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In addition to long-term regulation of blood pressure (BP), in the kidney resides the initial trigger for hypertension development due to an altered capacity to excrete sodium and water. Betaine is one of the major organic osmolytes, and its betaine/gamma-aminobutyric acid transporter (BGT-1) expression in the renal medulla relates to interstitial tonicity and urinary osmolality and volume. This study investigated altered water and sodium balance as well as changes in antidiuretic hormone (ADH) activity in female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats from their 3-5 weeks of age (prehypertensive phase) to SHR's 28-30 weeks of age (established hypertension-organ damage). Young prehypertensive SHRs showed a reduced daily urine output, an elevated urine osmolarity, and higher immunostaining of tubule BGT-1, alpha-1-Na-K ATPase in the outer medulla vs. age-matched WKY. ADH circulating levels were not different between young prehypertensive SHR and WKY, but the urine aquaporin2 (AQP2)/creatinine ratio and labeling of AQP2 in the collecting duct were increased. At 28-30 weeks, hypertensive SHR with moderate renal failure did not show any difference in urinary osmolarity, urine AQP2/creatinine ratio, tubule BGT-1, and alpha-1-Na-K ATPase as compared with WKY. These results suggest an increased sensitivity to ADH in prehypertensive female SHR. On this basis, a second series of experiments were set to study the role of ADH V1 and V2 receptors in the development of hypertension, and a group of female prehypertensive SHRs were treated from the 25th to 49th day of age with either V1 (OPC21268) or V2 (OPC 41061) receptor antagonists to evaluate the BP time course. OPC 41061-treated SHRs had a delayed development of hypertension for 5 weeks without effect in OPC 21268-treated SHRs. In prehypertensive female SHR, an increased renal ADH sensitivity is crucial for the development of hypertension by favoring a positive water balance. Early treatment with selective V2 antagonism delays future hypertension development in young SHRs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Verzicco, Tedeschi, Graiani, Bongrani, Carnevali, Dancelli, Zappa, Mattei, Bovino, Cavazzini, Rocco, Calvi, Palladini, Volpi, Cannone, Coghi, Borghetti and Cabassi.)

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum., (Copyright © 2014 by the American Society of Nephrology.)

Background: Idiopathic retroperitoneal fibrosis (IRF) is a rare fibro-inflammatory disorder characterized by a periaortic tissue which often encases the ureters causing acute renal failure. IRF histology shows fibrosis and a chronic inflammatory infiltrate with frequent tissue eosinophilia. We assessed a panel of molecules promoting eosinophilia and fibrosis in IRF patients and performed an immunogenetic study., Methods: Serum levels of eotaxin/CCL11, regulated and normal T-cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-5, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) were measured using a multiplex assay in 24 newly diagnosed, untreated IRF patients and 14 healthy controls. Retroperitoneal biopsies (available in 8/24 patients) were histologically evaluated to assess eosinophil infiltration, whereas mast cells (MCs) were identified by immunohistochemical analysis for human tryptase. Immunohistochemistry for eotaxin/CCL11 and its receptor CCR3 was also performed. Six single nucleotide polymorphisms (SNPs) within the CCL11 gene (rs6505403, rs1860184, rs4795896, rs17735961, rs16969415 and rs17809012) were investigated in 142 IRF patients and 214 healthy controls., Results: Serum levels of eotaxin/CCL11 were higher in IRF patients than in controls (P = 0.009). Eotaxin/CCL11 drives tissue infiltration of eosinophils and MCs, which can promote fibrosis. Eosinophilic infiltration was prominent (>5 cells/hpf) in five (62.5%) cases, and abundant tryptase-positive MCs were found in all cases; notably, MCs were in a degranulating state. Immunohistochemistry showed that CCL11 was highly produced by infiltrating mononuclear cells and that its receptor CCR3 was expressed by infiltrating eosinophils, MCs, lymphocytes and fibroblasts. None of the tested CCL11 SNPs showed disease association, but the TTCCAT haplotype was significantly associated with IRF (P = 0.0005)., Conclusions: These findings suggest that the eotaxin/CCL11-CCR3 axis is active in IRF and may contribute to its pathogenesis; the TTCCAT haplotype within the CCL11 gene is significantly associated with IRF.

