A kidney-specific fasting-mimicking diet induces podocyte reprogramming and restores renal function in glomerulopathy.

Cycles of a fasting-mimicking diet (FMD) promote regeneration and reduce damage in the pancreases, blood, guts, and nervous systems of mice, but their effect on kidney disease is unknown. In addition, a FMD has not been tested in rats. Here, we show that cycles of a newly developed low-salt FMD (LS-FMD) restored normal proteinuria and nephron structure and function in rats with puromycin-induced nephrosis compared with that in animals with renal damage that did not receive the dietary intervention. LS-FMD induced modulation of a nephrogenic gene program, resembling renal developmental processes in multiple kidney structures. LS-FMD also activated podocyte-lineage reprogramming pathways and promoted a quiescent state in mature podocytes in the rat kidney damage model. In a pilot clinical study in patients with chronic kidney disease, FMD cycles of 5 days each month for 3 months promoted renoprotection, including reduction of proteinuria and improved endothelial function, compared with that in patients who did not receive the FMD cycles. These results show that FMD cycles, which promote the reprogramming of multiple renal cell types and lead to glomerular damage reversal in rats, should be tested further for the treatment of progressive kidney diseases.

Revised version with tracked changes oral Magnesium reduces levels of pathogenic autoantibodies and skin disease in murine lupus.

BACKGROUND: Systemic Lupus Erythematosus (SLE) has a strong genetic susceptibility, but little is known about the impact of diet on disease severity. The Western diet is typically deficient in magnesium (Mg), and given the immunomodulatory effects of Mg, we hypothesized that the low Mg intake increases disease risk and that increasing Mg intake would reduce severity of murine lupus. Here, we placed 12-week old MRL/lpr female lupus mice on a normal (Mg500) or a high (Mg2800) Mg diet for 9 weeks. Urine and blood were collected during the study for quantification of urinary albumin, BUN, anti-dsDNA antibodies, and immune phenotyping. RESULTS: MRL/lpr lupus mice on high Mg2800 diet had significantly fewer skin lesions and less severe skin histology score, and reduced levels of pathogenic anti-dsDNA antibodies, compared with the Mg500 group (143.8±75.0 vs. 47.4±36.2 × 106U/ml; P < 0.05). The high Mg2800 group had a nearly two-fold increase in the percentage of CD4+FOXP3+ Treg cells compared to controls (19.9±5.4 vs. 11.4±5.5%; P < 0.05). Treg percentages inversely correlated with the concentration of anti-dsDNA. None of the mice developed arthritis during the observation period and there were no significant differences in weight, proteinuria, BUN or kidney histology. CONCLUSION: In conclusion, oral supplementation of Mg has a protective effect in a murine lupus model and may represent an inexpensive and safe adjuvant in the treatment of SLsE.

Reduced decay-accelerating factor expression promotes complement-mediated cystogenesis in murine ADPKD.

Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1-/- C3-/- mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1-/- C3-/- mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1-/- C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.

High-dimensional mass cytometry identified circulating natural killer T-cell subsets associated with protection from cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection is associated with poor kidney transplant outcomes. While innate and adaptive immune cells have been implicated in its prevention, an in-depth characterization of the in vivo kinetics of multiple cell subsets and their role in protecting against CMV infection has not been achieved. Here, we performed high-dimensional immune phenotyping by mass cytometry, and functional assays, on 112 serially collected samples from CMV seropositive kidney transplant recipients. Advanced unsupervised deep learning analysis was used to assess immune cell populations that significantly correlated with prevention against CMV infection and anti-viral immune function. Prior to infection, kidney transplant recipients who developed CMV infection showed significantly lower CMV-specific cell-mediated immune (CMI) frequencies than those that did not. A broad diversity of circulating cell subsets within innate and adaptive immune compartments were associated with CMV infection or protective CMV-specific CMI. While percentages of CMV (tetramer-stained)-specific T cells associated with high CMI responses and clinical protection, circulating CD3+CD8midCD56+ NK-T cells overall strongly associated with low CMI and subsequent infection. However, three NK-T cell subsets sharing the CD11b surface marker associated with CMV protection and correlated with strong anti-viral CMI frequencies in vitro. These data were validated in two external independent cohorts of kidney transplant recipients. Thus, we newly describe the kinetics of a novel NK-T cell subset that may have a protective role in post-transplantation CMV infection. Our findings pave the way to more mechanistic studies aimed at understanding the function of these cells in protection against CMV infection.

C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy.

