François Vrtovsnik

Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy.

Transferrin receptor (TfR) has been identified as a candidate IgA1 receptor expressed on human mesangial cells (HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangial IgA1 deposits, and is overexpressed in patients with IgA nephropathy (IgAN). Here, structural requirements of IgA1 for its interaction with mesangial TfR were analyzed. Polymeric but not monomeric IgA1 interacted with TfR on cultured HMC and mediates internalization. IgA1 binding was significantly inhibited (>50%) by soluble forms of both TfR1 and TfR2, confirming that TfR serves as mesangial IgA1 receptor. Hypogalactosylated serum IgA1 from patients with IgAN bound TfR more efficiently than IgA1 from healthy individuals. Serum IgA immune complexes from patients with IgAN containing aberrantly glycosylated IgA1 bound more avidly to TfR than those from normal individuals. This binding was significantly inhibited by soluble TfR, highlighting the role of TfR in mesangial IgA1 deposition. For addressing the potential role of glycosylation sites in IgA1-TfR interaction, a variety of recombinant dimeric IgA1 molecules were used in binding studies on TfR with Daudi cells that express only TfR as IgA receptor. Deletion of either N- or O-linked glycosylation sites abrogated IgA1 binding to TfR, suggesting that sugars are essential for IgA1 binding. However, sialidase and beta-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR-IgA1 interaction as initial steps in IgAN pathogenesis.

The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells.

Dendritic cell (DC) maturation and migration are events critical for the initiation of immune responses. After encountering pathogens, DCs upregulate the expression of costimulatory molecules and subsequently migrate to secondary lymphoid organs. Calcium (Ca2+) entry governs the functions of many hematopoietic cell types, but the role of Ca2+ entry in DC biology remains unclear. Here we report that the Ca2+-activated nonselective cation channel TRPM4 was expressed in and controlled the Ca2+ homeostasis of mouse DCs. The absence of TRPM4, which elicited Ca2+ overload, did not influence DC maturation but did considerably impair chemokine-dependent DC migration. Our results establish TRPM4-regulated Ca2+ homeostasis as crucial for DC mobility but not maturation and emphasize that DC maturation and migration are independently regulated.

Engagement of Transferrin Receptor by Polymeric IgA1: Evidence for a Positive Feedback Loop Involving Increased Receptor Expression and Mesangial Cell Proliferation in IgA Nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis in the world, is characterized by IgA immune complex-mediated mesangial cell proliferation. The transferrin receptor (TfR) was identified previously as an IgA1 receptor, and it was found that, in biopsies of patients with IgAN, TfR is overexpressed and co-localizes with IgA1 mesangial deposits. Here, it is shown that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (three- to four-fold increase) in quiescent human mesangial cells (HMC). IgA-induced but not cytokine-induced HMC proliferation is dependent on TfR engagement as it is inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. It is dependent on the continued presence of IgA1 rather than on soluble factors released during IgA1-mediated activation. In addition, pIgA1-induced IL-6 and TGF-beta production from HMC was specifically inhibited by mAb A24, confirming that pIgA1 triggers a TfR-dependent HMC activation. Finally, upregulation of TfR expression induced by sera from patients with IgAN but not from healthy individuals was dependent on IgA. It is proposed that deposited pIgA1 or IgA1 immune complexes could initiate a process of auto-amplification involving hyperexpression of TfR, allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation and activation, features that are commonly implicated in the chronicity of mesangial injuries observed in IgAN and that could explain the recurrence of IgA1 deposits in the mesangium after renal transplantation.

Inhibitory ITAM Signaling by FcαRI-FcRγ Chain Controls Multiple Activating Responses and Prevents Renal Inflammation

Inhibitory signaling is an emerging function of ITAM-bearing immunoreceptors in the maintenance of homeostasis. Monovalent targeting of the IgA Fc receptor (FcαRI or CD89) by anti-FcαRI Fab triggers potent inhibitory ITAM (ITAMi) signaling through the associated FcRγ chain (FcαRI-FcRγ ITAMi) that prevents IgG phagocytosis and IgE-mediated asthma. It is not known whether FcαRI-FcRγ ITAMi signaling controls receptors that do not function through an ITAM and whether this inhibition requires Src homology protein 1 phosphatase. We show in this study that FcαRI-Fcγ ITAMi signals depend on Src homology protein 1 phosphatase to target multiple non-ITAM-bearing receptors such as chemotactic receptors, cytokine receptors, and TLRs. We found that anti-FcαRI Fab treatment in vivo reduced kidney inflammation in models of immune-mediated glomerulonephritis and nonimmune obstructive nephropathy by a mechanism that involved decreased inflammatory cell infiltration and fibrosis development. This treatment also prevented ex vivo LPS activation of monocytes from patients with lupus nephritis or vasculitis, as well as receptor activation through serum IgA complexes from IgA nephropathy patients. These findings point to a crucial role of FcαRI-FcRγ ITAMi signaling in the control of multiple heterologous or autologous inflammatory responses. They also identify anti-FcαRI Fab as a new potential therapeutic tool for preventing progression of renal inflammatory diseases.

