Parietal epithelial cells and podocytes in glomerular diseases

In recent years, it has become apparent that parietal epithelial cells (PECs) play an important role within the renal glomerulus, in particular in diseased conditions. In this review, we examine current knowledge about the role of PECs and their interactions with podocytes in development and under physiological conditions. A particular focus is on the crucial role of PECs and podocytes in two major glomerular disease entities. In rapidly progressive glomerulonephritis, PECs and podocytes proliferate and obstruct the tubular outlet, resulting in loss of the affected nephron. In focal and segmental glomerulosclerosis, PECs become activated and invade a segment of the glomerular tuft via an adhesion. From this entry site, activated PECs displace podocytes and deposit matrix. Thus, activated PECs are involved in inflammatory as well as degenerative glomerular diseases, which both can lead to irreversible loss of renal function.     

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.     

Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli

Drebrins are actin-binding proteins (ABP) initially identified in and thought to be specific for neuronal cells, where they appear to contribute to the formation of cell processes. Recent studies have also detected the isoform drebrin E2 in a wide range of non-neuronal cell types, notably in and near actin-rich lamellipodia and filopodia. The present study demonstrates drebrin enrichment in renal glomeruli. Immunohistochemistry and double-label confocal laser scanning microscopy have shown intense drebrin reactions in the mesangial cells of diverse mammalian species. In adult human and bovine kidneys, drebrin is, in addition, markedly enriched in the foot processes of podocytes, as also demonstrable by immunoelectron microscopy. By contrast, the podocytes of rodent glomeruli appear to contain significant drebrin concentrations only during early developmental stages. In differentiated murine podocytes cultured in vitro, however, drebrin is concentrated in the cell processes, where it partially codistributes with actin and other ABP. In biochemical analyses using protein extracts from renal cortices, large (approximately 20S) complexes ("drebrosomes") were found containing drebrin and actin. These findings confirm and extend our hypothesis that drebrin is involved in the regulation of actin dynamics also outside the nervous system. Clearly, drebrin has to be added to the ensemble of ABP regulating the actomyosin system and the dynamics of mesangial cells and foot processes in podocytes.     

Ultrafiltration in single isolated human glomeruli

To determine the ultrafiltration properties of human glomeruli, we induced filtration in vitro and estimated the glomerular ultrafiltration coefficient, Kf or LpA, and the glomerular capillary hydraulic conductivity, Lp, in single glomeruli from 17 human kidneys retrieved for allotransplantation. Cadaver donors ranged in age from 2 to 46 years. Filtration was induced in individual isolated glomeruli by abruptly lowering the protein concentration of the medium surrounding a glomerulus to produce a transcapillary oncotic gradient. The events which occurred were recorded on videotape for analysis. Kf was calculated from the maximum rate of glomerular swelling during filtration. Initial glomerular diameter for the individuals studied ranged from 146 +/- 2 microns (age, 2 years) to 292 +/- 6 microns (age, 42 years). Kf ranged from 5.1 +/- 0.8 to 30.7 +/- 3.0 nl/min . mm Hg and varied directly with donor age and glomerular size. The glomerular filtering area was estimated from the formula A = 3 pi D2 and from morphometrically measured basement membrane surface density. Lp was calculated from Lp = Kf/A. Lp using A = 3 pi D2 (LpD) averaged 3.7 +/- 0.2 microliter/min . mm Hg . cm2. To compare the hydraulic conductivity of glomeruli from children and adults, Lp was also calculated using the total basement membrane area derived from measured surface density (LpS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bmp in podocytes is essential for normal glomerular capillary formation