The type and the extent of tissue damage inform the prognosis of chronic kidney disease (CKD), but kidney biopsy is not a routine test. Urinary tests that correlate with specific histological findings might serve as surrogates for the kidney biopsy. We used immunoblots and ARCHITECT-NGAL assays to define the immunoreactivity of urinary neutrophil gelatinase-associated lipocalin (NGAL) in CKD, and we used mass spectroscopy to identify associated proteins. We analyzed kidney biopsies to determine whether specific pathological characteristics associated with the monomeric NGAL species. Advanced CKD urine contained the NGAL monomer as well as novel complexes of NGAL. When these species were separated, we found a significant correlation between the NGAL monomer and glomerular filtration rate (r=-0.53, P<0.001), interstitial fibrosis (mild vs. severe disease; mean 54 vs. 167 μg uNGAL/g Cr, P<0.01), and tubular atrophy (mild vs. severe disease; mean 54 vs. 164 μg uNGAL/g Cr, P<0.01). Monospecific assays of the NGAL monomer demonstrated a correlation with histology that typifies progressive, severe CKD.

Background and Objectives: The discovery of different podocyte autoantibodies in membranous nephropathy (MN) raises questions about their pathogenetic and clinical meaning. This study sought to define antibody isotypes and correlations; to compare levels in MN, other glomerulonephritides, and controls; and to determine their association with clinical outcomes., Design, Setting, Participants, & Measurements: Serum IgG(1), IgG(3), and IgG(4) against aldose reductase (AR), SOD2, and α-enolase (αENO) were measured at diagnosis in 186 consecutive MN patients, in 96 proteinuric controls (36 with FSGS, and 60 with IgA nephropathy), and in 92 healthy people recruited in four Italian nephrology units. Anti-phospholipase A2 receptor (PLA2r) and anti-neutral endopeptidase (NEP) IgG(4) were titrated in the same specimens. Association with 1-year follow-up clinical parameters was studied in 120 patients., Results: IgG(4) was the most common isotype for all antibodies; IgG(1) and IgG(3) were nearly negligible. IgG(4) levels were positive in a significant proportion of MN patients (AR, 34%; SOD2, 28%; αENO, 43%). Antibody titers were higher in MN than in healthy and pathologic controls (P<0.005). Anti-NEP IgG(4) did not differ from normal controls (P=0.12). Anti-PLA2r IgG(4) was detected in 60% of patients and correlated with anti-AR, anti-SOD2, and anti-αENO IgG(4) (P<0.001). In MN patients negative for the whole antibody panel (20%), 1-year proteinuria was lower compared with patients with at least one antibody positivity (P<0.05)., Conclusions: Our data suggest that IgG(4) is the prevalent isotype for antibodies against cytoplasmic antigens of podocytes (AR, SOD2, αENO). Their levels were higher than in other proteinuric glomerulonephritides and in normal controls and were correlated with anti-PLA2r. Only baseline negativity for all known antibodies predicted lower 1-year proteinuria.

