Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway.

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

Anti-RNA polymerase III antibody-associated scleroderma renal crisis in a patient with limited cutaneous systemic sclerosis: A case report.

A 69-year-old Japanese man was presented with hypertensive crisis. Renal histology revealed malignant nephrosclerosis, including an onion skin pattern with fibrinoid necrosis of the small arteries from arterioles up to interlobular arteries. Immunological investigation clarified positive anti-RNA polymerase (RNAP) III antibody, and limited cutaneous systemic sclerosis (Lc SSc) was diagnosed by skin biopsy as the underlying disease causing scleroderma renal crisis (SRC). Angiotensin covering enzyme (ACE) inhibitor therapy and calcium antagonist were effective for his renal condition. Although an association between SRC and anti-RNAP III antibody has already been reported in patients with diffuse cutaneous SSc (Dc SSc), this case indicates that SRC with hypetensive emergency with malignant nephrosclerosis can also be diagnosed on patients with Lc SSc patients by the examination of anti-RNAP III antibody.

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis.

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1(rnu/rnu) ) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.

A human monoclonal antibody against the collagen type IV α3NC1 domain is a non-invasive optical biomarker for glomerular diseases.

Progressive kidney disease is a significant clinical problem. However, despite research aimed toward developing improved predictors of disease, the major tool to assess kidney ultrastructure damage is the kidney biopsy. Here we tested the capability of a labeled human monoclonal antibody (F1.1), directed against the NC1 domain of α3(IV) collagen, to detect pathologic kidney alterations in vivo using mouse models of nephrotoxic serum-induced nephritis and puromycin aminoglycoside nephrosis. The F1.1 antibody-fluorophore conjugate signal rapidly localized specifically to injured glomeruli in both the severe and mild kidney disease models while minimally labeling healthy kidney. This differential labeling is likely due to cryptic NC1-domain exposure as enzymatic or chemical treatment of healthy human or mouse kidney sections significantly increased F1.1 binding to the glomeruli. Finally, kidney tissue from patients with renal disease show significant glomerular staining by F1.1 indicating that exposure of the NC1 domain occurs in clinically relevant circumstances. Thus, NC1 domain exposure may represent an in situ biomarker for assessment of kidney injury. Our study suggests that F1.1 and similar antibodies may represent a new class of non-invasive renal imaging reagents.

Targeted expression of a pan-caspase inhibitor in tubular epithelium attenuates interstitial inflammation and fibrogenesis in nephritic but not nephrotic mice.

The caspase family of enzymes participates in apoptotic and proinflammatory reactions in any cell. Here we studied the role of caspase activation in the tubular epithelium of diseased kidneys using mice transgenic for the baculovirus pan-caspase inhibitor p35 gene held in a nonexpressed state (control mice) but target-expressed in the renal proximal tubule cells when crossed with mice expressing Cre recombinase under the control of the γ-glutamyltransferase promoter. Proinflammatory and profibrogenic parameters such as the number of monocytes and fibroblasts in the kidneys were significantly increased at 28 days in the control mice, but not in the renal tubule-targeted mice expressing p35 in a nephrotoxic serum nephritis model of disease. These cellular changes paralleled the number of apoptotic tubular cells and protein levels of active caspase-3 in the kidneys at 7 and 28 days of both the control and proximal tubule-targeted mice. Surprisingly, all of these parameters were not significantly affected at 7 and 28 days by targeted p35 expression in tubular epithelium when compared with nontargeted control mice in a model of adriamycin nephrosis. Thus, our study shows the critical role of caspase activation in the tubular epithelium in apoptosis along with proinflammatory and profibrogenic processes in nephrotoxic serum nephritis but not adriamycin nephrosis.

By homing to the kidney, activated macrophages potently exacerbate renal injury.

Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 x 10(6) to 5 x 10(6)) or activated (1 x 10(3) to 1 x 10(6)) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 x 10(4) macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 x 10(6) macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages.

