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BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45(+) immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Kidney resident macrophages (KRMs) are involved in homeostasis, phagocytosis, defense against infectious agents, response to insults, inflammation, and tissue repair. They also play critical roles in the pathogenesis and recovery from many kidney diseases such as acute kidney injury. KRMs historically have been studied as one homogenous population, but the wide-ranging roles and phenotypes observed suggest that there is greater heterogeneity than previously understood. Advancements in RNA sequencing technologies (single-cell RNA sequencing and spatial transcriptomics) have identified specific subsets of KRMs that are molecularly, functionally, and spatially distinct with dynamic changes after kidney injury. Multiple studies have identified unique markers that represent these subpopulations, permitting further characterization of the function and roles they play in the kidney. Understanding the diversity of KRM subpopulations will be key in the development of novel therapies used in treating kidney diseases and promoting kidney health.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species. NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

Although renal macrophages have been shown to contribute to cystic kidney disease in PKD animal models, it remains unclear if there is a specific macrophage subpopulation involved. Here we analyze changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We demonstrate that CD206+ resident macrophages are minimal in a normal adult kidney but accumulate in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and show this significantly reduces cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increases in kidneys and in the urine from ADPKD patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation and demonstrate that the CD206+ resident macrophages in urine may serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients.

Background There are increasing data on changes in intestinal inflammation and microbiome diversity during pregnancy in women with inflammatory bowel disease (IBD) with implications towards individual and offspring immune function. However, differences in intestinal inflammation, as measured by fecal calprotectin (FC) and microbial a-diversity, in consecutive pregnancies are not known. Methods We prospectively enrolled a cohort of women, 37 with IBD and 39 without IBD, during two consecutive pregnancies, and their offspring. We collected serial stool samples and clinical data, and measured FC and bacterial abundance during each trimester of each pregnancy. We further performed correlation analysis between FC in consecutive pregnancies and between microbial a-diversity in consecutive pregnancies among women with and without IBD. Results Compared to healthy controls, IBD pregnancies had significantly lower gestational age at birth and higher frequency of Cesarean section. Mode of delivery, the status of Group B Streptococcus (GBS) infection and GBS infection prophylaxis were significantly associated with the pregnancy order in both IBD and controls (Table). Furthermore, we observed strong correlations of FC (r=0.56, p-value=0.093) and microbial alpha-diversity assessed as operational taxonomic unit (OTU) richness (r=0.88, p=0.0087) between paired consecutive pregnancies in women with IBD, but not in those without IBD (Figure). There were no differences in microbial alpha-diversity using the Shannon and Simpson indices when comparing consecutive pregnancies of women with and without IBD. Conclusion In this study, we demonstrate that intestinal inflammation and microbiome diversity are more conserved in consecutive pregnancies of women with IBD compared to healthy controls. These findings may have implications towards understanding the impact of pregnancy on host-microbiome interactions in IBD as well as the potential impact on offspring health.

Although heart transplantation remains the gold standard for management of heart failure, ventricular assist devices (VAD) have emerged as viable alternatives. VAD implantation improves kidney function. However, whether the improvement is sustained or associated with improved outcomes is unclear. Herein we assess kidney function improvement, predictors of improvement, and associations with thromboembolism, hemorrhage, and mortality in VAD patients. Kidney function was defined using chronic kidney disease (CKD) stages: stage 1 (glomerular filtration rate [eGFR] ≥ 90 ml/min/1.73 m), stage 2 (eGFR 60-90 ml/min/1.73 m), stage 3a (eGFR 45-59 ml/min/1.73 m), stage 3b (eGFR 30-44 ml/min/1.73 m), stage 4 (eGFR 15-30 ml/min/1.73 m), and stage 5 (eGFR15 ml/min/1.73 m). Improvement in kidney function was defined as an improvement in eGFR that resulted in a CKD stage change to one of lesser severity. Kidney function improved post implant, and was maintained over 1 year for all patients, except those with baseline stage 5 CKD. Younger age at implantation (OR 0.93, 95% CI: 0.90-0.96, P0.0001) was associated with sustained improvement in kidney function. Poor kidney function was associated increased mortality but not with thromboembolism or hemorrhage. Compared to patients with baseline eGFR45 ml/min/1.73 m; patients with eGFR45 ml/min/1.73 m had a higher mortality risk (HR 3.32, 95% CI: 1.10-9.98, p = 0.03 for stage 3b; HR 4.07, 95% CI: 1.27-13.1, p = 0.02 for stage 4; and HR 4.01, 95% CI: 1.17-13.7, p = 0.03 for stage 5 CKD). Kidney function was not associated with thromboembolism or hemorrhage, and sustained improvement was not associated with lower risk of death. However, poor kidney function at implantation was associated with an increased risk of mortality.