Association of Kidney Function, Vitamin D Deficiency, and Circulating Markers of Mineral and Bone Disorders in CKD

BACKGROUND Vitamin D (25 hydroxyvitamin D [25(OH)D]) deficiency is common in patients with chronic kidney disease (CKD). Neither the relation of this deficiency to the decrease in glomerular filtration rate (GFR) nor the effects on CKD mineral and bone disorders (MBD) are clearly established. STUDY DESIGN Cross-sectional analysis of baseline data from a prospective cohort, the NephroTest Study. SETTING & PARTICIPANTS 1,026 adult patients with all-stage CKD not on dialysis therapy or receiving vitamin D supplementation. PREDICTORS For part 1, measured GFR (mGFR) using (51)Cr-EDTA renal clearance; for part 2, 25(OH)D deficiency at <15 ng/mL. OUTCOMES & MEASUREMENTS For part 1, 25(OH)D deficiency and several circulating MBD markers; for part 2, circulating MBD markers. RESULTS For part 1, the prevalence of 25(OH)D deficiency was associated inversely with mGFR, ranging from 28%-51% for mGFR ≥60-<15 mL/min/1.73 m(2). It was higher in patients of African origin; those with obesity, diabetes, hypertension, macroalbuminuria, and hypoalbuminemia; and during winter. After adjusting for these factors, ORs for 25(OH)D deficiency increased from 1.4 (95% CI, 0.9-2.3) to 1.4 (95% CI, 0.9-2.1), 1.7 (95% CI, 1.1-2.7), and 1.9 (95% CI, 1.1-3.6) as mGFR decreased from 45-59 to 30-44, 15-29, and <15 (reference, ≥60) mL/min/1.73 m(2) (P for trend = 0.02). For part 2, 25(OH)D deficiency was associated with higher age-, sex-, and mGFR-adjusted ORs of ionized calcium level <1.10 mmol/L (2.6; 95% CI, 1.2-5.9), 1,25 dihydroxyvitamin D concentration <16.7 pg/mL (1.8; 95% CI, 1.3-2.4), hyperparathyroidism (1.8; 95% CI, 1.3-2.4), and serum C-terminal cross-linked collagen type I telopeptides concentration >1,000 pg/mL (1.6; 95% CI, 1.0-2.6). It was not associated with hyperphosphatemia (phosphate >1.38 mmol/L). LIMITATIONS Cross-sectional analysis of the data prevents causal inferences. CONCLUSIONS 25(OH)D deficiency is related independently to impaired mGFR. Both mGFR decrease and 25(OH)D deficiency are associated with abnormal levels of circulating MBD biomarkers.

Therapeutic mTOR inhibition in autosomal dominant polycystic kidney disease: What is the appropriate serum level?

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disease, and sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to significantly retard cyst expansion in animal models. The optimal therapeutic dose of sirolimus is not yet defined. Here, we report the history of a previously unknown ADPKD deceased donor whose kidneys were engrafted in two different recipients. One of the two received an immunosuppressive regimen based on sirolimus for 5 years while the other did not. After transplantation, both patients developed severe transplant cystic disease. Donor DNA sequence identified a new hypomorphic mutation in PKD1. The rate of cyst growth was identical in the two patients regardless of the treatment. While sirolimus treatment reduced the activation of mTOR in peripheral blood mononuclear cells, it failed to prevent mTOR activation in kidney tubular cells, this could account for the inefficiency of treatment on cyst growth. Together, our results suggest that the dose of sirolimus required to inhibit mTOR varies according to the tissue.

