Neutral endopeptidase, a major brush border protein of the kidney proximal nephron, is directly targeted to the apical domain when expressed in Madin-Darby canine kidney cells

We have used a retroviral vector containing both the cDNA for rabbit neutral endopeptidase (EC 3.4.24.11; NEP) and the neomycin resistance gene to promote the expression of NEP in a polarized Madin-Darby canine kidney (MDCK) cell line. Cells resistant to G418 (a neomycin synthetic analog) were analyzed with a fluorescence-activated cell sorter to isolate a homogeneous population of cells which stably expressed NEP at their surface. When cells grown in Petri dishes were labeled with an antibody to NEP coupled to colloidal gold and examined under the electron microscope, a strong labeling of microvilli was observed, whereas very few particles were present on the basolateral domain, suggesting that the polarized distribution of this enzyme typical of proximal tubule cells is maintained in this MDCK cell population. To study more accurately the mechanism by which MDCK cells target NEP to the apical surface, cultures were grown to confluence on Costar Transwell chambers and used for pulse-chase experiments with [35S]methionine. Immunoprecipitation of recombinant NEP was then performed by adding an anti-NEP polyclonal antibody to the apical or basolateral surface of intact monolayers and by analyzing immunoprecipitates by gel electrophoresis and fluorography. Our results suggest that NEP is delivered directly to the apical domain and does not transit through the basolateral domain of the plasma membrane. This NEP-expressing MDCK cell line therefore constitutes a new model for investigating the molecular basis of apical membrane targeting in polarized epithelial cells.     

Neprilysin Is Identical to Skin Fibroblast Elastase: ITS ROLE IN SKIN AGING AND UV RESPONSES

Although human skin fibroblast (HSF) elastase has been characterized as a membrane-bound metalloproteinase, little is known about its structure, amino acid sequence, and encoding gene. As there are similarities in the molecular weights and inhibitory profiles of HSF elastase and neprilysin (neutral endopeptidase 24.11 (NEP)), in this study we tested the hypothesis that they are identical using immunoprecipitation and transfection methods. An immunoprecipitation study demonstrated that HSF elastase activity co-immunoprecipitated with anti-NEP in lysates of cultured HSF. Transfection of an NEP cDNA expression vector into COS-1 cells elicited the expression of HSF elastase and NEP activities in the transfected cells. These findings strongly suggest that HSF elastase is identical to NEP, which functions mainly in neuron-associated cells to degrade neuropeptides. Analysis of the expression pattern of NEP revealed that its expression was remarkably up-regulated at the gene, protein, and enzymatic activity levels during the replicative senescence of cultured HSF. Further, the activity of NEP was markedly enhanced in a pattern similar to elastase activity during the intrinsic aging of mouse skin, in UVA-exposed HSF as well as in HSF treated with conditioned medium from UVB-exposed human keratinocytes. Analysis of the cytokine profile for the stimulation of NEP and HSF elastase activities in HSF demonstrated that among the 11 cytokines tested, IL-1α, IL-1β, IL-6, IL-8, and GM-CSF had the potential to significantly stimulate both activities similarly, again supporting the identity of HSF elastase and NEP.     

The Calcineurin Inhibitor Tacrolimus Reduces Proteinuria in Membranous Nephropathy Accompanied by a Decrease in Angiopoietin-Like-4

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.     

Urinary Xist is a potential biomarker for membranous nephropathy

Membranous nephropathy (MN), a type of glomerular nephritis, is the most common cause of nephrotic syndrome in human adults. Changes in gene expression as a result of epigenetic dysregulation through long noncoding RNAs (lncRNAs) are increasingly being recognized as important factors in disease. Using an experimental MN mouse model, we identify the first dysregulated lncRNAs, Xist and NEAT1, whose levels are significantly upregulated in both tubular epithelial and glomerular cells. MN is also often characterized by glomerular podocyte injury. Treatment of a mouse podocyte cell line with lipopolysaccharides to induce injury resulted in the stable elevation of Xist, but not NEAT1 levels. In mice, the observed changes in Xist levels are specific: Xist can be effectively detected in urine, with a strong correlation to disease severity, but not serum in MN samples. We find that regulation of Xist may be controlled by post-translational modifications. H3K27me3 levels are significantly downregulated in mouse MN kidney, where chromatin immunoprecipitation experiments also showed decreased H3K27me3 at Xist promoter regions. Finally, we show that our findings in mice can be extended to human clinical samples. Urinary Xist is significantly elevated in urine samples from patients with different types of glomerular nephritis, including MN, compared to normal counterparts. Together, our results suggest that a reduction of H3K27me3 at Xist promoter regions leads to elevated levels of urinary Xist, which may be used as a biomarker to detect MN.     

