In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells ...seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.
Nephrotic syndrome (NS) is a clinical entity characterized by proteinuria, hypoalbuminemia, and peripheral edema. NS affects about 2-7 per 100,000 children aged below 18 years old yearly and is ...classified, based on the response to drugs, into steroid sensitive (SSNS), steroid dependent, (SDNS), multidrug dependent (MDNS), and multidrug resistant (MRNS). Forms of NS that are more difficult to treat are associated with a worse outcome with respect to renal function. In particular, MRNS commonly progresses to end stage renal failure requiring renal transplantation, with recurrence of the original disease in half of the cases. Histological presentations of NS may vary from minimal glomerular lesions (MCD) to focal segmental glomerulosclerosis (FSGS) and, of relevance, the histological patterns do not correlate with the response to treatments. Moreover, around half of MRNS cases are secondary to causative pathogenic variants in genes involved in maintaining the glomerular structure. The pathogenesis of NS is still poorly understood and therapeutic approaches are mostly based on clinical experience. Understanding of pathogenetic mechanisms of NS is one of the 'unmet needs' in nephrology and represents a significant challenge for the scientific community. The scope of the present review includes exploring relevant findings, identifying unmet needs, and reviewing therapeutic developments that characterize NS in the last decades. The main aim is to provide a basis for new perspectives and mechanistic studies in NS.
Nephrotic proteinuria is the hallmark of several glomerulonephritis determined by different pathogenetic mechanisms, including autoimmune, degenerative and inflammatory. Some conditions such as ...Minimal Change Nephropathy (MCN) and Focal Segmental Glomerulosclerosis (FSGS) are of uncertain pathogenesis. Chimeric anti-CD20 monoclonal antibodies have been used with success in a part of proteinuric conditions while some are resistant. New human and humanized monoclonal anti-CD 20 antibodies offer some advantages based on stronger effects on CD20 cell subtypes and have been already administered in hematology and oncology areas as substitutes of chimeric molecules. Here, we revised the literature on the use of human and humanized anti-CD 20 monoclonal antibodies in different proteinuric conditions, resulting effective in those conditions resistant to rituximab. Literature on the use of human anti-CD 20 monoclonal antibodies in different proteinuric diseases is mainly limited to ofatumumab, with several protocols and doses. Studies already performed with ofatumumab given in standard doses of 1,500 mg 1.73m
suggest no superiority compared to rituximab in children and young adults with steroid dependent nephrotic syndrome. Ofatumumab given in very high doses (300 mg/1.73m
followed by five infusion 2,000 mg/1.73 m
) seems more effective in patients who are not responsive to common therapies. The question of dose remains unresolved and the literature is not concordant on positive effects of high dose ofatumumab in patients with FSGS prior and after renal transplantation. Obinutuzumab may offer some advantages. In the unique study performed in patients with multidrug dependent nephrotic syndrome reporting positive effects, obinutuzumab was associated with the anti-CD38 monoclonal antibody daratumumab proposing the unexplored frontier of combined therapies. Obinutuzumab represent an evolution also in the treatment of autoimmune glomerulonephritis, such as membranous nephrotahy and lupus nephritis. Results of randomized trials, now in progress, are awaited to add new possibilities in those cases that are resistant to other drugs. The aim of the present review is to open a discussion among nephrologists, with the hope to achieve shared approaches in terms of type of antibodies and doses in the different proteinuric renal conditions.
Kidney disease is a global health and healthcare burden. Glomerulonephritis (Gn), both primary and secondary, is generally characterized by an inflammatory glomerular injury and may lead to end-stage ...renal disease. Kidney biopsy is fundamental to the diagnosis; however, kidney biopsy presents some concerns that may partly hamper the clinical process. Therefore, more accurate diagnostic tools are needed. Extracellular vesicles (EVs) are membranous vesicles released by cells and found in bodily fluids, including urine. EVs mediate intercellular signaling both in health and disease. EVs can have both harmful and cytoprotective effects in kidney diseases, especially Gn. Previous findings reported that the specific cargo of urinary EV contains an aerobic metabolic ability that may either restore the recipient cell metabolism or cause oxidative stress production. Here, we provide an overview of the most recent proteomic findings on the role of EVs in several aspects of glomerulopathies, with a focus on this metabolic and redox potential. Future studies may elucidate how the ability of EVs to interfere with aerobic metabolism and redox status can shed light on aspects of Gn etiology which have remained elusive so far.
