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.

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.

Proteomic Changes Induced by the Immunosuppressant Everolimus in Human Podocytes.

mTOR inhibitors (mTOR-Is) may induce proteinuria in kidney transplant recipients through podocyte damage. However, the mechanism has only been partially defined. Total cell lysates and supernatants of immortalized human podocytes treated with different doses of everolimus (EVE) (10, 100, 200, and 500 nM) for 24 h were subjected to mass spectrometry-based proteomics. Support vector machine and partial least squares discriminant analysis were used for data analysis. The results were validated in urine samples from 28 kidney transplant recipients receiving EVE as part of their immunosuppressive therapy. We identified more than 7000 differentially expressed proteins involved in several pathways, including kinases, cell cycle regulation, epithelial-mesenchymal transition, and protein synthesis, according to gene ontology. Among these, after statistical analysis, 65 showed an expression level significantly and directly correlated with EVE dosage. Polo-Like Kinase 1 (PLK1) content was increased, whereas osteopontin (SPP1) content was reduced in podocytes and supernatants in a dose-dependent manner and significantly correlated with EVE dose (p < 0.0001, FDR < 5%). Similar results were obtained in the urine of kidney transplant patients. This study analyzed the impact of different doses of mTOR-Is on podocytes, helping to understand not only the biological basis of their therapeutic effects but also the possible mechanisms underlying proteinuria.

Analysis of urinary exosomes applications for rare kidney disorders.

Introduction: Exosomes are nanovesicles that play important functions in a variety of physiological and pathological conditions. They are powerful cell-to-cell communication tool thanks to the protein, mRNA, miRNA, and lipid cargoes they carry. They are also emerging as valuable diagnostic and prognostic biomarker sources. Urinary exosomes carry information from all the cells of the urinary tract, downstream of the podocyte. Rare kidney diseases are a subset of an inherited diseases whose genetic diagnosis can be unclear, and presentation can vary due to genetic, epigenetic, and environmental factors. Areas covered: In this review, we focus on a group of rare and often neglected kidney diseases, for which we have sufficient available literature data on urinary exosomes. The analysis of their content can help to comprehend pathological mechanisms and to identify biomarkers for diagnosis, prognosis, and therapeutic targets. Expert opinion: The foreseeable large-scale application of system biology approach to the profiling of exosomal proteins as a source of renal disease biomarkers will be also useful to stratify patients with rare kidney diseases whose penetrance, phenotypic presentation, and age of onset vary sensibly. This can ameliorate the clinical management.

Autophagy Activation in Peripheral Blood Mononuclear Cells of Peritoneal Dialysis Patients.

Introduction: The complete systemic deregulated biological network in patients on peritoneal dialysis (PD) is still only partially defined. High-throughput/omics techniques may offer the possibility to analyze the main biological fingerprints associated with this clinical condition. Methods: We applied an innovative bioinformatic analysis of gene expression microarray data (mainly based on support vector machine (SVM) learning) to compare the transcriptomic profile of peripheral blood mononuclear cells (PBMCs) of healthy subjects (HS), chronic kidney disease (CKD) patients, and patients on PD divided into a microarray group (5 HS, 9 CKD, and 10 PD) and a validation group (10 HS, 15 CKD, and 15 PD). Classical well-standardized biomolecular approaches (western blotting and flow cytometry) were used to validate the transcriptomic results. Results: Bioinformatics revealed a distinctive PBMC transcriptomic profiling for PD versus CKD and HS (n = 419 genes). Transcripts encoding for key elements of the autophagic pathway were significantly upregulated in PD, and the autophagy related 5 (ATG5) reached the top level of discrimination [-Log10 P-value = 11.3, variable importance in projection (VIP) score = 4.8, SVM rank:1]. Protein levels of ATG5 and microtubule associated protein 1 light chain 3 beta (LC3B), an important constituent of the autophagosome, validated microarray results. In addition, the incubation of PBMCs of HS with serum of patients on PD upregulated both proteins. Autophagy in PBMCs from patients on PD was attenuated by N-acetyl-cysteine or Resatorvid treatment. Conclusions: Our data demonstrated, for the first time, that the autophagy pathway is activated in immune-cells of patients on PD, and this may represent a novel therapeutic target.

Immune abnormalities in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and it is characterized by mesangial IgA deposition. Asymptomatic hematuria with various degrees of proteinuria is the most common clinical presentation and up to 20%-40% of patients develop end-stage kidney disease within 20 years after disease onset. The pathogenesis of IgAN involves four sequential processes known as the "four-hit hypothesis" which starts with the production of a galactose-deficient IgA1 (gd-IgA1), followed by the formation of anti-gd-IgA1 IgG or IgA1 autoantibodies and immune complexes that ultimately deposit in the glomerular mesangium, leading to inflammation and injury. Although several key questions about the production of gd-IgA1 and the formation of anti-gd-IgA1 antibodies remain unanswered, a growing body of evidence is shedding light on the innate and adaptive immune mechanisms involved in this complex pathogenic process. Herein, we will focus on these mechanisms that, along with genetic and environmental factors, are thought to play a key role in disease pathogenesis.

Sphingomyelin and Medullary Sponge Kidney Disease: A Biological Link Identified by Omics Approach.

Background: Molecular biology has recently added new insights into the comprehension of the physiopathology of the medullary sponge kidney disease (MSK), a rare kidney malformation featuring nephrocalcinosis and recurrent renal stones. Pathogenesis and metabolic alterations associated to this disorder have been only partially elucidated. Methods: Plasma and urine samples were collected from 15 MSK patients and 15 controls affected by idiopathic calcium nephrolithiasis (ICN). Plasma metabolomic profile of 7 MSK and 8 ICN patients was performed by liquid chromatography combined with electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Subsequently, we reinterrogated proteomic raw data previously obtained from urinary microvesicles of MSK and ICN focusing on proteins associated with sphingomyelin metabolism. Omics results were validated by ELISA in the entire patients' cohort. Results: Thirteen metabolites were able to discriminate MSK from ICN (7 increased and 6 decreased in MSK vs. ICN). Sphingomyelin reached the top level of discrimination between the two study groups (FC: -1.8, p < 0.001). Ectonucleotide pyrophophatase phosphodiesterase 6 (ENPP6) and osteopontin (SPP1) resulted the most significant deregulated urinary proteins in MSK vs. ICN (p < 0.001). ENPP6 resulted up-regulated also in plasma of MSK by ELISA. Conclusion: Our data revealed a specific high-throughput metabolomics signature of MSK and indicated a pivotal biological role of sphingomyelin in this disease.