High serological response to pneumococcal vaccine in nephrotic children at disease onset on high-dose prednisone.

Our objective was to demonstrate that nephrotic children at disease onset and under high-dose prednisone respond to vaccination with 23-valent pneumococcal vaccine (PV). We compared the serological response after PV in 30 children with nephrotic syndrome, directly after initiation of prednisone therapy (60 mg/m2 body surface area) at disease onset (group 1), with the response in 13 patients who received the vaccine while in remission (group 2). Safety was studied, comparing disease course in group 1 with those in patients who did not receive any PV (group 3). In group 1, 23-valent PV antibody (Ab) levels increased tenfold after 1 month and remained increased after 1 year (P < 0.01). Ab response in the short term and in the long term was not different from that of patients in group 2. Serum albumin, age, or immunosuppressive drugs did not influence Ab response. Disease courses in groups 1 and 3 were not different. In conclusion, nephrotic children on high-dose glucocorticoid therapy respond to a 23-valent anti-PV. Children with steroid dependent/resistant forms acquire high Ab levels, even if early relapses delay the tapering of steroids or if immunosuppressive agents are introduced. Patients who relapse during the tapering of steroids already have increased anti-pneumococcal Ab at the time of relapse.

Partial depletion of macrophages by ED7 reduces renal injury in Adriamycin nephropathy.

BACKGROUND: Because macrophages are considered to be possible effectors of disease in Adriamycin (ADR) nephrosis, we hypothesized that depletion of macrophages might protect against the initiation of renal injury. In the present study, a monoclonal antibody (ED7) directed against CD11b/CD18 integrin, which is expressed by macrophages, was used to investigate the pathogenetic effects of macrophages in ADR nephropathy. METHODS: Male Wistar rats were treated with ED7 antibody, starting 1 day prior to ADR (7.5 mg/kg) treatment, or 7 days post-ADR when overt proteinuria was established. RESULTS: Circulating ED7-positive cells were reduced by approximately 30% in rats with ADR nephrosis by the ED7 antibody, while the number of macrophages in the renal cortex of ADR rats was reduced by nearly 50% with the ED7 treatment, whether administered before or after ADR. Creatinine clearance was significantly ameliorated by ED7 when commenced pre-ADR (P < 0.05), but not when commenced post-ADR (P = NS) in comparison to untreated ADR rats. However, proteinuria was not alleviated by either ED7 treatment. Morphometric analysis showed less glomerular sclerosis when ED7 was commenced pre-ADR compared with ADR alone (P < 0.01), but not when commenced post-ADR (P = NS). Tubular atrophy was reduced by ED7 when it was commenced pre-ADR (tubular cell height and tubular diameter: P < 0.01 and P < 0.001, respectively), as was interstitial expansion (P < 0.01) compared with ADR alone. Cortical macrophage infiltration was reduced by 50% compared with ADR alone by the ED7 commenced before or after ADR. The number of cortical CD4+ T cells fell with ED7 starting pre-ADR, but not with the ED7 treatment commencing after ADR. CONCLUSION: Partial macrophage depletion starting before but not after ADR protected both renal function and structure in this model of chronic proteinuric renal disease.

Treatment of idiopathic nephrosis by immunophillin modulation.

