Cutting Edge: Neutrophil Complement Receptor Signaling Is Required for BAFF-Dependent Humoral Responses in Mice.

T cell-independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer's patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF.

The repertoire of testicular extracellular vesicle cargoes and their involvement in inter-compartmental communication associated with spermatogenesis.

BACKGROUND: Spermatogenesis is regulated by a complex network of intercellular communication processes. Extracellular vesicles (EVs) are one of the important mediators in intercellular communication. Previous reports have demonstrated the involvement of EVs from the epididymis and prostate in sperm maturation and function. However, the presence of EVs in the testis and their potential involvement in spermatogenesis has not been explored. Here, we have established a testis dissociation protocol that allows the isolation and characterization of testicular EVs. RESULTS: We show that testicular EVs are specifically and efficiently taken up by somatic cells and germ cells, including the spermatozoa in the interstitial space and the seminiferous tubule compartments. We profiled the proteome of testicular EVs and probed the cell types that release them, revealing the potential contributions from the Leydig cells and testicular macrophages. Moreover, we sequenced the small RNA cargoes of testicular EVs and identified sets of small non-coding RNAs that were overlooked in the testis transcriptome. Selected miRNA candidates in testicular EVs were found in sperm RNA payload and demonstrated specific resistance towards ribonuclease A independent of the vesicle membrane. Small molecule inhibition of EV secretion perturbed spermatogenesis via inter-compartmental communication. CONCLUSIONS: Together, our study provides a valuable resource on the repertoire of cargoes carried by testicular EVs and uncovers a physiological function of testicular EVs in inter-compartmental communication associated to spermatogenesis.

The single-cell landscape of kidney immune cells reveals transcriptional heterogeneity in early diabetic kidney disease.

The pathogenesis of diabetic kidney disease (DKD) involves multifactorial processes that converge to initiate and advance the disease. Although DKD is not typically classified as an inflammatory glomerular disease, mounting evidence supports the involvement of kidney inflammation as a key contributor in DKD pathogenesis, particularly through macrophages. However, detailed identification and corresponding phenotypic changes of macrophages in DKD remain poorly understood. To capture the gene expression changes in specific macrophage cell subsets in early DKD, we performed single-cell transcriptomic analysis of CD45-enriched kidney immune cells from type 1 diabetic OVE26 mice at two time points during the disease development. We also undertook a focused analysis of mononuclear phagocytes (macrophages and dendritic cells). Our results show increased resident and infiltrating macrophage subsets in the kidneys of mice with diabetes over time, with heightened expression of pro-inflammatory or anti-inflammatory genes in a subset-specific manner. Further analysis of macrophage polarization states in each subset in the kidneys showed changes consistent with the continuum of activation and differentiation states, with gene expression tending to shift toward undifferentiated phenotypes but with increased M1-like inflammatory phenotypes over time. By deconvolution analysis of RNAseq samples and by immunostaining of biopsies from patients with DKD, we further confirmed a differential expression of select genes in specific macrophage subsets essentially recapitulating the studies in mice. Thus, our study provides a comprehensive analysis of macrophage transcriptomic profiles in early DKD that underscores the dynamic macrophage phenotypes in disease progression.

The Sperm Small RNA Transcriptome: Implications beyond Reproductive Disorder.

Apart from the paternal half of the genetic material, the male gamete carries assorted epigenetic marks for optimal fertilization and the developmental trajectory for the early embryo. Recent works showed dynamic changes in small noncoding RNA (sncRNA) in spermatozoa as they transit through the testicular environment to the epididymal segments. Studies demonstrated the changes to be mediated by epididymosomes during the transit through the adluminal duct in the epididymis, and the changes in sperm sncRNA content stemmed from environmental insults significantly altering the early embryo development and predisposing the offspring to metabolic disorders. Here, we review the current knowledge on the establishment of the sperm sncRNA transcriptome and their role in male-factor infertility, evidence of altered offspring health in response to the paternal life experiences through sperm sncRNA species and, finally, their implications in assisted reproductive technology in terms of epigenetic inheritance.

