CD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease.

In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI:http://dx.doi.org/10.7554/eLife.01149.001.

Anti-glomerular basement membrane (GBM) nephritis is characterized by circulating anti-GBM antibodies and crescentic glomerulonephritis (GN) with deposition of IgG along the GBM. In a limited number of cases, glomerular immune complexes have been identified in anti-GBM nephritis. A 38-year-old female presented azotemia, hematuria, and proteinuria without any pulmonary symptoms. A renal biopsy showed crescentic GN with linear IgG deposition along the GBM and mesangial IgA deposition. The patient was diagnosed as concurrent anti-GBM nephritis and IgA nephropathy. Therapies with pulse methylprednisolone and cyclophosphamide administration were effective. Concurrent cases of both anti-GBM nephritis and IgA nephropathy are rare among cases of anti-GBM diseases with deposition of immune complexes. This rare case of concurrent anti-GBM nephritis and IgA nephropathy with literature review is noteworthy.

The thickening of the glomerular basement membrane in rats after Vacor ingestion was examined by electron microscopy. This study was performed to elucidate which biochemical components changed in the glomerular basement membrane after Vacor-induced diabetic glomerulopathy.

Anti-glomerular basement membrane (anti-GBM) disease is a rare but severe autoantibody-mediated immune disorder. The typical clinical presentation includes rapidly progressive glomerulonephritis and often concurrent pulmonary hemorrhage. The present study is aimed to investigate the therapeutic effects of rituximab either used alone or with other immunosuppressants.

The first study that estimated single-nephron GFR (SNGFR) in patients with membranous nephropathy (MN). Associations of SNGFR with MN staging by electron microscopy and clinicopathologic findings were cross-sectionally investigated. This study illustrates a role for disease-specific GBM structural lesions as determinants of SNGFR in patients with MN.

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor-β (TGF-β) cytokine family and an inflammation-associated protein. Here, we investigated the role of GDF15 in murine anti-glomerular basement membrane (GBM) glomerulonephritis. Glomerulonephritis induction in mice induced systemic expression of GDF15. Moreover, we demonstrate the protective effects for GDF15, as GDF15-deficient mice exhibited increased proteinuria with an aggravated crescent formation and mesangial expansion in anti-GBM nephritis. Herein, GDF15 was required for the regulation of T-cell chemotactic chemokines in the kidney. In addition, we found the upregulation of the CXCR3 receptor in activated T-cells in GDF15-deficient mice. These data indicate that CXCL10/CXCR3-dependent-signaling promotes the infiltration of T cells into the organ during acute inflammation controlled by GDF15. Together, these results reveal a novel mechanism limiting the migration of lymphocytes to the site of inflammation during glomerulonephritis.

Infection has long been suspected as a trigger of autoimmune diseases, and molecular mimicry mechanism was hypothesized in this study. Microbe originated peptides were searched from the Uniprot database based on a previous defined critical amino acid motif within α3129-150, isoleucine137, tryptophan140, glycine142, phenylalanine 143 and phenylalanine 145. 23826 microbial peptides were identified using our searching strategy, among which seven were related with human infections. Circulating IgG and IgM antibodies against the seven microbial peptides were detected using ELISA in 76 patients with anti-GBM disease. Four peptides were recognized by both IgG and IgM antibodies, and one peptide was recognized by IgG antibodies only. Peptides from Bacteroides, Saccharomyces cerevisiae, and Bifidobacterium thermophilum possessed the highest recognition frequency with the prevalence of 73.7%, 61.8% and 67.1% for IgG, 56.6%, 44.7% and 67.1% for IgM in anti-GBM patients. Patients with antibodies against these microbial peptides showed more severe kidney injury, including higher serum creatinine and higher percentage of crescent formation. In conclusion, antibodies against microbial peptides were identified in the circulation of anti-GBM patients, implying its etiological role in eliciting autoimmune response against α3(IV)NC1 through molecular mimicry.