Until 1985, glucocorticoids and cytotoxic drugs were the only treatments available for idiopathic nephrotic syndrome (nephrosis), that is, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Trials of cyclosporine (CsA) treatment of nephrosis, the rationale of which was based on pathophysiologic considerations, have shown that this immunophillin modulator is effective in inducing and maintaining remission in patients suffering from idiopathic nephrotic syndrome. It appears that the best results, in the order of 80% remission rate, are obtained in steroid-sensitive cases, essentially MCD, and that in steroid-resistant FSGS the drug obtains remission in no more than 20% of the cases. Addition of glucocorticoids increases the success rate to approximately 30% of cases. Renal toxicity is proportional to previous impairment of renal function, primary renal disease (FSGS vs MCD) dosage >5.5 mg/kg/day and duration of treatment. The better bioavailability of the new formulation of CsA (Neoral), implies that the former dosage recommendations be reconsidered for distinctly lower figures. Repeat renal biopsy after 1 year of continuous CsA treatment is advisable, as stable serum creatinine levels may be falsely reassuring. CsA dependency is the rule during the first year of treatment. However, in some 25% of cases stable remission may be maintained after slow tapering off following 3-4 years of treatment. Other immunophillin modulators have been tried in the treatment of idiopathic nephrotic syndrome. Despite few preliminary reports indicating some success of tacrolimus the effects of this drug do not seem convincingly superior to CsA in terms of remission rate, toxicity and dependency. Rapamycin has not been tried in the treatment of nephrosis. Anecdotal cases of de novo FSGS induced by rapamycin in transplanted patients might indicate that this drug is in fact contraindicated in the treatment of nephrosis.

Glomerular endothelial cytotoxicity and dysfunction in nephrosis with focal segmental glomerulosclerosis.

Glomerular endothelial cell dysfunction (GED) with defective release of vasodilator has been delineated in nephrosis (NS) in vivo and in vitro studies. In NS with focal segmental glomerulosclerosis (FSGS), an immunocirculatory balance may be impaired due to defective anti-inflammatory cytokine. This study aimed at simultaneous determination of both proinflammatory cytokine (tumor necrosis factor alpha) and an anti-inflammatory cytokine (interleukin-10) in NS with FSGS. An endothelial cell cytotoxicity (ECC) was also examined using nephrotic serum. It was shown that (1) the initial endothelial cell cytotoxicity was significantly different from the control, (2) ratio between tumor necrosis alpha and interleukin-10 was significantly elevated, and (3) intrarenal hemodynamics was changed significantly.

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.

BACKGROUND: Decay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF. METHODS: Function-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats. RESULTS: DAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab')2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were "planted" on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model. CONCLUSION: In cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC.

Transient and sequential expression of chemokine mRNA in glomeruli in puromycin aminonucleoside nephrosis.

Chemokines are a large family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. We previously reported that among six chemokines, the expression of mRNAs for MCP-1, MCP-3, TCA3, and MIP-1alpha, but not for MIP-1beta and RANTES, was markedly elevated in the renal cortex of rats with puromycin aminonucleoside induced nephrosis. In this study we have determined the glomerular expression of the chemokine mRNAs in this model using quantitative competitive reverse-transcriptase polymerase chain reaction. After an injection of puromycin aminonucleoside, the number of monocytes/macrophages and CD4+ and CD8+ cells markedly increased by day 5 and increased thereafter until day 10. The levels of mRNAs for MCP-1, MCP-3, and lymphotactin increased on day 5 and returned to their normal levels by day 7. The level of TCA3 mRNA increased on day 3, and that of MIP-1alpha mRNA increased on day 7, but both returned to their normal levels within 2 days. No increase in the mRNAs of MIP-1beta or RANTES was observed until day 10. These results indicate that the expression pattern of the chemokine mRNAs in glomeruli resembles that in renal cortex, but is more transient and sequential.

Immunosuppressant pharmacodynamics on lymphocytes from healthy subjects and patients with chronic renal failure, nephrosis, and psoriasis: possible implications for individual therapeutic efficacy.