The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial ß1-adrenergic receptor expression and signaling.

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial ß1-adrenergic receptors (ß1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the ß1AR. ß1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of ß1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the ß1AR and failed to deubiquitinate the ß1AR. USP20-KO mice showed normal baseline systolic function but impaired ß1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on ß1AR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial ß1AR expression in USP20-KO was drastically reduced, whereas ß2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in ß1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates ß1AR signaling in vitro and in vivo Additionally, ß1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize ß1AR expression and signaling during pathological insults to the myocardium.

Acute kidney injury and maladaptive tubular repair leading to renal fibrosis.

PURPOSE OF REVIEW: Despite improvements in acute kidney injury (AKI) detection, therapeutic options to halt the progression of AKI to chronic kidney disease (CKD) remain limited. In this review, we focus on recent discoveries related to the pathophysiology of the AKI to CKD continuum, particularly involving the renal tubular epithelial cells, and also discuss related ongoing clinical trials. While our focus is on injured renal tubular epithelial cells as initiators of the cascade of events resulting in paracrine effects on other cells of the kidney, the summation of maladaptive responses from various kidney cell types ultimately leads to fibrosis and dysfunction characteristic of CKD. RECENT FINDINGS: Recent findings that we will focus on include, but are not limited to, characterizations of: the association between cell cycle arrest and cellular senescence in renal tubular epithelial cells and its contribution to renal fibrosis, chronic inflammation with persistent cytokine production and lymphocyte infiltration among unrepaired renal tubules, mitochondrial dysfunction and a unique role of cytosolic mitochondria DNA in fibrogenesis, prolyl hydroxylase domain proteins as potential therapeutic targets, and novel mechanisms involving the Hippo/yes-associated protein/transcriptional coactivator with PDZ-binding pathway. SUMMARY: Potential therapeutic options to address CKD progression will be informed by a better understanding of fibrogenic pathways. Recent advances suggest additional drug targets in the various pathways leading to fibrosis.

Autosomal Dominant Tubulointerstitial Kidney Disease Due to MUC1 Mutation.

Mucin 1 kidney disease, previously referred to as medullary cystic kidney disease type 1, is a rare hereditary kidney disease. It is one of several diseases now termed autosomal dominant tubulointerstitial kidney disease, as proposed by a KDIGO (Kidney Disease: Improving Global Outcomes) consensus report in 2014. Autosomal dominant tubulointerstitial kidney diseases share common clinical findings, such as autosomal dominant inheritance, bland urinary sediment, absent to mild proteinuria, and progressive loss of kidney function. Although the pathophysiology of mucin 1 kidney disease is still under investigation, genetic testing has been developed to detect the most well-known mutation, a single cytosine insertion into a string of 7 cytosines in the variable-number tandem repeat (VNTR) region of the MUC-1 gene. With this diagnostic tool, nephrologists can offer genetic counseling to affected families and monitor closely for progression of disease. We report a Hispanic patient with a strong family history of chronic kidney disease who tested positive for the MUC1 mutation.

Circulating Exosomes Induced by Cardiac Pressure Overload Contain Functional Angiotensin II Type 1 Receptors.

BACKGROUND: Whether biomechanical force on the heart can induce exosome secretion to modulate cardiovascular function is not known. We investigated the secretion and activity of exosomes containing a key receptor in cardiovascular function, the angiotensin II type I receptor (AT1R). METHODS AND RESULTS: Exosomes containing AT1Rs were isolated from the media overlying AT1R-overexpressing cells exposed to osmotic stretch and from sera of mice undergoing cardiac pressure overload. The presence of AT1Rs in exosomes was confirmed by both electron microscopy and radioligand receptor binding assays and shown to require ß-arrestin2, a multifunctional adaptor protein essential for receptor trafficking. We show that functional AT1Rs are transferred via exosomes in an in vitro model of cellular stretch. Using mice with global and cardiomyocyte conditional deletion of ß-arrestin2, we show that under conditions of in vivo pressure overload the cellular source of the exocytosis of exosomes containing AT1R is the cardiomyocyte. Exogenously administered AT1R-enriched exosomes target cardiomyocytes, skeletal myocytes, and mesenteric resistance vessels and are sufficient to confer blood pressure responsiveness to angiotensin II infusion in AT1R knockout mice. CONCLUSIONS: AT1R-enriched exosomes are released from the heart under conditions of in vivo cellular stress to likely modulate vascular responses to neurohormonal stimulation. In the context of the whole organism, the concept of G protein-coupled receptor trafficking should consider circulating exosomes as part of the reservoir of functional AT1Rs.

