCR5-positive T cells might play an important role during progressive loss of renal function, and are potential therapeutic targets in human glomerular diseases.

The role of podocytes in glomerular pathobiology.

Podocytes are unique cells with a complex cellular organization. With respect to their cytoarchitecture, podocytes may be divided into three structurally and functionally different segments: cell body, major processes, and foot processes (FPs). The FPs of neighboring podocytes regularly interdigitate, leaving between them the filtration slits that are bridged by an extracellular structure, known as the slit diaphragm (SD). Podocytes cover the outer aspect of the glomerular basement membrane (GBM). They therefore form the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria. Chronic podocyte injury may lead to podocyte detachment from the GBM. Our knowledge of the molecular structure of the SD has been remarkably improved in the past few years. Several molecules, including nephrin, CD2AP, FAT, ZO-1, P-cadherin, Podocin, and Neph 1-3 have all been shown to be associated with the SD complex, and some of these molecules are critical for its integrity. Podocytes are injured in many forms of human and experimental glomerular disease. The early events are characterized either by alterations in the molecular composition of the SD without visible changes in morphology or, more obviously, by a reorganization of FP structure with the fusion of filtration slits and the apical displacement of the SD. Based on recent insights into the molecular pathology of podocyte injury, at least four major causes have been identified that lead to the uniform reaction of FP effacement and proteinuria: (1) interference with the SD complex and its lipid rafts; (2) direct interference with the actin cytoskeleton; (3) interference with the GBM or with podocyte-GBM interaction; and (4) interference with the negative surface charge of podocytes. There is also evidence, in focal segmental glomerular sclerosis (FSGS) and in idiopathic nephrotic syndrome in humans and rats, that podocyte damage may be caused by circulating albuminuric factors. Ongoing studies in many laboratories are aiming at an understanding of the dynamic relationship between SD proteins, the actin cytoskeleton, and the dynamics of FP structure in nephrotic syndrome and FSGS. These studies should provide us with a better understanding of the biological mechanism underlying the podocyte response to injury. Such studies will potentially translate into more refined treatment and the prevention of proteinuria and progressive glomerular disease.

Accumulation of cholesterol in the lesions of focal segmental glomerulosclerosis.

Intraglomerular deposition of low-density lipoprotein (LDL) and oxidized LDL has been described in various human glomerular diseases. Yet it is not clear whether esterified cholesterol (EC) and unesterified cholesterol (UC) carried in LDL are mobilized from deposited LDL particles or accumulate in the diseased human glomeruli, particularly in the segmentally sclerotic lesions. To address this issue, frozen sections of renal biopsies were first immunostained to localize apolipoprotein B (apo B) and then oil red O (ORO) stained to colocalize neutral lipids. By using 124 ORO-positive biopsies and nine ORO-negative ones, UC was visualized directly with filipin staining, and EC was visualized after its enzymatic hydrolysis and staining with filipin. Seventy-seven biopsies (58%) showed filipin staining of accumulated EC and/or UC in the glomeruli. Of these, 11 showed heavy filipin staining for both EC and UC in the segmentally sclerotic lesions. In a group with UC deposits in the sclerotic segments, the percentage of the glomeruli affected by sclerosis and the intensity of filipin fluorescence for UC were significantly higher than biopsies with only mesangial UC deposits. Most filipin-positive biopsies showed apo B staining mainly in the mesangium. Yet in the sclerotic segments, apo B staining was rarely noted. Accumulated apo B-stained lipoprotein was not coincident with ORO-stained lipid in the diseased glomeruli. These results suggest that both EC and UC accumulate in the sclerotic glomerular segments as the glomerular lesions are advanced, and that these EC and UC appear to be derived from altered LDL with progressive loss of apo B.