BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in nonreduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A 2 receptor (PLA 2 R). Reactive serum specimens recognized recombinant PLA 2 R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA 2 R antibody. Anti-PLA 2 R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA 2 R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA 2 R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA 2 R. PLA 2 R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA 2 R is a major antigen in this disease.

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M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.

Serial creatinine clearances (5 to 14 studies) were obtained for 446 normal volunteers in the Baltimore Longitudinal Study of Aging followed between 1958 and 1981. When those subjects with possible renal or urinary tract disease and subjects on diuretics and antihypertensives were removed from the study, leaving a group of 254 "normal" subjects, the mean decrease in creatinine clearance was 0.75 ml/min/year. The slopes of the creatinine clearance vs. time fell into a normal (Gaussian) distribution around this mean. One third of all subjects followed had no absolute decrease in renal function (positive slope of creatinine clearance vs. time) and there was a small group of patients who showed a statistically significant increase (P less than 0.05) in creatinine clearance with age.

Longitudinal Studies on the Rate of Decline in Renal Function with Age

Part I highlights the mechanisms of glomerular filtration and tubular reabsorption of plasma proteins, selected characteristics of urinary proteins based upon electrophoretic properties and recent advances in clinical laboratory analysis of proteinuria. Both structural characteristic of the glomerular capillary wall and molecular properties of plasma proteins are important determinants of glomerular filtration. Proteins filtered by the glomerulus subsequently appear in urine only after escaping the efficient mechanisms of tubular reabsorption. Albumin is one such protein and constitutes the major protein in normal urine although trace amounts of alpha, beta, and gamma globulins are also detectable. Several techniques of protein analysis have thus been developed to specifically measure albumin as well as other plasma proteins. Other methods have been adapted to measure total urinary protein content enabling the clinician to readily monitor renal function in health and disease. The second part of this review will consider conditions associated with proteinuria in both asymptomatic individuals and patients with renal disease. Asymptomatic proteinuria encompasses states of excess protein excretion during conditions of orthostasis, exercise, travel to high altitude of fever. Proteinuria during renal disease has received considerable interest as a means to monitor kidney function. It is therefore classified according to the type of damage incurred: (1) glomerular-type where large molecular weight proteins are excreted (2) tubular-type where small molecular weight proteins are excreted and (3) mixed-type characterized by both large and small molecular weight proteinuria.

Normal and abnormal aspects of proteinuria: Part I: Mechanisms, characteristics and analyses of urinary protein. Part II: Clinical considerations

The nephritogenic antigen of Heymann's nephritis (HN) was previously purified from tubular brush-border fractions of rat kidney and found to be a 330,000- mol-wt glycoprotein (gp330). This study was conducted to determine whether gp330 is also present in the rat glomerulus, and, if so, to establish where in the glomerulus it is located. Rabbit polyclonal and mouse monoclonal antibodies were raised against purified gp330, which specifically immunoprecipitated gp330 from solubilized brush-border fractions and specifically stained microvilli and coated invaginations (located at the base of the microvilli) of proximal tubule cells. Accordingly, they were used to localize gp330 by immunoprecipitation and immunocytochemistry in glomeruli of normal Lewis rats. 

For immunoprecipitation, purified glomerular fractions were prepared from [(35)S]-methionine-labeled kidneys, extracted with Triton X-100, and the extract was used for immunoprecipitation with affinity-purified rabbit polyclonal, or mouse monoclonal, anti-gp330 IgG. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography indicated that a band corresponding in mobility to gp330 was specifically precipitated. 

When unfixed cryostat sections were incubated for indirect immunofluorescence with monoclonal or affinity-purified polyclonal IgG, a fine granular fluorescent staining was seen throughout the glomerulus. When aldehyde-fixed cryostat sections were incubated for indirect immunoperoxidase, reaction product was detected only in the epithelial cells and was not seen in the GBM, endothelium, or mesangium. Within the epithelium it was localized to the endoplasmic reticulum, occasional Golgi elements, multivesicular bodies, and coated pits at the cell surface. The reactive coated pits were distributed all along the cell membrane, including the sides and base of the foot processes. Reaction product was detected in the latter location only in sections that had been digested with neuraminidase before antibody incubation. When rats were given rabbit anti-gp330 IgG by intravenous injection and their kidneys stained for direct immunoperoxidase 3 d later, rabbit IgG was seen to be deposited beneath the slit diaphragms and in the coated pits at the base of the foot processes. 