Myeloid cell populations play critical roles in the context of acute kidney injury and recovery. This chapter provides a description of acute kidney injury and an explanation of the various model systems used to study the mononuclear phagocyte system, with a focus on ischemia–reperfusion injury. The role of cellular damage caused by the initial insult and function of infiltrating versus resident cell populations through injury and recovery, or if there is a failure to restore homeostasis, are discussed.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

Background: Acute kidney injury (AKI) is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. Methods: We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized COVID-19 patients with (n=5) or without AKI (n=3) as well as four non-COVID-19 AKI patients (n=4) to assess differences in cellular composition and gene expression during AKI. Results: Analysis of 30,076 cells revealed a diverse array of cell types, most of which were kidney, urothelial, and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared to those without AKI. ACE2 and TMPRSS2 expression were highest in urothelial cells amongst cell types recovered. Notably, in one patient we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with anti-viral and anti-inflammatory pathways. Conclusions: We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally, we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases.

Meals rich in oxalate are associated with calcium oxalate (CaOx) kidney stone disease. Hydroxy-L-proline (HLP) is an oxalate precursor found in milk and collagen-containing foods. HLP has been shown to induce CaOx crystal formation in rodents. The purpose of this study was to evaluate the effect of HLP-induced oxalate levels on inflammation and renal leukocytes during crystal formation. Male Sprague-Dawley rats (6-8 weeks old) were fed a control diet containing no oxalate for 3 days before being randomized to continue the control diet or 5% HLP for up to 28 days. Blood, 24-hour urine, and kidneys were collected on Days 0, 7, 14, or 28. Urinary oxalate levels, crystal deposition, and renal macrophage markers were evaluated using ion chromatography-mass spectrometry, immunohistochemistry, and qRT-PCR. Renal leukocytes were assessed using flow cytometry and RNA-sequencing. HLP feeding increased urinary oxalate levels and renal crystal formation in animals within 7 days. HLP also increased renal macrophage populations on Days 14 and 28. Transcriptome analysis revealed that renal macrophages from animals fed HLP for 7 days were involved in inflammatory response and disease, stress response to LPS, oxidative stress, and immune cell trafficking. Renal macrophages isolated on Day 14 were involved in cell-mediated immunological pathways, ion homeostasis, and inflammatory response. Collectively, these findings suggest that HLP-mediated oxalate levels induce markers of inflammation, leukocyte populations, and reprograms signaling pathways in macrophages in a time-dependent manner. Additional studies investigating the significance of oxalate on renal macrophages could aid in our understanding of kidney stone formation.

BACKGROUND: Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12–14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. METHODS: We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. RESULTS: Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11c(lo)) to R2a (CD11c(hi)) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. CONCLUSIONS: These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.

Background Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. Methods As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell–negative CD45 + innate immune cells in mouse, rat, pig, and human kidney tissue. Results We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. Conclusions Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.