Bone morphogenetic protein (BMP) 4 exerts multiple biological effects on kidney and ureter development. To examine the role of BMP4 in glomerular morphogenesis, we generated transgenic mice with altered BMP4 function in podocytes by conferring tissue-specificity with the nephrin (Nphs1) promoter. At birth, Tg(Nphs1-Nog) mice, which had loss of BMP4 function in podocytes, were found to have glomerular microaneurysms, collapsed glomerular capillary tufts, enlarged Bowman's capsules, and fewer normal proximal tubules. Conversely, Tg(Nphs1-Bmp4) mice, which had increased BMP4 function in podocytes, demonstrated defects in glomerular capillary formation, but podocytes were not appreciably affected. The Tg(Nphs1-Nog) and Tg(Nphs1-Bmp4) mice shared morphological characteristics with the previously reported podocyte-specific Vegf-A over-expressing and knockout mice, respectively. Consistent with the morphological similarity, in situ hybridization revealed an intense signal for podocyte expression of Vegf in Tg(Nphs1-Nog) mice, whereas the signal was markedly suppressed in Tg(Nphs1-Bmp4) mice. However, in vitro studies with metanephroi failed to demonstrate a direct interaction between BMP4 or Noggin and VEGF in podocytes. Instead, immunostaining showed that phosphorylated Smads, the mediators of BMP signaling, are present in endothelial and/or mesangial cells, but not in podocytes, within the developing glomeruli. Therefore, this study suggests that podocyte-derived BMP plays an important role in glomerular capillary formation, perhaps by acting on non-podocyte glomerular cells in a paracrine fashion.     

Resolved: normal glomeruli filter nephrotic levels of albumin

The glomerular filtration barrier has long been thought largely impermeable to albumin. Startling new data suggest it may not be especially important in this process. Much of the argument hinges on whether charge selectivity really exists, how feasible it is for enormous quantities of albumin to instead be reclaimed by the proximal tubule, and the technical merits of previous experiments. Three experts in the field debate the merits of this argument.     

Acetylcholine increases the free intracellular calcium concentration in podocytes in intact rat glomeruli via muscarinic M(5) receptors

The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca2+](i)) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca2+](i) increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca2+ concentrations. The [Ca2+](i) plateau was not affected by the L-type Ca2+ channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 microM) blocked the plateau. Diphenylacetoxy-N:-methylpiperidine methiodide (100 nM), an M(3)/M(5) receptor antagonist, and pirenzepine (1 microM), an M(1) receptor antagonist, completely inhibited the effect of ACh. [Ca2+](i) measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca2+](i) was increased in single podocytes. Immunohistochemical studies did not demonstrate M(3) receptor expression in glomerular cells. M(1) receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M(5) receptors were found only in podocytes. The data show that ACh increases [Ca2+](i) in podocytes of intact glomeruli, most likely via muscarinic M(5) receptors.     

Ultrafiltration coefficients of glomeruli from human biopsies

Ultrafiltration coefficient (Kf) was measured in vitro using glomeruli isolated from 28 human renal biopsies in order to asses both the relationship between glomerular structure and filtration characteristics and the relationship between Kf and patients' clinical state. The patients, ages 1 to 72 years, had a wide variety of renal diseases and serum creatinines of 1 to 16 mg/dl. Glomeruli were examined by light, immunofluorescence and electron microscopy, and glomerular alterations were measured. Filtration was induced in isolated glomeruli by an oncotic gradient and Kf calculated. Glomerular diameter (D), averaged for each patient, varied from 131 to 315 microns, and Kf varied from 5.7 to 51 nl/min mm Hg. Hydraulic conductivity (Lp) in 15 biopsies averaged 1.45 mu.min-1.mm Hg-1.cm-2. In order to identify the significant predictors of Kf and delineate their relationships, stepwise multiple regression analysis was performed. Kf increased with increasing glomerular size and with increasing degree of glomerular hypercellularity. D, in turn, increased with body surface area, urinary protein, and degree of capillary damage, and decreased with percent senescent glomeruli and degree of epithelial foot process broadening. Kf did not significantly correlate with clinical measures of renal function.     

Correlation of percentage of normal glomeruli with renal outcome in Wegener's granulomatosis