The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Efficacy of combined rituximab and daratumumab treatment in posttransplant recurrent focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is one of the leading causes of kidney failure and it is characterized by a high rate of recurrence after kidney transplant. Moreover, FSGS recurrence is worsened by an increased risk of graft failure. Common therapies for FSGS recurrence mostly consist of plasma exchange treatments, also for prolonged time, and rituximab, with variable efficacy. We report 5 cases of early FSGS recurrence after kidney transplant, resistant to plasma exchange and rituximab treatment that subsequently resolved after combined therapy with rituximab and daratumumab. All cases were negative for genetic FSGS. The combined treatment induced a complete response in all the cases and was well tolerated. We also performed a comprehensive flow cytometry analysis in 2 subjects that may suggest a mechanistic link between plasma cells and disease activity. In conclusion, given the lack of viable treatments for recurrent FSGS, our reports support the rationale for a pilot trial testing the safety/efficacy profile of combined rituximab and daratumumab in posttransplant FSGS recurrence.

Endogenous erythropoietin has immunoregulatory functions that limit the expression of autoimmune kidney disease in mice.

Background: Administration of recombinant erythropoietin (EPO), a kidney-produced hormone with erythropoietic functions, has been shown to have multiple immunoregulatory effects in mice and humans, but whether physiological levels of EPO regulate immune function in vivo has not been previously evaluated. Methods: We generated mice in which we could downregulate EPO production using a doxycycline (DOX)-inducible, EPO-specific silencing RNA (shEPOrtTAPOS), and we crossed them with B6.MRL-Faslpr/J mice that develop spontaneous lupus. We treated these B6.MRL/lpr shEPOrtTAPOS with DOX and serially measured anti-dsDNA antibodies, analyzed immune subsets by flow cytometry, and evaluated clinical signs of disease activity over 6 months of age in B6.MRL/lpr shEPOrtTAPOS and in congenic shEPOrtTANEG controls. Results: In B6.MRL/lpr mice, Epo downregulation augmented anti-dsDNA autoantibody levels and increased disease severity and percentages of germinal center B cells compared with controls. It also increased intracellular levels of IL-6 and MCP-1 in macrophages. Discussion: Our data in a murine model of lupus document that endogenous EPO reduces T- and B-cell activation and autoantibody production, supporting the conclusion that EPO physiologically acts as a counterregulatory mechanism to control immune homeostasis.

The Gut and Kidney Crosstalk in Immunoglobulin A Nephropathy.

Immunoglobulin A nephropathy(IgAN) is the most common primary glomerulonephritis worldwide. The working model for the pathogenesis of IgAN involves a multistep process starting from the production of galactose-deficient and polymeric immunoglobulin A-1 (gd-IgA1) that enters systemic circulation from gut-associated lymphoid tissue (GALT). Galactose-deficient IgA are targeted by endogenous IgG, leading to the formation of circulating immune complexes that deposit in the mesangium and resulting in glomerular inflammation. Disease onset and relapses are often associated with gut infections, supporting the hypothesis that the gut plays an important pathogenic role. In the presence of microbial pathogens or food antigens, activated dendritic cells in the gut mucosa induce T cell dependent and independent B cell differentiation into IgA secreting plasma cells. In IgAN patients, this promotes the systemic release of mucosal gd-IgA1. Not all bacterial strains have the same capacity to elicit IgA production, and little is known about the antigen specificity of the pathogenic gd-IgA1. However, efficacy of treatments targeting gut inflammation support a pathogenic link between the bowel immune system and IgAN. Herein, we review the evidence supporting the role of gut inflammation in IgAN pathogenesis.

Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO's effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor-knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

Early calcineurin-inhibitor to belatacept conversion in steroid-free kidney transplant recipients.

Background: Belatacept (Bela) was developed to reduce nephrotoxicity and cardiovascular risk that are associated with the chronic use of Calcineurin inhibitors (CNIs) in kidney transplant recipients. The use of Bela with early steroid withdrawal (ESW) and simultaneous CNI avoidance has not been formally evaluated. Methods: At 3 months post-transplant, stable kidney transplant recipients with ESW on Tacrolimus (Tac) + mycophenolate (MPA) were randomized 1:1:1 to: 1) Bela+MPA, 2) Bela+low-dose Tac (trough goal <5 ng/mL), or 3) continue Tac+MPA. All patients underwent surveillance graft biopsies at enrollment and then at 12, and 24 months post-transplant. Twenty-seven recipients were included; 9 underwent conversion to Bela+MPA, 8 to Bela+low-dose Tac and 10 continued Tac+MPA. Serial blood samples were collected for immune phenotyping and gene expression analyses. Results: The Bela+MPA arm was closed early due to high rate of biopsy proven acute rejection (BPAR). The incidence of BPAR was 4/9 in Bela+MPA, 0/8 in Bela+low dose Tac and 2/10 in Tac+MPA, P= 0.087. The Bela+low-dose Tac regimen was associated with +8.8 mL/min/1.73 m2 increase in eGFR compared to -0.38 mL/min/1.73 m2 in Tac+MPA, P= 0.243. One graft loss occurred in the Bela+MPA group. Immunophenotyping of peripheral blood monocyte count (PBMC) showed that CD28+CD4+ and CD28+CD8+ T cells were higher in Bela+MPA patients with acute rejection compared to patients without rejection, although the difference did not reach statistical significance. Conclusions: Our data indicate that, in steroid free regimens, low-dose Tac maintenance is needed to prevent rejection when patients are converted to Bela, at least when the maneuver is done early after transplant.