Acute kidney injury (AKI) remains a significant clinical problem through its diverse etiologies, the challenges of robust measurements of injury and recovery, and its progression to chronic kidney disease (CKD). Bridging the gap in our knowledge of this disorder requires bringing together not only the technical resources for research but also the investigators currently endeavoring to expand our knowledge and those who might bring novel ideas and expertise to this important challenge. The University of Alabama at Birmingham-University of California-San Diego O’Brien Center for Acute Kidney Injury Research brings together technical expertise and programmatic and educational efforts to advance our knowledge in these diverse issues and the required infrastructure to develop areas of novel exploration. Since its inception in 2008, this O’Brien Center has grown its impact by providing state-of-the-art resources in clinical and preclinical modeling of AKI, a bioanalytical core that facilitates measurement of critical biomarkers, including serum creatinine via LC-MS/MS among others, and a biostatistical resource that assists from design to analysis. Through these core resources and with additional educational efforts, our center has grown its investigator base to include >200 members from 51 institutions. Importantly, this center has translated its pilot and catalyst funding program with a $37 return per dollar invested. Over 500 publications have resulted from the support provided with a relative citation ratio of 2.18 ± 0.12 (iCite). Through its efforts, this disease-centric O’Brien Center is providing the infrastructure and focus to help the development of the next generation of researchers in the basic and clinical science of AKI. This center creates the promise of the application at the bedside of the advances in AKI made by current and future investigators.

The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis., Bone marrow Tregs regulate stromal cell function to maintain hematopoiesis through IL-10 and provide a potential therapeutic avenue for stromal manipulation.

Background Preliminary evidence suggests changes in breast milk cytokines in women with inflammatory bowel disease (IBD) compared to healthy controls, with potential implications toward offspring immunological development. However, changes in breast milk cytokine profiles in consecutive pregnancies are not known. Methods In this pilot study, we prospectively enrolled 11 pregnant women with, and 10 without IBD during two consecutive pregnancies and collected clinical data during each pregnancy and post birth. We collected breast milk samples at two weeks post birth and obtained the expression levels of 92 cytokines using the Olink proteomic platform. We further analyzed the correlation of cytokine profiles within each sample, in paired breast milk samples from consecutive pregnancies, and in random two unpaired breast milk samples, of women with and without IBD. Results The baseline characteristics of women with and without IBD were comparable (Table). The cytokine profiles were significantly correlated between paired breast milk samples from consecutive pregnancies compared to unpaired breast milk samples from women with or without IBD. The overall correlations of cytokine profiles in paired IBD pregnancies were significantly higher than the controls (Figure). Conclusion Our pilot study results suggest that the breast milk cytokine signatures are more conserved in consecutive pregnancies of women with IBD compared to those without IBD. Future analysis will test if our findings have implications toward familial clustering of immune functions in offspring.

Background & Aims The effect of pregnancy on inflammatory bowel disease (IBD) remains poorly understood. We aimed to monitor intestinal inflammation using fecal calprotectin (FC) in pregnant women and their babies during early life. Methods Pregnant women with or without IBD and their infants were prospectively enrolled. FC levels were measured at each trimester of pregnancy and in babies throughout the first 3 years of life. Repeated-measures analysis was applied to investigate changes in FC levels while adjusting for confounders. The FC levels were correlated with the bacterial abundance in both mothers and babies. Results Six hundred and fourteen fecal samples from 358 mothers (98 with IBD) and 1005 fecal samples from 289 infants (76 born to IBD mothers) were analyzed. Pregnant Patients with IBD maintained higher FC levels through pregnancy compared with controls (P = 7.5 × 10–54). FC gradually increased in controls and declined in Patients with IBD throughout pregnancy (P for interaction = 5.8 × 10–7). Babies born to mothers with IBD presented with significantly higher FC levels than those born to controls up to 3 years of age, after adjusting for sex, delivery mode, feeding behavior, and antibiotics exposure (2 weeks to 3 months of age, P = .015; 12–36 months of age, P = .00003). Subdoligranulum, Roseburia, Fusicatenibacter, and Alistipes negatively correlated, and Streptococcus, Prevotella, Escherichia-Shigella, and Bifidobacterium positively correlated with maternal FC levels at T3. Faecalibacterium, Bifidobacterium, and Alistipes showed negative correlations, and Streptococcus were positively correlated with FC levels within 3 months of birth. Conclusions Pregnancy is associated with decreased inflammatory activity in mothers with IBD. Higher FC levels in babies born to mothers with IBD suggest subclinical inflammation in early life, the long-term consequences of which are uncertain.