The human albumin gene, the most abundant serum protein, is located in the long arm of chromosome 4, near the centromere, position 4q11-3. It is divided by 14 intervening introns into 15 exons, the ...last of which is untranslated. To date, 74 nucleotide substitutions (mainly missense) have been reported, determining the circulating variants of albumin or pre-albumin. In a heterozygous state, this condition is known as alloalbuminaemia or bisalbuminaemia (OMIM # 103600). The genetic variants are not associated with disease, neither in the heterozygous nor in the homozygous form. Only the variants resulting in familial dysalbuminaemic hyperthyroxinaemia and hypertriiodothyroninaemia are of clinical relevance because affected individuals are at risk of inappropriate treatment or may have adverse drug effects. In 28 other cases, the pathogenic variants (mainly affecting splicing, nonsense, and deletions), mostly in the homozygous form, cause a premature stop in the synthesis of the protein and lead to the condition known as congenital analbuminaemia. In this review, we will summarize the current knowledge of genetic and molecular aspects, functional consequences and potential therapeutic uses of the variants. We will also discuss the molecular defects resulting in congenital analbuminaemia, as well as the biochemical and clinical features of this rare condition.
Type I interferons (IFNs) are pleiotropic cytokines with potent antiviral properties that also promote protective T cell and humoral immunity. Paradoxically, type I IFNs, including the widely ...expressed IFNβ, also have immunosuppressive properties, including promoting persistent viral infections and treating T-cell-driven, remitting-relapsing multiple sclerosis. Although associative evidence suggests that IFNβ mediates these immunosuppressive effects by impacting regulatory T (Treg) cells, mechanistic links remain elusive. Here, we found that IFNβ enhanced graft survival in a Treg-cell-dependent murine transplant model. Genetic conditional deletion models revealed that the extended allograft survival was Treg cell-mediated and required IFNβ signaling on T cells. Using an in silico computational model and analysis of human immune cells, we found that IFNβ directly promoted Treg cell induction via STAT1- and P300-dependent Foxp3 acetylation. These findings identify a mechanistic connection between the immunosuppressive effects of IFNβ and Treg cells, with therapeutic implications for transplantation, autoimmunity, and malignancy.
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•IFNβ synergizes with CTLA-4 Ig to prolong allograft survival by promoting Treg cells•IFNβ directly acts on T cells to enhance Treg cell induction•IFNβ activates STAT1 signaling to promote P300 expression and increase Foxp3 acetylation
The mechanism by which IFNβ, a pro-inflammatory cytokine, mediates its paradoxical immunosuppressive effects in autoimmunity and chronic viral infections remains elusive. Fueyo-González et al. use a transplant model to show that IFNβ synergizes with CTLA-4 Ig to prolong allograft survival. Mechanistically, IFNβ directly promotes Treg cell induction via activation of STAT1 signaling and P300-dependent Foxp3 acetylation.