The identification of glomerular auto-antigens in idiopathic human membranous glomerulonephritis (MGN) is a crucial step towards the definition of the mechanisms of the disease. Recent 'in vivo' studies demonstrated a heterogeneous composition of glomerular immune-deposits in MGN biopsies only a part of which have been characterized. We studied with a proteomical approach IgGs eluted from laser capture microdissected glomeruli of 8 MGN patients and showed the existence of other three immune proteins in MGN glomeruli (α-enolase, elongation factor 2 and Glycyl Aminoacyl-tRNA Synthetase). One of these, i.e. α-enolase, fulfilled all criteria for being considered an auto-antigen. Specific IgG₁ and IgG₄ reacting with podocyte α-enolase were, in fact, eluted from microdissected glomeruli and Confocal- and Immuno Electron-Microscopy showed co-localization of α-enolase with IgG₄ and C5b-9 in immune-deposits. Serum levels of anti a-enolase IgG4 were determined in 131 MGN patients and were found elevated in 25% of cases. Overall, our data demonstrate that glomerular α-enolase is a target antigen of autoimmunity in human MGN. Circulating anti α-enolase auto-antibodies can be detected in sera of a significant quota of MGN patients. Like other auto-antigens, α-enolase may be implicated in the pathogenesis of human MGN., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Renal auto-immune diseases represent a major source of morbidity in humans. For many years the knowledge on mechanisms of auto-immunity involving the kidney has been uniquely based on animal models. However, these findings often could not be readily translated to humans owing to notably difference in antigen expression by human podocytes. One example is Heymann nephritis (HN), the experimental model of human membranous glomerulonephritis (MGN), which is obtained in rats by injecting antibodies against megalin, a protein that is not present in human glomeruli. Human studies could not be done in the past since sequencing required too much material exceeding what obtainable from tissue biopsies in vivo. Research is now on the way to identify auto-antigens and isolate specific auto-antibodies in humans. New technology developments based on tissue microdissection and proteomical analysis have facilitated the recent discoveries, allowing direct analysis of human tissue in vivo. Major advances on the pathogenesis of MGN, the prototype for the formation and glomerular deposition of auto-antibodies, are now in progress. Two independent groups have, in fact, demonstrated the existence of specific IgG(4) against phospholipase A2 receptor, aldose reductase and Mn-superoxide dismutase in glomerular eluates and in plasma of a prominent part of patients with MGN, suggesting a major role of these proteins as auto-antigens in human MGN. This review will focalize these aspects outlining the contribution of proteomics in most recent developments., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic periaortitis (CP) is a rare disease hallmarked by the presence of a periaortic retroperitoneal fibro-inflammatory tissue which can often cause obstructive uropathy. CP is isolated in most cases but it may also be associated with other sclerosing inflammatory and immune-mediated diseases. We here present the case of a patient who was initially diagnosed as having CP and subsequently developed membranous nephropathy and chronic sclerosing sialoadenitis of the right parotid gland. As these conditions were all characterized by either pronounced infiltration of IgG4-positive plasma cells or marked IgG4 tissue deposition, we hypothesize that they are part of the same disease spectrum, and discuss the immune-mediated pathogenetic mechanisms potentially shared by these conditions. In particular, we consider the role of Th2-mediated immune reactions and of immunogenetic factors such as HLA genotype as common determinants of these disorders.

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Nephrosis and a rapid decline in kidney function characterize HIV-associated nephropathy (HIVAN). Histologically, HIVAN is a collapsing focal segmental glomerulosclerosis with prominent tubular damage. We explored the expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of tubular injury, to determine whether this protein has the potential to aid in the noninvasive diagnosis of HIVAN. We found that expression of urinary NGAL was much higher in patients with biopsy-proven HIVAN than in HIV-positive and HIV-negative patients with other forms of chronic kidney disease. In the HIV-transgenic mouse model of HIVAN, NGAL mRNA was abundant in dilated, microcystic segments of the nephron. In contrast, urinary NGAL did not correlate with proteinuria in human or in mouse models. These data show that marked upregulation of NGAL accompanies HIVAN and support further study of uNGAL levels in large cohorts to aid in the noninvasive diagnosis of HIVAN and screen for HIVAN-related tubular damage.