BACKGROUND: In organ transplantation, patients with peripheral blood mononuclear cells (PBMCs) that exhibit resistance to cyclosporine (INN, ciclosporin) or glucocorticoids in vitro are refractory to therapy based on these drugs in vivo. However, detection or distribution of the resistant patients with immunologic disorders remains to be documented. METHODS: Drug sensitivity tests were performed with PBMCs from four subject groups: 69 healthy subjects, 100 patients with chronic renal failure, 38 patients with nephrosis, and 51 patients with psoriasis. The values for the concentration that produces 50% lymphocyte-mitosis inhibition (IC50) of the drugs on PBMC blastogenesis were estimated, and individual variations or group differences in the IC50 values were examined. RESULTS: The median cyclosporine IC50 values of the four subject groups were similar, but large individual deviations in the IC50 values were observed. Individual differences in prednisolone IC50 values were spread from 1 to 3500 ng/ml. When compared with healthy subjects, a significantly large number of the patients with chronic renal failure group exhibited low responses to prednisolone (p < 0.04). In contrast, no significant difference in the methylprednisolone IC50 was observed among the groups. Normal upper thresholds for IC50 values of these drugs were estimated from the mean + 2 standard deviations (SD) of the IC50 values of healthy PBMCs, and the patients with IC50 values above these levels were considered to be resistant. The incidence of resistant patients with nephrosis or psoriasis was similar to that of healthy subjects; however, the incidence of cyclosporine- or prednisolone-resistant subjects with chronic renal failure was significantly higher (p < 0.04). Significant correlations between PBMC sensitivity to cyclosporine in vitro and clinical efficacy of the drug in vivo were observed in renal transplant recipients and in patients with psoriasis. CONCLUSIONS: A large subset of patients with chronic renal failure showed PBMC resistance to cyclosporine and prednisolone. Hyperresistant patients have a high risk of being refractory to immunosuppressive therapy with one of these drugs. Alternative treatment should be considered according to the individual drug-sensitivity data.

The racial prevalence of glomerular lesions in nephrotic adults.

We retrospectively evaluated the prevalence of primary glomerular lesions in adults who had a renal biopsy for nephrotic proteinuria. From July 1975 to May 1994, 340 patients undergoing renal biopsies evaluated at Rush-Presbyterian-St Lukes Medical Center had the primary glomerular lesions of minimal-change disease, focal segmental glomerular sclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephropathy, immunoglobulin A nephropathy, and immunotactoid glomerulopathy. The patients had a mean age of 43 +/- 17 years, 57% were male, and 50% were white, 36% were black, 7% were of other race, and 7% were of unknown race. The distribution of lesions for black patients was minimal-change disease 14%, FSGS 57%, MGN 24%, membranoproliferative glomerulonephritis 2%, immunoglobulin A 2%, and immunotactoid glomerulopathy 1%; for white patients, the distribution of lesions was minimal-change disease 20%, FSGS 23%, MGN 36%, membranoproliferative glomerulonephropathy 6%, immunoglobulin A 8%, and immunotactoid glomerulopathy 6%. The prevalence of FSGS was significantly greater (P < 0.0001) and that for MGN, immunoglobulin A, and immunotactoid glomerulopathy was significantly less (P < 0.05) for black patients compared with white patients. In a logistic regression analysis, race remained the only significant predictor of FSGS (P = 0.0001), with black patients four times as likely to have FSGS as white patients. The distribution of lesions among white patients was similar based on gender, age, and biopsy period. For black patients the distribution was also similar based on gender and age, but over 20 years the incidence of FSGS increased from 39% (1975 to 1984) to 64% (1985 to 1994) (P < 0.01). Thus, significant racial differences in the distribution of primary glomerular lesions exists. This has important prognostic and therapeutic implications for nephrotic adults.

Altered localization of antigen recognized by proteinuria-inducing monoclonal antibody in experimental nephrosis.

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.

Immunohistochemical study of complement S protein (Vitronectin) in normal and diseased human kidneys: relationship to neoantigens of the C5b-9 terminal complex.