Expression of nestin in the podocytes of normal and diseased human kidneys

The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.     

Nuclear pore complexes deposited in the glomerular basement membrane are associated with autoantibodies in a case of membranous nephritis

Massive deposits of organelles, morphologically identical with nuclear pore complexes (NP), were identified in the glomerular basement membrane of an individual with membranous nephritis. Analysis by immunofluorescence demonstrated that the patient's serum contained autoantibodies that became bound within intact cells to cellular components, including centrosomes or centrioles and filaments of the cytoskeleton. Furthermore, these antibodies became bound to numerous small structures at the nuclear periphery. The above evidence suggests that among the autoantibodies were some specifically directed toward the NP, and that the glomerular deposits were NP, possibly in association with IgG.     

Glomerular Endothelial Cell Injury and Focal Segmental Glomerulosclerosis Lesion in Idiopathic Membranous Nephropathy

Background

Focal segmental glomerulosclerosis (FSGS) lesions have often been discussed as a negative predictor in idopathic membranous nephropathy (MN). The mechanism of the development of FSGS lesion in MN is still uncertain.

Methods

From 250 cases of MN, 26 cases contained FSGS lesion. We compared the clinicopathological characteristics between MN cases with FSGS lesion [MN-FSGS(+)] and MN without FSGS lesion [MN-FSGS(−)], matched for gender, age, stage of MN.

Results

The glomerular filtration rate (eGFR) was significantly lower in MN-FSGS(+) cases compared to MN-FSGS(−), although nephrotic syndrome, hematuria, and systolic blood pressure levels were not significantly different between the two groups. Pathologically, glomeruli in MN-FSGS(+) cases showed narrowing and loss of glomerular capillaries with separating from GBM or disappearance of CD34+ endothelial cells, and accumulation of extracellular matrix (ECM) in capillary walls, indicating the development of glomerular capillary injury. These findings of endothelial injury were seen even in MN-FSGS(−) cases, but they were more prominent in MN-FSGS(+) than MN-FSGS(−) by computer assessed morphometric analysis. In MN-FSGS(+) cases, 44 out of 534 glomeruli (8.2%) contained FSGS lesions (n = 31, NOS lesion; n = 13, perihilar lesion). Significant thickness of GBM with ECM accumulation was evident in MN-FSGS(+) cases. Podocyte injury with effacement of foot processes was also noted, but the expression of VEGF on podocytes was not different between the two groups, which suggests that the significant thickness of capillary walls may influence the function of VEGF from podocyte resulting in the glomerular capillary injury that contribute to the development of FSGS lesion in MN.

Conclusion

Glomerular capillary injury was seen in all MN cases. Furthermore, the prominent injuries of glomerular capillaries may be associated with the deterioration of eGFR and the formation of FSGS lesions in MN.     

Mouse and Human Neuronal Pentraxin 1 (NPTX1): Conservation, Genomic Structure, and Chromosomal Localization

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1–q25.2 and chromosome 11e2–e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.     

Ectoenzymes of peptidic metabolism in renal glomerular and vascular cells]

The ectoenzymes acting in the metabolism of peptides play an essential role in renal cell-cell communication. We have studied four of these ectoenzymes, aminopeptidases N and A (APN, APA), dipeptidylpeptidase IV (DPP IV) and neutral endopeptidase (NEP) in cultured human glomerular mesangial and epithelial cells and cultured rabbit renal cortical vascular smooth muscle cells. APN is present at the surface of both mesangial and epithelial cells with identical characteristics. Its expression (enzyme activity and immunoreactive protein) is induced by phorbol-esters and other protein kinase C-stimulating agents. APA is present only in glomerular epithelial cells. Its expression is induced by glucocorticoids and cyclic AMP-stimulating agents. DPP IV is also present only in glomerular epithelial cells. Its expression (enzyme activity, immunoreactive protein and mRNA) is induced by interferon gamma. NEP is present in glomerular epithelial cells and vascular smooth muscle cells. The expression of the latter enzyme is inhibited in the presence of serum via the combined effect of Ca2+i and PKC-stimulating agents. In contrast, glucocorticoids and cyclic GMP induce its expression. NEP plays a major role in the catabolism by these cells of atrial natriuretic factor. All these data emphasize the multiplicity of the mechanisms controlling ectopeptidase expression in cultured glomerular and renal vascular cells.     