Lewisite and many other similar arsenicals are warfare vesicants developed and weaponized for use in World Wars I and II. These chemicals, when exposed to the skin and other epithelial tissues, cause rapid severe inflammation and systemic damage. Here, we show that topically applied arsenicals in a murine model produce significant acute kidney injury (AKI), as determined by an increase in the AKI biomarkers NGAL and KIM-1. An increase in reactive oxygen species and ER stress proteins, such as ATF4 and CHOP, correlated with the induction of these AKI biomarkers. Also, TUNEL staining of CHOP-positive renal tubular cells suggests CHOP mediates apoptosis in these cells. A systemic inflammatory response characterized by a significant elevation in inflammatory mediators, such as IL-6, IFN-α, and COX-2, in the kidney could be the underlying cause of AKI. The mechanism of arsenical-mediated inflammation involves activation of AMPK/Nrf2 signaling pathways, which regulate heme oxygenase-1 (HO-1). Indeed, HO-1 induction with cobalt protoporphyrin (CoPP) treatment in arsenical-treated HEK293 cells afforded cytoprotection by attenuating CHOP-associated apoptosis and cytokine mRNA levels. These results demonstrate that topical exposure to arsenicals causes AKI and that HO-1 activation may serve a protective role in this setting.

Altered inflammation and tissue remodeling are cardinal features of cardiovascular disease and cardiac transplant rejection. Neutrophils have increasingly been understood to play a critical role in acute rejection and early allograft failure; however, discrete mechanisms that drive this damage remain poorly understood. Herein, we demonstrate that early acute cardiac rejection increases allograft prolyl endopeptidase (PE) in association with de novo production of the neutrophil proinflammatory matrikine proline-glycine-proline (PGP). In a heterotopic murine heart transplant model, PGP production and PE activity were associated with early neutrophil allograft invasion and allograft failure. Pharmacologic inhibition of PE with Z-Pro-prolinal reduced PGP, attenuated early neutrophil graft invasion, and reduced proinflammatory cytokine expression. Importantly, these changes helped preserve allograft rejection-free survival and function. Notably, within 2 independent patient cohorts, both PGP and PE activity were increased among patients with biopsy-proven rejection. The observed induction of PE and matrikine generation provide a link between neutrophilic inflammation and cardiovascular injury, represent a potential target to reduce allogenic immune responses, and uncover a mechanism of cardiovascular disease that has been previously unrecognized to our knowledge.

Chronic kidney disease (CKD) is a condition with significant morbidity and mortality that affects 15% of adults in the United States. One cause of CKD is acute kidney injury (AKI), which commonly occurs secondary to sepsis, ischemic events, and drug-induced nephrotoxicity. Unilateral ischemia-reperfusion injury (UIRI) without contralateral nephrectomy (CLN) and repeated low-dose cisplatin (RLDC) models of AKI to CKD demonstrate responses characteristic of the transition; however, previous studies have not effectively compared the pathogenesis. We demonstrate both models instigate renal dysfunction, inflammatory cytokine responses, and fibrosis. However, the models exhibit differences in urinary excretory function, inflammatory cell infiltration, and degree of fibrotic response. UIRI without CLN demonstrated worsening perfusion and function, measured with 99mTc-mercaptoacetyltriglycine-3 imaging, and physiologic compensation in the contralateral kidney. Furthermore, UIRI without CLN elicited a robust inflammatory response that was characterized by a prolonged polymorphonuclear cell and natural killer cell infiltrate and an early expansion of kidney resident macrophages, followed by T-cell infiltration. Symmetrical diminished function occurred in RLDC kidneys and progressively worsened until day 17 of the study. Surprisingly, RLDC mice demonstrated a decrease in inflammatory cell numbers relative to controls. However, RLDC kidneys expressed increased levels of kidney injury molecule-1 (KIM-1), high mobility group box-1 ( HMGB1), and colony stimulating factor-1 ( CSF-1), which likely recruits inflammatory cells in response to injury. These data emphasize how the divergent etiologies of AKI to CKD models affect the kidney microenvironment and outcomes. This study provides support for subtyping AKI by etiology in human studies, aiding in the elucidation of injury-specific pathophysiologic mechanisms of the AKI to CKD transition.