The localization of S protein (Vitronectin) antigen was studied by indirect immunofluorescence and immunoelectron microscopy in normal adult human kidneys and in biopsy specimens from patients with a wide range of renal diseases, and compared with that of neoantigens of the C5b-9 terminal complement complex. S protein antigen was diffusely present in arteriolar perimyocytic matrices, the glomerular basement membrane and mesangial matrix, and tubular basement membranes in the cortex of normal and diseased kidneys without superimposable staining for C5b-9 neoantigens. Cell remnants embedded in normal and sclerotic extracellular matrices expressed S protein antigen and also stained for C5b-9 neoantigens. Several lines of evidence suggested that S protein present in connective matrices most likely represents S protein or C5b-9 complexes trapped from the circulation. Glomerular immune deposits and arteriolar hyalin deposits which contained C5b-9 neoantigens also contained S protein antigen in the same location. In a few specimens from patients with membranous nephritis stage I and IgA nephropathy, immune deposits contained neither detectable C5b-9 neoantigens nor S protein. The observed strong co-staining of immune deposits for S-protein and C5b-9 caution against the generalization that C5b-9 within glomerular immune deposits represent membrane-bound cytolytic complement complexes.

Immunopathological findings in idiopathic nephrosis: clinical significance of glomerular "immune deposits".

Idiopathic nephrosis (IN), which includes minimal change (MCD), diffuse mesangial proliferation (DMP) and focal segmental glomerular sclerosis (FSGS), is classically characterized by the absence of significant deposits by immunofluorescence microscopy (IF), except for the focal lesions of segmental sclerosis and/or hyalinosis of FSGS, which fix IgM and C3 antiserums. Since IF is available in most centres, an increasing number of unexpected findings has been reported. In order to evaluate the clinical significance of the glomerular deposits revealed by IF in some instances, we reviewed the renal biopsy findings of 222 consecutive children presenting with IN and in whom IF microscopy was available. By light microscopy, 122 patients showed MCD, 10 DMP, and 90 FSGS with DMP (11 cases) or without (79 cases). By IF, 125 specimens were negative and served as controls; 54 showed mesangial IgM deposits, 24 mesangial IgG deposits (associated with Clq deposits in 16), 15 scattered granules of C3 and 4 predominant deposits of mesangial IgA. We correlated these findings with initial response to steroid therapy and outcome and could find no significant difference between the various categories defined by IF and the control group. Repeat biopsies, performed in 21 cases, showed the persistence of deposits in 11 and their transformation in 10. The particular problem raised by the patients who present with IN and mesangial IgA deposits is discussed. Our results demonstrate that patients presenting with IN and "positive IF", whether showing IgM, IgG and Clq, C3 or IgA, do not represent distinct clinicopathological entities.

Charge selectivity of proteinuria in diabetic glomerulopathy.

Differential macromolecule clearances were used to elucidate the mechanism of proteinuria in patients with diabetic glomerulopathy. Uncharged dextrans of graded size, combined with albumin and IgG separated into narrow fractions of varying charge by preparative electrofocusing, were used to probe the filtration barrier. Analysis of the fractional clearance profile of dextrans in the 30- to 60-A interval revealed a small fraction of filtrate volume (0.0023-0.0097) permeating large nonrestrictive glomerular pores and correlating strongly with the fractional clearances of albumin (r = .88, P less than .001) or IgG (r = .91, P less than .001). The fractional clearance of the most anionic species of albumin [isoelectric point (pI) 4.0-4.5] significantly exceeded that of less anionic species (pI 4.5-5.5) at all levels of proteinuria. A corresponding increase in fractional clearance of anionic (pI 4.5-5.0) over neutral (pI 7.0-7.5) IgG species was observed in patients with subnephrotic-range proteinuria. We conclude that a loss of barrier size selectivity underlies proteinuria in diabetic glomerulopathy. In addition, either facilitated filtration of polyanions or preferential tubular reabsorption of polycations can be invoked to explain the final composition of urinary protein. Similar loss of size selectivity combined with enhanced fractional clearance of anionic IgG in a group of nondiabetic patients with nephrotic syndrome indicates that the foregoing abnormality of renal protein handling is not unique to diabetic glomerulopathy.