In patients presenting with anti-glomerular basement membrane (GBM) disease with advanced isolated kidney involvement, the benefit of intensive therapy remains controversial due to adverse events, particularly infection. We aim to describe the burden of severe infections (SI) (requiring hospitalization or intravenous antibiotics) and identify predictive factors of SI in a large cohort of patients with anti-GBM disease. Among the 201 patients (median [IQR] age, 53 [30-71] years) included, 74 had pulmonary involvement and 127 isolated glomerulonephritis. A total of 161 SI occurred in 116 patients during the first year after diagnosis. These infections occurred during the early stage of care (median [IQR] time, 13 [8-19] days after diagnosis) with mainly pulmonary (45%), catheter-associated bacteremia (22%) and urinary tract (21%) infections. In multivariable analysis, positive ANCA (HR [95\% CI] 1.62 [1.07--2.44]; p = 0.02) and age at diagnosis (HR [95% CI] 1.10 [1.00-1.21]; p = 0.047) remained independently associated with SI. Age-adjusted severe infection during the first three months was associated with an increased three-year mortality rate (HR [95% CI] 3.13 [1.24-7.88]; p = 0.01). Thus, SI is a common early complication in anti-GBM disease, particularly in the elderly and those with positive anti-neutrophil cytoplasmic antibodies (ANCA). No significant association was observed between immunosuppressive strategy and occurrence of SI.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin β2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

We report the overall and renal outcome in a French nationwide multicenter cohort of 119 patients with anti-glomerular basement membrane (anti-GBM) disease. Sixty-four patients (54%) had an exclusive renal involvement, 7 (6%) an isolated alveolar hemorrhage and 48 (40%) a combined renal and pulmonary involvement. Initial renal replacement therapy (RRT) was required in 78% of patients; 82% received plasmapheresis, 82% cyclophosphamide, and 9% rituximab. ANCA positive (28%) patients were older (70 vs. 47 years, p < 0.0001), less frequently smokers (26 vs. 54%, p = 0.03), and had less pulmonary involvement than ANCA- patients. The 5 years overall survival was 92%. Risk factors of death (n = 11, 9.2%) were age at onset [HR 4.10 per decade (1.89-8.88) p = 0.003], hypertension [HR 19.9 (2.52-157 0.2) p = 0.005], dyslipidemia [HR 11.1 (2.72-45) p = 0.0008], and need for mechanical ventilation [HR 5.20 (1.02-26.4) p = 0.047]. The use of plasmapheresis was associated with better survival [HR 0.29 (0.08-0.98) p = 0.046]. At 3 months, 55 (46%) patients had end-stage renal disease (ESRD) vs. 37 (31%) ESRD-free and 27 (23%) unevaluable with follow-up < 3 months. ESRD patients were older, more frequently female and had a higher serum creatinine level at presentation than those without ESRD. ESRD-free survival was evaluated in patients alive without ESRD at 3 months (n = 37) using a landmark approach. In conclusion, this large French nationwide study identifies prognosis factors of renal and overall survival in anti-GBM patients.

Neuropilin1 (Nrp1) is a co-receptor best known to regulate the development of endothelial cells and is a target of anticancer therapies. However, its role in other vascular cells including pericytes is emergent. The kidney is an organ with high pericyte density and cancer patients develop severe proteinuria following administration of NRP1B-neutralizing antibody combined with bevacizumab. Therefore, we investigated whether Nrp1 regulates glomerular capillary integrity after completion of renal development using two mouse models; tamoxifen-inducible NG2Cre to delete Nrp1 specifically in pericytes and administration of Nrp1-neutralizing antibodies. Specific Nrp1 deletion in pericytes did not affect pericyte number but mutant mice developed hematuria with glomerular basement membrane defects. Despite foot process effacement, albuminuria was absent and expression of podocyte proteins remained unchanged upon Nrp1 deletion. Additionally, these mice displayed dilation of the afferent arteriole and glomerular capillaries leading to glomerular hyperfiltration. Nidogen-1 mRNA was downregulated and collagen4α3 mRNA was upregulated with no significant effect on the expression of other basement membrane genes in the mutant mice. These features were phenocopied by treating wild-type mice with Nrp1-neutralizing antibodies. Thus, our results reveal a postdevelopmental role of Nrp1 in renal pericytes as an important regulator of glomerular basement membrane integrity. Furthermore, our study offers novel mechanistic insights into renal side effects of Nrp1 targeting cancer therapies.