Quantitative detection of eryptosis in human erythrocytes using tunable resistive pulse sensing and annexin-V-beads.

Toxicological assessments of human red blood cells (RBCs) are important in human health because RBCs are the most abundant cell type in our body. Erythrotoxicology testing guidelines using hemolysis have been established as a standard (e.g. by the ASTM International). However, many xenobiotics promote eryptosis (apoptosis in human RBCs) without causing hemolysis. Based on the major features of eryptosis, i.e. cell shrinkage and translocation of phosphatidylserine (PS) to the outer lipid bilayer of the plasma membrane, we report here a novel approach utilizing the quantitative tunable resistive pulse sensing (TRPS) technology, a widely adopted technique for characterizing nanoparticles in the field of nanotechnology, to measure the degree of eryptosis in a non-optical manner. With the TRPS system, we were able to determine PS externalization with microbeads functionalized with annexin-V for PS binding, cell swelling and shrinkage in physiological buffers (cell volume: 86 ± 12 fL) and solutions of different osmolarities with or without apoptotic trigger. After setting these standards, we then evaluated the toxicity of Polyphyllin D (PD), a potential anti-cancer drug that kills more liver cancer cells with multi-drug resistance, in erythrocytes to prove our concept. Data revealed that PD induced PS externalization and shrinkage in RBCs in a dose-dependent manner. Moreover, another feature of eryptosis, as small as 5 fL, was detected thus showing the PD-induced erythrotoxicity in human cells. Taken together, our results indicate that our approach using annexin-V-beads and TRPS is simple, safe and convenient, using only a small volume (35 µL) to evaluate the erythrotoxicity of xenobiotics.

Back to the beginning: Perceptions of older Singaporean couples living alone.

Singapore has an aging population that is projected to increase by 32% in 2100. The majority of older couples live alone. "Aging in Place" is an initiative that is widely promoted by the government, in which older people are supported to live in their own homes. In this qualitative study, we explored the perceptions of older people living with their spouse in Singapore. Fifteen community-dwelling older participants were interviewed. Four themes emerged from the thematic analysis: (i) maintaining health and mobility; (2) maintaining relationships with spouse; (iii) maintaining relationship with others; and (iv) living the last leg of the race. The older people living on their own were generally content about maintaining their interdependence and complementary roles. Insights gathered from this study have relevance to implementation of the Aging-in-Place policy to ensure that older people receive the support they need to maintain their physical and psychosocial well-being while living on their own.

Monitoring bacterial growth using tunable resistive pulse sensing with a pore-based technique.

A novel bacterial growth monitoring method using a tunable resistive pulse sensor (TRPS) system is introduced in this study for accurate and sensitive measurement of cell size and cell concentration simultaneously. Two model bacterial strains, Bacillus subtilis str.168 (BSU168) and Escherichia coli str.DH5α (DH5α), were chosen for benchmarking the growth-monitoring performance of the system. Results showed that the technique of TRPS is sensitive and accurate relative to widely used methods, with a lower detection limit of cell concentration measurement of 5 × 105 cells/ml; at the same time, the mean coefficient of variation from TRPS was within 2 %. The growth of BSU168 and DH5α in liquid cultures was studied by TRPS, optical density (OD), and colony plating. Compared to OD measurement, TRPS-measured concentration correlates better with colony plating (R = 0.85 vs. R = 0.72), which is often regarded as the gold standard of cell concentration determination. General agreement was also observed by comparing TRPS-derived cell volume measurements and those determined from microscopy. We have demonstrated that TRPS is a reliable method for bacterial growth monitoring, where the study of both cell volume and cell concentration are needed to provide further details about the physical aspects of cell dynamics in real time.