Gastrointestinal bleeding (GIB) is common in patients with continuous-flow left ventricular assist devices (CF-LVADs) possibly because of changes in blood flow. We aimed to test the hypothesis that a low pulsatility index (PI) is associated with an increased hazard of overt GIB in patients with CF-LVADs. We conducted a retrospective cohort study of patients who had a HeartMate II (HMII) CF-LVAD implanted at our center. The study end-point was the first overt GIB causing or occurring during a hospitalization between 6 days and 6 months after HMII implantation. HMII PI was recorded at 48 hours and at 1, 3, and 6 month intervals after implantation. We analyzed the associations of PI and clinical variables with the hazard of overt GIB. Ninety-five patients met eligibility criteria. PI ranged from 2.5 to 5.9 (low PI < 4.15 and high PI ≥ 4.15 on the basis of receiver operating characteristic curve analysis). Seventeen (18%) patients experienced overt GIB. In a multivariable model, only lower baseline hemoglobin was a significant predictor of an increased hazard of overt GIB. After adjusting for the baseline hemoglobin, low PI was independently associated with an increased hazard of overt GIB in our cohort of HMII recipients.

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1−/− mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1−/− cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1−/− PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1−/− PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.

Anticoagulation control has been associated with risk of thromboembolism and hemorrhage. Herein, we explore the relationship between anticoagulation control achieved in left ventricular assist device (LVAD) patients and evaluate the association with risk of thromboembolism and hemorrhage. Patients (19 years old or older) with a continuous flow LVAD placed from 2006 to 2012. Percent time spent in target range (PTTR) for international normalized ratio (INR) was estimated with target range of 2.0-3.0. Proportion of time spent in target range was categorized into PTTR60%, PTTR ≥ 5060%, and PTTR50%. The relationship between PTTR and thromboembolism and hemorrhage was assessed. One hundred fifteen participants contributed 624.5 months of follow-up time. Only 20% of patients achieved anticoagulation control (PTTR60% for INR range of 2-3). After adjusting for chronic kidney disease, history of diabetes, history of atrial fibrillation, and age at implant, compared with patients with PTTR50%, the relative risk of thromboembolism in patients with PTTR ≥ 60% (hazard ratio [HR]: 0.37; 95% confidence interval [CI]: 0.14-0.96; p = 0.042) was significantly lower, but not for patients with a PTTR of ≥ 5060% (HR: 0.21; 95% CI: 0.02-1.82; p = 0.16). The relative risk for hemorrhage was also significantly lower among patients with a PTTR ≥ 60% (HR: 0.45; 95% CI: 0.21-0.98; p = 0.045), but not among those with PTTR of ≥ 5060% (HR: 0.47; 95% CI: 0.14-1.56; p = 0.22). This current study demonstrates that LVAD patients remain in the INR target range an average of 42.9% of the time. To our knowledge, this is the first report with regard to anticoagulation control as assessed by PTTR and its association with thromboembolism, hemorrhage, or death among patients with ventricular assist devices (VADs).