Both IgA nephropathy and alcoholic cirrhosis feature abnormally glycosylated IgA1 and soluble CD89–IgA and IgG–IgA complexes: common mechanisms for distinct diseases

Abnormalities of IgA arise in alcoholic cirrhosis, including mesangial IgA deposits with possible development of secondary IgA nephropathy (IgAN). Since little is known about circulating immune complexes in cases of secondary IgAN, we analyzed IgA-associated parameters in the serum of 32 patients with compensated or advanced alcoholic cirrhosis. Galactose deficiency and decreased sialylation of IgA1, as well as increased amounts of abnormally glycosylated polymeric IgA1, were detected in the serum of patients with advanced alcoholic cirrhosis. Moreover, aberrant IgA1 formed complexes with IgG and soluble CD89 in serum of patients with advanced alcoholic cirrhosis, similar to those found in primary IgAN. The IgA1 of alcoholic cirrhosis, however, had a modified N-glycosylation, not found in primary IgAN. In patients with alcoholic cirrhosis and IgAN, IgA deposits were associated with CD71 overexpression in mesangial areas, suggesting that CD71 might be involved in deposit formation. Although the IgA1 found in alcoholic cirrhosis bound more extensively to human mesangial cells than control IgA1, they differ from primary IgAN by not inducing mesangial cell proliferation. Thus, abnormally glycosylated IgA1 and soluble CD89-IgA and IgA-IgG complexes, features of primary IgAN, are also present in alcoholic cirrhosis. Hence, common mechanisms appear to be shared by diseases of distinct origins, indicating that common environmental factors may influence the development of IgAN.

The IgA1 immune complex–mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.

Fcα receptor I activation induces leukocyte recruitment and promotes aggravation of glomerulonephritis through the FcRγ adaptor

Myeloid cells bear Fc receptors (FcR) that mediate inflammatory signaling through the ITAM‐containing FcRγ adaptor. They express FcRγ‐associated FcαRI, which modulate either activating or inhibitory signaling depending on the type of ligand interaction. The role of FcαRIγ in disease progression remains unknown, notably in IgA nephropathy (IgAN), one of major causes of end‐stage renal disease, in which large amounts of circulating IgA‐immune complexes (IC) may mediate receptor activation. To analyze the involvement of FcαRI activation in glomerulonephritis (GN), we generated Tg mice expressing a mutated, signaling‐incompetent, human FcαRIR209L that cannot associate with FcRγ. Like FcαRIwt‐Tg mice, they developed mesangial IgA deposits but not macrophage infiltration. FcαRI activation in FcαRIwt, but not in FcαRIR209L, Tg mice resulted in marked inflammation with severe proteinuria and leukocyte infiltration in spontaneous IgAN or anti‐glomerular basement membrane Ab‐induced GN models. Receptor triggering of syngenically transferred FcαRIwt Tg macrophages into non‐Tg animals induced their recruitment into injured kidneys during GN development. FcαRIwt cross‐linking on macrophages activated MAP kinases and production of TNF‐α and MCP‐1. Moreover, IgA‐IC from IgAN patients activated FcαRI and induced TNF‐α production. Thus, FcαRI activation mediates GN progression by initiating a cytokine/chemokine cascade that promotes leukocyte recruitment and kidney damage.

Urinary ammonia and long-term outcomes in chronic kidney disease

Recent studies suggest that alkalinizing treatments improve the course of chronic kidney disease (CKD), even in patients without overt metabolic acidosis. Here, we tested whether a decreased ability in excreting urinary acid rather than overt metabolic acidosis may be deleterious to the course of CKD. We studied the associations between baseline venous total CO2 concentration or urinary ammonia excretion and long-term CKD outcomes in 1065 patients of the NephroTest cohort with CKD stages 1-4. All patients had measured glomerular filtration rate (mGFR) by (51)Cr-EDTA renal clearance. Median mGFR at baseline was 37.6 ml/min per 1.73 m(2). Urinary ammonia excretion decreased with GFR, whereas net endogenous acid production did not. After a median follow-up of 4.3 years, 201 patients reached end-stage renal disease (ESRD) and 114 died before ESRD. Twenty-six percent of the patients had mGFR decline rate greater than 10% per year. Compared with patients in the highest tertile of urinary ammonia excretion, those in the lowest tertile had a significantly increased hazard ratio for ESRD, 1.82 (95% CI, 1.06-3.13), and a higher odds ratio of fast mGFR decline, 1.84 (0.98-3.48), independent of mGFR and other confounders. Patients in the lowest tertile of venous total CO2 had significantly increased risk of fast mGFR decline but not of ESRD. None of these biomarkers was associated with mortality. Thus, these results suggest that the inability to excrete the daily acid load is deleterious to renal outcomes.