Effects of surface material, ventilation, and human behavior on indirect contact transmission risk of respiratory infection.

Infectious particles can be deposited on surfaces. Susceptible persons who contacted these contaminated surfaces may transfer the pathogens to their mucous membranes via hands, leading to a risk of respiratory infection. The exposure and infection risk contributed by this transmission route depend on indoor surface material, ventilation, and human behavior. In this study, quantitative infection risk assessments were used to compare the significances of these factors. The risks of three pathogens, influenza A virus, respiratory syncytial virus (RSV), and rhinovirus, in an aircraft cabin and in a hospital ward were assessed. Results showed that reducing the contact rate is relatively more effective than increasing the ventilation rate to lower the infection risk. Nonfabric surface materials were found to be much more favorable in the indirect contact transmission for RSV and rhinovirus than fabric surface materials. In the cases considered in this study, halving the ventilation rate and doubling the hand contact rate to surfaces and the hand contact rate to mucous membranes would increase the risk by 3.7-16.2%, 34.4-94.2%, and 24.1-117.7%, respectively. Contacting contaminated nonfabric surfaces may pose an indirect contact risk up to three orders of magnitude higher than that of contacting contaminated fabric surfaces. These findings provide more consideration for infection control and building environmental design.

Viral Glomerulopathy.

Background: The association between viral infections and glomerular diseases, commonly known as "viral glomerulopathies," has been described in various clinical scenarios for decades. Despite advancements in diagnostic tools, it remains challenging to establish a causative link fully. Summary: Data from mouse models have substantiated clinical observations and implicate direct viral infection in the pathogenesis of viral glomerulopathy, particularly in human immunodeficiency virus-associated nephropathy. In addition to the traditional concept of direct viral effects on kidneys, other factors such as APOL1 risk alleles can further modify the clinical outcomes or presentations of different viral glomerulopathies. Newly developed antiviral drugs are now applicable to a wider range of patients with lower kidney function and fewer side effects. Key Message: Efforts focusing on vaccines and antiviral treatments have significantly reduced the incidence of viral glomerulopathies. However, the most recent pandemic caused by severe acute respiratory syndrome coronavirus 2 infection complicated by COVID-associated nephropathy illustrates our susceptibility to novel viruses. Ongoing research is pivotal to deciphering the mechanisms behind viral glomerulopathies and discovering therapeutics in a collaborative approach.

Aged glomeruli: a link between PD-1 and podocytes.

Understanding the loss of kidney function resulting from kidney aging has become an emerging research focus that will facilitate the future development of antisenolytic treatments. In this issue of the JCI, Pippin et al. first identified PD-1 upregulation in the aged mouse podocyte via unbiased RNA-seq analysis. Overexpression of PD-1 in immortalized mouse podocytes induced cell death and a senescence-associated secretory phenotype, suggesting the pathological role of PD-1 upregulation in aged podocytes. Blocking PD-1 signaling via a neutralizing anti-PD-1 antibody reversed the aged phenotype in the aged mice and ameliorated proteinuria in an experimental focal segmental glomerulosclerosis (FSGS) mouse model. These findings highlight the role of PD-1 signaling in kidney aging and its therapeutic potential for human clinical trials.

Happy gut, happy kidneys? Restoration of gut microbiome ameliorates acute and chronic kidney disease.

In a new study, Zhu et al. (2021) show that mitigating dysbiosis by the probiotic L. casei Zhang reduces kidney inflammation via restoring short-chain fatty acid-producing gut microbiome and nicotinamide metabolism. These findings shed light on the underlying mechanisms of probiotics in treating human kidney diseases.

IgA Nephropathy After SARS-CoV-2 Vaccination.