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80(hi)-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow–derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII(–) KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Background and aimsPrenatal and early life bacterial colonisation is thought to play a major role in shaping the immune system. Furthermore, accumulating evidence links early life exposures to the risk of developing IBD later in life. We aimed to assess the effect of maternal IBD on the composition of the microbiome during pregnancy and on the offspring’s microbiome.MethodsWe prospectively examined the diversity and taxonomy of the microbiome of pregnant women with and without IBD and their babies at multiple time points. We evaluated the role of maternal IBD diagnosis, the mode of delivery, antibiotic use and feeding behaviour on the microbiome composition during early life. To assess the effects of IBD-associated maternal and infant microbiota on the enteric immune system, we inoculated germ-free mice (GFM) with the respective stool and profiled adaptive and innate immune cell populations in the murine intestines.ResultsPregnant women with IBD and their offspring presented with lower bacterial diversity and altered bacterial composition compared with control women and their babies. Maternal IBD was the main predictor of the microbiota diversity in the infant gut at 7, 14, 30, 60 and 90 days of life. Babies born to mothers with IBD demonstrated enrichment in Gammaproteobacteria and depletion in Bifidobacteria. Finally, GFM inoculated with third trimester IBD mother and 90-day infant stools showed significantly reduced microbial diversity and fewer class-switched memory B cells and regulatory T cells in the colon.ConclusionAberrant gut microbiota composition persists during pregnancy with IBD and alters the bacterial diversity and abundance in the infant stool. The dysbiotic microbiota triggered abnormal imprinting of the intestinal immune system in GFM.

Significance: Acute kidney injury (AKI) and chronic kidney disease (CKD) represent a considerable burden in healthcare. The heme oxygenase (HO) system plays an important role in regulating oxidative stress and is protective in a variety of human and animal models of kidney disease. Preclinical studies of the HO system have led to the development of several clinical trials targeting the enzyme or its products. Recent Advances: Connection of HO, ferritin, and other proteins involved in iron regulation has provided important insight into mechanisms of damage in AKI. Also, HO-1 expression is important in the pathogenesis of hypertension, diabetic kidney disease, and progression to end-stage renal disease. Critical Issues: Despite intriguing discoveries, no drugs targeting the HO system have been translated to the clinic. Meanwhile, treatments for AKI and CKD are urgently needed. Many factors have likely contributed to challenges in clinical translation, including variation in animal models, difficulties in obtaining human tissue, and complexity of the disease processes being studied. Future Directions: The HO system represents a promising avenue of investigation that may lead to targeted therapeutics. Tissue-specific gene modulation, widening the scope of animal studies, and continued clinical research will provide valuable insight into the role HO plays in kidney homeostasis and disease. Antioxid. Redox Signal. 25, 165–183.

Ventricular assist device patients (VAD) are at increased risk for thromboembolism. Biomarkers of hemolysis, such as lactate dehydrogenase (LDH) and poorly controlled international normalized ratio (INR) has been identified as predictors of thromboembolism. Patients aged 19 years and older who had a continuous flow VAD placed from 2006 to 2012 were included in this study (N = 115). We assessed the relationship of LDH elevation (≥600 IU/L) at different time points and thromboembolism. Over the 51.3 person-years of follow-up, a total of 23 first thromboembolic events occurred. Patients with elevated LDH on the day of VAD implantation had an increased risk for thromboembolism (hazard ratio [HR]: 4.72, 95% confidence interval [CI]: 1.44-15.4; p = 0.01). There was an increased risk of thromboembolism with early LDH elevation within the first month post-VAD (HR: 4.95, 95% CI: 1.69-14.4; p = 0.003) and estimated glomerular filtration rate