Modulation of antibody-mediated glomerular injury in vivo by bacterial lipopolysaccharide, tumor necrosis factor, and IL-1

We have investigated the effects of LPS, human rTNF (hrTNF) and human rIL-1 beta (hrIL-1 beta) pretreatment on the intensity of antibody-mediated injury in vivo by using a passive model of anti-glomerular basement membrane (GBM) antibody-mediated nephritis in rats. The experiments show that all three pretreatments exacerbate injury in this model whether judged by albuminuria or the prevalence of glomerular capillary thrombi. The effect on albuminuria was dose dependent with all three treatments. The lowest effective dose of LPS was 0.025 microgram while those for hrTNF and hrIL-1 beta were 0.4 microgram and 0.5 microgram, respectively. All three pretreatments also increased the prevalence of glomerular capillary thrombi which were rare in rats injected with anti-GBM antibodies without pretreatment. LPS pretreatment appeared to be more effective in causing glomerular capillary thrombi than hrTNF or hrIL-1 beta and this was reflected in the correlations between albuminuria and the proportion of glomeruli with capillary thrombi. This relation was linear for all three pretreatments but the slope was appreciably greater for rats pretreated with LPS (0.37) when compared with results from rats given either hrTNF (0.22) or hrIL-1 beta (0.29). Pretreatment of nephritic rats with both cytokines increased the slope to 0.42 demonstrating a synergistic effect. The synergism of hrTNF with hrIL-1 beta was also demonstrated by the effective doses needed to induce albuminuria which was evident in rats treated with 0.05 microgram of IL-1 beta and 0.4 microgram of TNF. Neither the cytokines nor LPS caused clinical, morphologic, or biochemical evidence of renal toxicity when given alone in the dose used here but they did cause a transient increase in the number of neutrophils marginated in glomerular capillaries. Pretreatment of rats with LPS or cytokines increased the glomerular neutrophil influx after anti-GBM antibodies by roughly sixfold. Our experiments show that TNF and IL-1 can increase the severity of glomerular injury in nephritis; they may be important in modulating glomerular injury clinically.     

Human neprilysin-2 (NEP2) and NEP display distinct subcellular localisations and substrate preferences

Neprilysin-2 (NEP2) is a novel metallopeptidase homologous to neprilysin (NEP), an enzyme involved in regulation of neuropeptide signalling. NEP2 exists as two alternatively spliced isoforms, NEP2 and NEP2(Delta). In this study, we cloned and expressed both human isoforms. Human NEP2 exists as a membrane-bound and soluble enzyme, whereas human NEP2(Delta) exists as two membrane-bound glycoforms, localised to the ER and plasma membrane. Surprisingly, NEP2 substrate specificity and inhibitor binding was distinct from that of human NEP, suggesting that NEP and NEP2 play distinct physiological roles in humans, and human NEP2 differs markedly from its rodent homologues.     

Experimental model of membranous nephropathy in mice: sequence of histological and biochemical events

An experimental model of membranous nephropathy (MN) has not been established fully in mice. We characterized the time course of MN in a murine MN model induced by cationic bovine serum albumin (cBSA). Preimmunized mice received cBSA intravenously for six weeks to induce MN and were then sacrificed at different times. Metabolic profiles, renal histopathology, lymphocyte subsets, serum anti-cBSA immunoglobulins (Igs), antibody subclasses and circulating immune complexes (CIC) were evaluated to study the severity and mechanisms of disease initiation and progression. Clinical symptoms of overt proteinuria, hypoalbuminaemia and hypercholesterolaemia were observed from week 4, and typical histological findings of diffuse thickening of the glomerular basement membrane and subepithelial deposition were identified after week 6. Granular fluorescent staining for IgG and complement C3 were observed as early as week 4. Total splenocyte number increased, but the percentages of CD4+ and CD8+ cells did not change as the disease progressed. The predominant isotype of anti-cBSA Igs was IgG1, suggesting a T-helper 2 cell-prone immune response in the development of MN. The strong positive immunofluorescent staining of the immune complex concomitant with higher concentrations of Igs in serum but no significant change in CIC levels before week 4 suggest the involvement of in situ deposition of immune complex in the process of MN. This murine model resembles the clinical and pathological features of human MN and may provide a tool for investigating MN; this model may also have potential applications in gene-knockout or transgenic mouse technologies.     