The immunocytochemical and immunoprecipitation data indicate, in confirmation of the results of others, that the nephritogenic HN antigen is present in renal glomeruli as well as in proximal tubular brush borders. The immunocytochemical results further demonstrate that gp330 is an epithelial, rather than a glomerular basement membrane, antigen. It appears to be synthesized by glomerular epithelial cells and subsequently becomes concentrated in coated pits. As both the endogenous antigen (gp330) and exogenously administered anti-gp330 antibody were localized to coated pits, it seems likely that coated pits located at the base of the foot processes are the sites where the HN antigen (gp330) and circulating antibodies directed against gp330 meet and where immune complexes are formed.

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Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats

THE process of urine formation begins at the walls of the glomerular capillaries with the separation of as much as a third of the plasma entering the glomeruli of each kidney into a solution having the properties of a nearly ideal ultrafiltrate. Driven by a hydraulic force created by the pumping action of the heart, ultrafiltrate flows across these capillary walls at an enormous rate, when considered in biologic terms, exceeding by at least two orders of magnitude that across other membranes studied thus far.1 Despite their extremely low intrinsic resistance to the flow of water, however, these same glomerular . . .

Molecular basis of proteinuria of glomerular origin.

The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.

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Experimental glomerulonephritis in the isolated perfused rat kidney.

Crescentic glomerulonephritis without immune deposits: clinicopathologic features.

Autologous immune complex nephropathy. I. Sequential study of immune complex deposition, ultrastructural changes, proteinuria, and alterations in glomerular sialoprotein.

The effect of increased capillary permeability on glomerular immune complex localization was studied in rats immunized with proximal tubular antigen (Fx1A) to induce autologous immune complex nephropathy (AICN). AICN rats were made proteinuric by injection or unilateral renal perfusion with aminonucleoside of puromycin (PA) before developing subepithelial complex deposits. Control AICN kidneys developed diffuse granular deposits of IgG and Fx1A on the subepithelial surface of the glomerular basement membrane (GBM) at 3 wk by immunofluorescence and electron microscopy, and deposits increased in subsequent weekly biopsies. In contrast, PA-nephrotic AICN kidneys developed few or no GBM deposits and a significant increase in mesangial localization of IgG and Fx1A during the period of PA-induced proteinuria. These alterations in complex localization were documented both in rats with PA nephrosis and in unilaterally PA-nephrotic kidneys compared with contralateral controls in the same animals, thus excluding any effect of PA on the immunopathogenetic mechanism in AICN as an explanation for these findings. The absence of GBM deposits closely correlated with reduced staining for polyanionic glomerular sialoprotein in proteinuric kidneys, since PA-perfused kidneys studied 2 wk after resolution of proteinuria demonstrated return of normal staining for sialoprotein and development of subepithelial complex deposits similar to those in contralateral control kidneys. These studies demonstrate that properties of the glomerulus itself play an important role in determining the site of complex deposition in experimental AICN and suggest that electrophysical characteristics of the glomerular capillary wall may influence complex localization on the GBM.

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Effect of aminonucleoside nephrosis on immune complex localization in autologous immune complex nephropathy in rats.

The effect of anti-GBM antibody on protein excretion was studied in isolated rat kidneys perfused with 20 mg of sheep anti-rat GBM (experimental) or nonantibody sheep IgG (control). Six control kidneys excreted 176 +/- 31 micrograms/min of BSA initially, rising to 296 +/- 111 micrograms/min at 2 h. Fractional clearance of BSA rose from 0.51 to 1.70%. Eight experimental kidneys excreted 211 +/- 56 micrograms/min of BSA, increasing to 1,924 +/- 804 micrograms/min at 2 h. Fractional BSA clearance increased from 0.56 to 11.49%. After 60 min, BSA excretion in anti-GBM-perfused kidneys exceeded controls by a factor of 6.5-7.9 (P less than 0.05) and fractional BSA clearance exceeded controls by a factor of 5.8-7.1 (P less than 0.05). Studies with fluorescent markers indicated proteinuria to be of glomerular origin in antibody-perfused kidneys. There were no significant differences between anti-GBM-perfused and control kidneys in perfusion pressures, perfusate flow rates, urine flow rates, inulin clearance, or sodium reabsorption. Antibody to GBM can induce a marked increase in glomerular permeability to BSA and IgG without participation of other systemic humoral or cellular mediation systems.

Magda M. Stilmant

Papers

Experimental glomerulonephritis in the isolated perfused rat kidney.

Crescentic glomerulonephritis without immune deposits: clinicopathologic features.

Autologous immune complex nephropathy. I. Sequential study of immune complex deposition, ultrastructural changes, proteinuria, and alterations in glomerular sialoprotein.

Effect of aminonucleoside nephrosis on immune complex localization in autologous immune complex nephropathy in rats.

Anti-GBM antibody-induced proteinuria in isolated perfused rat kidney