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn’s disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Cardiac surgery causes direct trauma to cardiac tissue, breaches the pericardium, and disrupts the normal composition of the fluid largely produced from the myocardial interstitium and epicardial and visceral pericardium. This leaves the heart exposed to pericardial fluid (PCF) and mediastinal contents comprising inflammatory cells and their products that now bathe the heart. This can potentially have adverse effects on the thin-walled atria leading to postoperative atrial fibrillation (AF).1 After cardiac surgery, the pericardium remains open, and chest drains are routinely placed to prevent fluid accumulation around the heart. Here, we describe the concentration and trajectory of blood proinflammatory factors in the PCF after cardiac surgery over time. The study protocol was approved by the University of Alabama at Birmingham. Institutional Review Board approval and informed consent were obtained from all patients. PCF (n=19) was collected immediately after pericardiotomy (time 0) and from the pericardial drains at times 4, 12, 24, and 48 hours after surgery. The patient population (mean age, 60±3 years) included 26.3% women and 26.3% blacks undergoing cardiac surgery (coronary artery bypass graft, n=14; coronary artery bypass graft+valve procedure, n=3; valve procedure alone, n=2). Patients with ventricular assist devices, AF surgery, thoracic aorta surgery, and AF within 6 months prior were excluded. All participants who had valve replacement (with or without coronary artery bypass graft) underwent on-pump surgeries. Of the patients undergoing coronary artery bypass …

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency.

Atherosclerosis and valvular heart disease often require treatment with corrective surgery to prevent future myocardial infarction, ischemic heart disease, and heart failure. Mechanisms underlying the development of the associated complications of surgery are multifactorial and have been linked to inflammation and oxidative stress, classically as measured in the blood or plasma of patients. Post-operative pericardial fluid (PO-PCF) has not been investigated in depth with respect to the potential to induce oxidative stress. This is important since cardiac surgery disrupts the integrity of the pericardial membrane surrounding the heart, and causes significant alterations in the composition of the pericardial fluid (PCF). This includes contamination with hemolyzed blood and high concentrations of oxidized hemoglobin, which suggests that cardiac surgery results in oxidative stress within the pericardial space. Accordingly, we tested the hypothesis that PO-PCF is highly pro-oxidant and that the potential interaction between inflammatory cell-derived hydrogen peroxide with hemoglobin is associated with oxidative stress. Blood and PCF were collected from 31 patients at the time of surgery and postoperatively from 4 to 48 hours after coronary artery bypass grafting, valve replacement, or valve repair (mitral or aortic). PO-PCF contained high concentrations of neutrophils and monocytes which are capable of generating elevated amounts of superoxide and hydrogen peroxide through the oxidative burst. In addition, PO-PCF primed naïve neutrophils resulting in an enhanced oxidative burst upon stimulation. The PO-PCF also contained increased concentrations of cell-free oxidized hemoglobin which was associated with elevated levels of F2α-isoprostanes and prostaglandins, consistent with both oxidative stress and activation of cyclooxygenase. Lastly, protein analysis of the PO-PCF revealed evidence of protein thiol oxidation and protein carbonylation. We conclude that PO-PCF is highly pro-oxidant and speculate that it may contribute to the risk of post-operative complications.

The immune cellular compartment of the kidney is involved in organ development and homeostasis, as well as in many pathological conditions. Little is known about the mechanisms that drive intrarenal immune responses in the presence of renal tubular and interstitial cell death. However, it is known that tissue-resident leukocytes have the potential to have distinct roles compared with circulating cells. We used a parabiosis model in C57BL/6 CD45 congenic and green fluorescent protein transgenic mice to better understand the dynamics of immune cells in the kidney. We found F4/80Hi intrarenal macrophages exhibit minimal exchange with the peripheral circulation in two models of parabiosis, whether mice were attached for 4 or 16 weeks. Other intrarenal inflammatory cells demonstrate near total exchange with the circulating immune cell pool in healthy kidneys, indicating that innate and adaptive immune cells extensively traffic through the kidney interstitium during normal physiology. Neutrophils, dendritic cells, F4/80Low macrophages, T cells, B cells, and NK cells are renewed from the circulating immune cell pool. However, a fraction of double-negative T (CD4- CD8-) and NKT cells are long-lived or tissue resident. This study provides direct evidence of leukocyte sub-populations that are resident in the renal tissue, cells which demonstrate minimal to no exchange with the peripheral blood. In addition, the data demonstrate continual exchange of other sub-populations through uninflamed tissue.