BACKGROUND/AIMS: We examined the correlations between renal biopsy findings in Wegener's granulomatosis and renal function at baseline and 1 year in 22 patients who presented between 1988 and 1999. METHODS: Renal histology was independently reviewed by 2 pathologists who were masked to the clinical data. The primary outcome was the relationship of the percentage of normal glomeruli to reciprocal serum creatinine at baseline and 1 year. Other histologic data were collected using a series of ordinal rating scales. Acute and chronic sum scores were calculated. RESULTS: The median serum creatinine (SCr) at baseline was 3.9 mg/dl (0.8-14 mg/dl). Seven patients initially required hemodialysis, of whom 4 subsequently regained independent renal function. By 1 year of follow-up, the median (SCr) for patients with independent renal function was 1.9 mg/dl (0.9-6.8 mg/dl). Reciprocal SCr at baseline correlated with the percentage of normal glomeruli (r = 0.584, p = 0.005), crescent score (r = -0.595, p = 0.003), interstitial fibrosis as a percentage of cortical surface area (r = -0.669, p = 0.002), interstitial inflammation (r = -0.439, p = 0.041), eosinophil (r = -0.495, p = 0.019), neutrophil score (r = -0.557, p = 0.005), and acute sum score (r = -0.499, p = 0.018). However, only the percentage of normal glomeruli (r = 0.493, p = 0.023), the crescent score (r = -0.452, p = 0.035), and interstitial fibrosis as a percentage of cortical surface area (r = -0.466, p = 0.052) correlated with reciprocal SCr at 1 year of follow-up. CONCLUSION: In this relatively small data set, the percentage of normal glomeruli, the crescent score, and interstitial fibrosis as a percentage of cortical surface area correlated with renal function at both baseline and 1 year of follow-up.     

No complement receptor 1 stumps on podocytes in human glomerulopathies

BACKGROUND: Type one complement receptor (CR1) is the only physiological inhibitor of complement on podocytes. CR1 is lost in different glomerulopathies, in particular in lupus nephritis, in which it has been suggested that CR1 is removed by proteolysis from the cell membrane. METHODS: To define whether proteolytic cleavage of CR1 on podocytes is a general phenomenon, we analyzed the expression of CR1 in different glomerulopathies using a monoclonal antibody against epitopes present on the extracellular portion of the molecule and a polyclonal antibody directed at the intracellular tail of CR1. The two antibodies were applied on sequential serial histologic sections of renal biopsy. RESULTS: In normal glomeruli, the two antibodies provided similar results, that is, strong staining of podocytes, and both were shown to recognize specifically CR1. Decreased expression of the extracellular portion of CR1 was observed in lupus nephritis (8/8), focal and segmental glomerulosclerosis (FSGS; 7/7), IgA nephritis (6/6), membranous glomerulonephritis (3/3), and minimal change disease (3/3). In each case, the decreased expression was accompanied by a simultaneous decrease of the expression of the intracellular tail of CR1 (Spearman's correlation coefficient rs = 0.951, P < 0.001). This observation was confirmed by analyzing focal glomerular lesions on sequential serial sections. CONCLUSION: These data indicate that there are no CR1 stumps on podocytes, even in lupus nephritis, and suggest that the CR1 loss on podocytes is not due to consumption but to decreased synthesis. A loss of CR1 synthesis might render podocytes highly sensitive to complement attack.     

Histochemical and immunohistochemical studies of diseased human glomeruli

In order to study the localization of Lentil lectin (LCH)-binding glycoresidues in glomeruli from patients with a variety of glomerulopathies, and to elucidate the relationship between LCH-binding sugars and the components of the extracellular matrix, laminin and type IV collagen, investigations of formalin-fixed, paraffin-embedded kidney tissues digested with trypsin were carried out by the direct and indirect immunofluorescence microscopy techniques. The glomerular basement membrane (GBM) and the mesangium reacted well with LCH, whereas areas with sclerotic lesions exhibited a decreased reactivity. The pattern of LCH binding to the GBM in various glomerulopathies was similar to that of laminin but different from that of type IV collagen. The pattern of localization of LCH-reacting sites and of laminin in the GBM included the double linear lines in diabetic nephropathy, inner linear line with outer projections (spikes) in membranous nephropathy, and reduplicated basement membrane in membranoproliferative glomerulonephritis. The results obtained by enzyme-linked immunoadsorbent assay showed that LCH had a stronger reactivity for laminin than for type IV collagen or fibronectin. These findings suggest that LCH is more reactive with laminin than with other components of the glomerular extracellular matrix.     

The significance of monocytes in glomeruli of human renal transplants

A total of 50 biopsies from 42 renal transplants obtained during a 30-month period were studied for the presence of monocytes in the glomeruli using the nonspecific esterase reaction. Eleven biopsies from ten grafts were positive. Immune deposition was light or absent. The prognosis for the grafts containing glomerular monocytes was significantly worse during the six months after the biopsy than for those without such cells present.