Adaptation of Avian Influenza A Virus Polymerase in Mammals To Overcome the Host Species Barrier

Avian influenza A viruses, such as the highly pathogenic avian H5N1 viruses, sporadically enter the human population but often do not transmit between individuals. In rare cases, however, they establish a new lineage in humans. In addition to well-characterized barriers to cell entry, one major hurdle which avian viruses must overcome is their poor polymerase activity in human cells. There is compelling evidence that these viruses overcome this obstacle by acquiring adaptive mutations in the polymerase subunits PB1, PB2, and PA and the nucleoprotein (NP) as well as in the novel polymerase cofactor nuclear export protein (NEP). Recent findings suggest that synthesis of the viral genome may represent the major defect of avian polymerases in human cells. While the precise mechanisms remain to be unveiled, it appears that a broad spectrum of polymerase adaptive mutations can act collectively to overcome this defect. Thus, identification and monitoring of emerging adaptive mutations that further increase polymerase activity in human cells are critical to estimate the pandemic potential of avian viruses.     

Impact of mitochondrial DNA on radiation sensitivity of transformed human fibroblast cells: clonogenic survival, micronucleus formation and cellular ATP level

The purpose of this study was to evaluate the impact of mitochondrial DNA (mtDNA) on the radiation sensitivity of transformed human fibroblast cells. The p+ and p0 human fibroblast cell lines were used, which carry wild-type mtDNA and no mtDNA, respectively. Clonogenic radiosensitivity was evaluated by colony formation assay and micronucleus (MN) formation assay. The ATP assay was then used to address the discrepancy between the results of the former two assays. Despite the lack of a significant difference in survival in the colony formation assay, p+ and p0 cells exhibited high and low radiosensitivities, respectively, in the MN formation assay (P < 0.003). This difference in MN formation correlated with high and low levels of cellular ATP content in p+ and p0 cells (P = 0.004). The addition of antimycin A suppressed differences in both MN formation and cellular ATP content. In the transformed human fibroblast cells we used, mtDNA played an important role in radiation-induced MN formation that was correlated with the levels of cellular ATP content. These results may imply the presence of an MN expression pathway that is dependent on the intrinsic ATP level and that may be compensated and lead to an equivalent level of clonogenic survival.     

Zinc Inhibits Apoptosis and Maintains NEP Downregulation, Induced by Ropivacaine, in HaCaT Cells

Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15????) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2?mM) (p?<?0.05) as well as apoptosis induced by the drug (0.5 and 1?mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0?mM) downregulated NEP expression in the presence of zinc (15????) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.     

Resveratrol Ameliorates Renal Damage,Increases Expression of Heme Oxygenase-1, and Has Anti-Complement,Anti-Oxidative,and Anti-Apoptotic Effects in a Murine Model of Membranous Nephropathy

BackgroundIdiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and a common cause of nephrotic syndrome in adults. There are limited available treatments for MN. We assessed the efficacy of resveratrol (RSV) therapy for treatment of MN in a murine model of this disease.MethodsMurine MN was experimentally induced by daily subcutaneous administration of cationic bovine serum albumin, with phosphate-buffered saline used in control mice. MN mice were untreated or given RSV. Disease severity and pathogenesis was assessed by determination of metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, apoptosis, and production of heme oxygenase-1 (HO1).ResultsMN mice given RSV had significantly reduced proteinuria and a marked amelioration of glomerular lesions. RSV also significantly attenuated immunofluorescent staining of C3, although there were no changes of serum immunoglobulin levels or immunocomplex deposition in the kidneys. RSV treatment of MN mice also reduced the production of reactive oxygen species (ROS), reduced cell apoptosis, and upregulated heme oxygenase 1 (HO1). Inhibition of HO1 with tin protoporphyrin IX partially reversed the renoprotective effects of RSV. The HO1 induced by RSV maybe via Nrf2 signaling.ConclusionOur results show that RSV increased the expression of HO1 and ameliorated the effects of membranous nephropathy in a mouse model due to its anti-complement, anti-oxidative, and anti-apoptotic effects. RSV appears to have potential as a treatment for MN.     

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.