Here we present the first case of newly diagnosed IgA nephropathy (IgAN) after a SARS-CoV-2 vaccination. A 30-year-old man with no known past medical history presented with gross hematuria and subnephrotic proteinuria 24 hours after the second dose of the mRNA-1273 SARS-CoV-2 vaccine. A kidney biopsy showed IgAN. He was started on an angiotensin receptor blocker, resulting in proteinuria reduction. Similar to natural infection of SARS-CoV-2, persons who receive 2 mRNA-based vaccines demonstrate robust antibodies against the receptor-binding domain (RBD) of the S1 protein. Given the uniqueness of glycosylation of RBD and potent stimulation of immune response from mRNA-based vaccine compared to other vaccines, we hypothesize that our patient developed de novo antibodies, leading to IgA-containing immune-complex deposits. This case highlights the urgency of understanding the immunological responses to novel mRNA-based SARS-CoV-2 vaccines in more diverse populations. Despite the lack of clear causality, nephrologists should be alerted if any new-onset hematuria or proteinuria is observed.

Two-component mixed and patterned films on carbon surfaces through the photografting of arylazides.

Organic films have been grafted to glassy carbon surfaces by the photolysis of arylazides. Atomic force microscopy and electrochemical measurements reveal that the films are loosely packed. The methodology was expanded to prepare two-component thin films incorporating either a reactive tether species and a nonreactive background film or two different reactive tethers. Strategies were developed to generate both continuous mixed films and surfaces presenting patterns of two components. For patterning, the arylazide derivative was grafted onto previously modified glassy carbon surfaces. In this case, the first modification step is not limited to photografting, which increases the scope of the methods. For all grafted surfaces, the reactivity of tether species was confirmed by coupling electroactive targets to the tethers, followed by electrochemical monitoring. The ease of preparing surfaces with spatially controlled functionality offers promise for the design of sensing platforms on graphitic carbon substrates.

Complement, a Therapeutic Target in Diabetic Kidney Disease.

Currently available treatments of diabetic kidney disease (DKD) remain limited despite improved understanding of DKD pathophysiology. The complement system is a central part of innate immunity, but its dysregulated activation is detrimental and results in systemic diseases with overt inflammation. Growing evidence suggests complement activation in DKD. With existent drugs and clinical success of treating other kidney diseases, complement inhibition has emerged as a potential novel therapy to halt the progression of DKD. This article will review DKD, the complement system's role in diabetic and non-diabetic disease, and the potential benefits of complement targeting therapies especially for DKD patients.

Promotion of cadmium uptake and cadmium-induced toxicity by the copper transporter CTR1 in HepG2 and ZFL cells.

Cadmium (Cd2+) is considered a human carcinogen as it causes oxidative stress and alters DNA repair responses. However, how Cd2+ is taken up by cells remains unclear. We hypothesized that Cd2+ could be transported into cells via a membrane copper (Cu) transporter, CTR1. CTR1 expression was not affected by Cd2+ exposure at the mRNA or protein level. Stable cell lines overexpressing either hCTR1, in the human liver cell line HepG2, or zCTR1, in the zebrafish liver cell line ZFL, were created to study their responses to Cd2+ insult. It was found that both HepG2 and ZFL cells overexpressing CTR1 had higher Cd2+ uptake and thus became sensitive to Cd2+. In contrast, hCTR1 knockdown in HepG2 cells led to a reduced uptake of Cd2+, making the cells relatively resistant to Cd2+. Localization studies revealed that hCTR1 had a clustered pattern after Cd2+ exposure, possibly in an attempt to reduce both Cd2+ uptake and Cd2+-induced toxicity. These in vitro results indicate that CTR1 can transport Cd2+ into the cell, resulting in Cd2+ toxicity.

Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells.

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) (n = 42) or end-stage kidney disease (ESKD) (n = 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-1ß 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 (TFH) was similar amongst groups, ESKD had reduced frequency of TFH1 (CCR6-CXCR3+CXCR5+PD1+CD4+CD8-), but increased TFH2 (CCR6-CXCR3-CXCR5+PD1+CD4+CD8-), and plasmablasts (CD3-CD56-CD19+CD27highCD38highCD138-). In conclusion, kidney failure is associated with pro-inflammatory markers, exhausted T cell phenotype, and upregulated TFH2, 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.