Background: Type IV collagen is a major structural component of the normal kidney glomerulus. However, its role in chronic acquired glomerulopathies has been only partially elucidated., Methods: Urinary levels of col(IV)alpha1, col(IV)alpha3 and col(IV)alpha5 collagen chains were analyzed in 107 patients with chronic acquired glomerulopathies. In a subgroup of 33 patients, tissue mRNA levels, protein expression and urinary excretion were evaluated for all col(IV)alpha chains, from col(IV)alpha1 to col(IV)alpha5. The renal specimens were examined to get a semiquantitative score of the acute and chronic activity of the histological lesions. Urines obtained from 13 healthy subjects and 10 normal renal tissue samples were used as controls., Results: Urinary levels of col(IV)alpha1, col(IV)alpha3, col(IV)alpha5 chains were significantly higher in patients than in controls [p < 0.01 for all], while only col(IV)alpha1 and col(IV)alpha3 urinary excretion correlated with the degree of chronic histological damage [col(IV)alpha1 R = 0.44, p < 0.001; col(IV)alpha3: R = 0.47, p < 0.001]. Compared with controls, patients showed a renal expression of mRNA for col(IV)alpha5 chain significantly higher [p = 0.001], while having a significantly lower protein expression of col(IV)alpha3, col(IV)alpha4 and col(IV)alpha5 chains [p < 0.01 for all]., Conclusion: Patients with chronic acquired glomerulopathies show important alterations in the col(IV)alpha chain network mimicking some molecular features of the X-linked Alport's syndrome. Further studies are needed to show whether urinary levels of the col(IV)alpha chains may be used as markers for monitoring renal injury., (Copyright 2007 S. Karger AG, Basel.)

Background/aims: To verify whether human IgG induces proinflammatory activation of human proximal tubular epithelial cells (PTEC) independent of the metabolic overload of protein reabsorption., Methods: Cultured PTEC were incubated with normal IgG, IgG from systemic lupus erythematosus (SLE) patients, albumin or transferrin. IL-6 secretion and extracellular regulated kinase (ERK) activation (dual-phosphorylated ERK) were measured by ELISA and by Western blotting of PTEC extracts, respectively; renal biopsy specimens from patients with IgG and non-IgG proteinuria were analyzed by immunohistochemistry and in situ hybridization to detect ERK-P and IL-6., Results: Normal and SLE IgG, but not albumin or transferrin, induced an early significant increase in IL-6 secretion by PTECs. Also ERK activation was found after 1-hour incubation of PTEC with IgG, but not with control medium and albumin-treated PTEC. Activated ERK and IL-6 were found to colocalize in tubular cells in the kidney specimens of patients with IgG proteinuria only., Conclusion: IgG-dependent early activation of ERK and increased IL-6 secretion in PTEC suggest that IgG filtered during nonselective proteinuria may play a specific role in tubulointerstitial disease. Such a role could be particularly relevant in diseases associated with abnormal IgG pool compositions, such as SLE. Preliminary results on human renal biopsy specimens suggest that our in vitro observations may also be relevant in vivo.

The Authors discuss the etiologic, pathogenetic and immunopathologic aspects of Heymann nephritis, in order to compare the numerous acquisitions concerning this nephropathy with the scanty knowledge of human membranous nephropathy, of which it represents the experimental counterpart. This rat disease can be obtained by inoculation of tubular brush border preparations (active form) or of the relevant antibodies (passive form); after an initial hypothesis of glomerular deposition of circulating immune complexes, studies on its pathogenetic mechanisms, instead demonstrated that in situ immunoaggregates, caused by an interaction between circulating antibodies and fixed glomerular antigens, are formed. Recent investigations have led to the identification of a major nephritogenic antigen (gp330), which is a tubular brush border glycoprotein expressed by coated pits located at the glomerular epithelial cell surface. Studies on antigen-antibody interactions at this level have demonstrated that there is a quick redistribution and accumulation of the so-formed immune complexes, and when polyclonal antibodies were utilized, growth of subepithelial electron dense deposits was observed. Although other tubulo-glomerular antigens, which can also be expressed by endothelial cells, play an uncertain role, they seem to favour transmembrane passing of anti-gp330 antibodies. Immune complex formation gives rise to the onset of proteinuria through complement system activation, without leukocyte involvement: in particular a MAC and C9 fraction lytic effect was demonstrated on cultured epithelial cells. In conclusion, studies on Heymann nephritis contribute to our understanding of the etiopathogenetic mechanisms regarding human membranous nephropathy, and emphasize a possible role played by tubular antigens and in situ formed immune complexes.