Individuals with kidney failure are at increased risk of cardiovascular events, as well as infections and malignancies, but the associated immunological abnormalities are unclear. We hypothesized ...that the uremic milieu triggers a chronic inflammatory state that, while accelerating atherosclerosis, promotes T cell exhaustion, impairing effective clearance of pathogens and tumor cells. Clinical and demographic data were collected from 78 patients with chronic kidney disease (CKD) (
= 42) or end-stage kidney disease (ESKD) (
= 36) and from 18 healthy controls (HC). Serum cytokines were analyzed by Luminex. Immunophenotype of T cells was performed by flow cytometry on peripheral blood mononuclear cells. ESKD patients had significantly higher serum levels of IFN-γ, TNF-α, sCD40L, GM-CSF, IL-4, IL-8, MCP-1, and MIP-1β than CKD and HC. After mitogen stimulation, both CD4
and CD8
T cells in ESKD group demonstrated a pro-inflammatory phenotype with increased IFN-γ and TNF-α, whereas both CKD and ESKD patients had higher IL-2 levels. CKD and ESKD were associated with increased frequency of exhausted CD4
T cells (CD4
KLRG1
PD1
CD57
) and CD8
T cells (CD8
KLRG1
PD1
CD57
), as well as anergic CD4
T cells (CD4
KLRG1
PD1
CD57
) and CD8
T cells (CD8
KLRG1
PD1
CD57
). Although total percentage of follicular helper T cell (T
) was similar amongst groups, ESKD had reduced frequency of T
(CCR6
CXCR3
CXCR5
PD1
CD4
CD8
), but increased T
(CCR6
CXCR3
CXCR5
PD1
CD4
CD8
), and plasmablasts (CD3
CD56
CD19
CD27
CD38
CD138
). In conclusion, kidney failure is associated with pro-inflammatory markers, exhausted T cell phenotype, and upregulated T
, especially in ESKD. These immunological changes may account, at least in part, for the increased cardiovascular risk in these patients and their susceptibility to infections and malignancies.
Abstract
Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array ...covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.
Preclinical tests in animal models are key steps in early drug development. Consequently, the International Society of Nephrology held a consensus meeting that connected experts in the global kidney ...community in order to provide guidance on optimal management of translational animal studies for the development of new drugs to treat kidney disease, entitled “TRANSFORM; TRAnslational Nephrology Science FOR new Medications.” The meeting covered various themes, including the following: (i) selection of disease model; (ii) pharmacokinetics; (iii) interventions in late preclinical models; (iv) choice of animal; (v) statistical power; (vi) organoids and organ-on-a-chip models; and (vii) reporting of results. This guidance is the first to be provided on the optimal conduct of translational animal studies for the development of new drugs to treat kidney disease. These recommendations are designed to accelerate development of new drugs for efficacious treatment of kidney diseases, and to improve the prognosis and quality of life of patients with a variety of kidney diseases.
Glomerulonephritis are renal disorders resulting from different pathogenic mechanisms (i.e., autoimmunity, complement, inflammatory activation, etc.). Clarifying details of the pathogenic cascade is ...basic to limit the progression from starting inflammation to degenerative stages. The balance between tissue injury, activation of protective systems and renal tissue repair determines the final outcome. Induction of an oxidative stress is part of glomerular inflammation and activation of protective antioxidant systems has a crucial role in reducing tissue effects. The generation of highly reactive oxygen species can be evaluated in vivo by tracing the inner-layer content of phosphatidyl ethanolamine and phosphatidyl serine in cell membranes. Albumin is the major antioxidant in serum and the level of oxidized albumin is another indirect sign of oxidative stress. Studies performed in Gn, specifically in FSGS, showed a high degree of oxidation in most contexts. High levels of circulating anti-SOD2 antibodies, limiting the detoxyfing activity of SOD2, have been detected in autoimmune Gn(lupus nephritis and membranous nephropathy) in association with persistence of proteinuria and worsening of renal function. In renal transplant, high levels of circulating anti-Glutathione S-transferase antibodies have been correlated with chronic antibody rejection and progressive loss of renal function. Annexins, mainly ANXA1 and ANXA2, play a general anti-inflammatory effect by inhibiting neutrophil functions. Cytosolic ANXA1 is decreased in apoptotic neutrophils of patients with glomerular polyangitis in association with delayed apoptosis that is considered the mechanism for polyangitis. High circulating levels of anti-ANXA1 and anti-ANXA2 antibodies characterize lupus nephritis implying a reduced anti-inflammatory effect. High circulating levels of antibodies targeting Macrophages (anti-FMNL1) have been detected in Gn in association with proteinuria. They potentially modify the intra-glomerular presence of protective macrophages (M2a, M2c) thus acting on the composition of renal infiltrate and on tissue repair.
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