CyTOF-Enabled Analysis Identifies Class-Switched B Cells as the Main Lymphocyte Subset Associated With Disease Relapse in Children With Idiopathic Nephrotic Syndrome.

B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.

Erythropoietin Reduces Auto- and Alloantibodies by Inhibiting T Follicular Helper Cell Differentiation.

BACKGROUND: Although high-affinity IgG auto- and alloantibodies are important drivers of kidney inflammation that can result in ESKD, therapeutic approaches that effectively reduce such pathogenic antibodies remain elusive. Erythropoietin (EPO) has immunomodulatory functions, but its effects on antibody production are unknown. METHODS: We assessed the effect and underlying mechanisms of EPO/EPO receptor (EPOR) signaling on primary and secondary, T cell-dependent and T-independent antibody formation using in vitro culture systems, murine models of organ transplantation and lupus nephritis, and mice conditionally deficient for the EPOR expressed on T cells or B cells. RESULTS: In wild-type mice, recombinant EPO inhibited primary, T cell-dependent humoral immunity to model antigens and strong, polyclonal stimuli, but did not alter T-independent humoral immune responses. EPO also significantly impaired secondary humoral immunity in a potent allogeneic organ transplant model system. The effects required T cell, but not B cell, expression of the EPOR and resulted in diminished frequencies of germinal center (GC) B cells and T follicular helper cells (TFH). In vitro and in vivo experiments showed that EPO directly prevented TFH differentiation and function via a STAT5-dependent mechanism that reduces CD4+ T cell expression of Bcl6. In lupus models, EPO reduced TFH, GC B cells, and autoantibody production, and abrogated autoimmune glomerulonephritis, demonstrating clinical relevance. In vitro studies verified that EPO prevents differentiation of human TFH cells. CONCLUSIONS: Our findings newly demonstrate that EPO inhibits TFH-dependent antibody formation, an observation with potential implications for treating antibody-mediated diseases, including those of the kidney.

Erythropoietin administration expands regulatory T cells in patients with autoimmune hepatitis.

OBJECTIVE: Autoimmune hepatitis (AIH) is a chronic liver disease associated with impaired regulatory T cell (Treg) number and function. Erythropoietin (EPO) is a kidney-produced erythropoietic hormone that has known immune-modulating effects, including Treg induction. Whether EPO administration increases Treg in patients with AIH is unknown. METHODS: We treated six stable AIH patients with a single 1000 IU dose of EPO and comprehensively characterized changes in Treg overall and in Treg subsets before and at 4 and 12 weeks after treatment using mass cytometry (CyTOF) combined with an unbiased clustering approach (Phenograph) based on 22 Treg-relevant cell-surface markers. RESULTS: EPO was well-tolerated and no patients showed significant changes in hematological parameters, liver enzymes, or IgG levels from baseline to 12 weeks following EPO administration. Total Treg and Treg/CD8+ T cell ratios significantly increased at 4 weeks and returned to baseline levels at 12 weeks after EPO injection. We identified 17 Treg subsets of which CD4+CD25HICD127NEG HLADR+ Treg had the highest increase and the most favorable Treg/CD8+ ratio upon EPO treatment. At 12 weeks after EPO administration, the HLADR+ Treg subset also returned to values comparable to those at baseline. Ex vivo assays documented that Treg were functional and the ones isolated at 12 weeks after EPO injection were significantly more suppressive than the ones isolated at baseline. In Treg-depleted assays, EPO did not show a significant effect on IFN-γ+, IL-2+, and IL-17+ CD4+ T cells. CONCLUSION: In stable AIH patients, EPO increases overall Treg and particularly those expressing the high function marker HLA-DR. These results provide the rationale for future studies testing the hypothesis that EPO or EPO analogues improve outcomes of AIH patients by increasing Treg.