Podocytes are lost by detachment from the GBM as viable cells; details are largely unknown. We studied this process in the rat after growth stimulation with FGF-2. Endothelial and mesangial cells responded by hyperplasia, podocytes underwent hypertrophy, but, in the long run, developed various changes that could either be interpreted showing progressing stages in detachment from the GBM or stages leading to a tighter attachment by foot process effacement (FPE). This occurred in microdomains within the same podocyte; thus, features of detachment and of reinforced attachment may simultaneously be found in the same podocyte. (1) Initially, hypertrophied podocytes underwent cell body attenuation and formed large pseudocysts, i.e., expansions of the subpodocyte space. (2) Podocytes entered the process of FPE starting with the retraction of foot processes (FPs) and the replacement of the slit diaphragm by occluding junctions, thereby sealing the filtration slits. Successful completion of this process led to broad attachments of podocyte cell bodies to the GBM. (3) Failure of sealing the slits led to gaps of varying width between retracting FPs facilitating the outflow of the filtrate from the GBM. (4) Since those gaps are frequently overarched by broadened primary processes, the drainage of the filtrate into the Bowman's space may be hindered leading to the formation of small pseudocysts associated with bare areas of GBM. (5) The merging of pseudocysts created a system of communicating chambers through which the filtrate has to pass to reach Bowman's space. Multiple flow resistances in series likely generated an expansile force on podocytes contributing to detachment. (6) Such a situation appears to proceed to complete disconnection generally of a group of podocytes owing to the junctional connections between them. (7) Since such groups of detaching podocytes generally make contact to parietal cells, they start the formation of tuft adhesions to Bowman's capsule.

While pirfenidone has been established as an effective anti-fibrosis remedy, whether or not its antifibrotic effect contributes to a reduction of proteinuria remains unclear. We investigated the renoprotective properties of pirfenidone in an anti-glomerular basement membrane (GBM) glomerulonephritis model both prophylactically and therapeutically to determine its profile against proteinuria. In the prophylactic regimen, pirfenidone was treated immediately after anti-serum injection. We observed a significant reduction in the progression of proteinuria (P<0.05) and decline in renal function (P<0.01) and also noted histological improvement in renal injury. These effects appeared to be due to the maintained expression of nephrin and podocin on podocytes as well as the reduced expression of profibrotic factors like transforming growth factor-β (TGF-β). The expression of nephrin mRNA was strongly negatively correlated with the amount of urinary protein excretion (R=-0.84, P<0.001), implicating podocyte damage in the outcome of proteinuria (R(2)=0.70). These results suggest that preservation of podocytes with the pirfenidone treatment may have resulted in the decrease of proteinuria. In contrast, when the therapeutic regimen was initiated 2 weeks after nephritis induction, pirfenidone had little effect on the progression of proteinuria, although the decline of renal function and fibrosis were suppressed. Taken together, present findings suggested that pirfenidone prevented the progression of proteinuria only when administered prophylactically but was still able to ameliorate the decline of renal function independent of proteinuria. In conclusion, pirfenidone as a prophylactic regimen reduces proteinuria in anti-GBM nephritis via preservation of podocytes with markedly reduced efficacy when administered as a therapeutic regimen.

A new enzyme immunoassay has been developed for the demonstration of antiglomerular basement membrane antibodies. Magnetically responsive polyacrylamide-agarose beads (Magnogel) activated with glutaraldehyde were used to bind sonicated insoluble rat glomerular basement membranes. Both the collagenous and the non-collagenous moieties were demonstrated to be fixed on the beads. Sera from brown Norway rats with anti-glomerular basement membrane antibodies induced by HgCl2 injections were incubated with the beads. After washing, the fixed rat IgG were revealed using alkaline phosphatase labelled Fab fragments from anti-rat IgG sheep, IgGs. Comparison with a radioimmunoassay showed that results were reliable. This enzyme immunoassay has several advantages which may render this assay of considerable clinical usefulness.

Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer-trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions.

Matrix metalloproteinase (MMP)9/gelatinase B is increased in various nephropathies. To investigate its role, we used a genetic approach. Adult MMP9-deficient (MMP9(-/)-) mice showed normal renal histology and function at 3 mo. We investigated the susceptibility of 3-mo-old mice to the accelerated model of anti-glomerular basement membrane nephritis, in which fibrin is an important mediator of glomerular injury and renal impairment. Unexpectedly, nephritis was more severe in MMP9(-/)- than in control mice, as attested by levels of serum creatinine and albuminuria, and the extent of crescents and fibrin deposits. Circulating or deposited immunoglobulin G, interleukin (IL)-1beta, or IL-10 were the same in MMP9(-/-) and MMP9(+/+) mice. However, we found that fibrin is a critical substrate for MMP9, and in its absence fibrin accumulated in the glomeruli. These data indicate that MMP9 is required for a novel protective effect on the development of fibrin-induced glomerular lesions.

The combination of anti-glomerular basement membrane (GBM) disease and immunoglobulin A nephropathy (IgAN) has been well documented in sporadic cases, but lacks overall assessment in large collections. Herein, we investigated the clinical and immunological characteristics and outcome of this entity.

Here we report estimates of glomerular basement membrane (GBM) thickness in the Brazilian population performed using direct (DM) and orthogonal interception methods (OIM), and comment on potential sources of variation among estimates made by different laboratories.