Macrophage Depletion Protects Against Cisplatin-Induced Ototoxicity and Nephrotoxicity.

Cisplatin is a widely used and highly effective anti-cancer drug with significant side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced ototoxicity and nephrotoxicity, we used PLX3397, an FDA-approved inhibitor of the colony-stimulating factor 1 receptor (CSF1R), to eliminate tissue-resident macrophages during the course of cisplatin administration. Mice treated with cisplatin alone (cisplatin/vehicle) had significant hearing loss (ototoxicity) as well as kidney injury (nephrotoxicity). Macrophage ablation using PLX3397 resulted in significantly reduced hearing loss measured by auditory brainstem responses (ABR) and distortion-product otoacoustic emissions (DPOAE). Sensory hair cells in the cochlea were protected against cisplatin-induced death in mice treated with PLX3397. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis as well as reduced plasma blood urea nitrogen (BUN) and neutrophil gelatinase-associated lipocalin (NGAL) levels. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together our data indicate that ablation of tissue-resident macrophages represents a novel strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

Readmission and Mortality After Hospitalization With Acute Kidney Injury.

RATIONALE & OBJECTIVE: Acute kidney injury (AKI) carries high rates of morbidity and mortality. This study quantified various short- and long-term outcomes after hospitalization with AKI. STUDY DESIGN: Retrospective propensity score (PS)-matched cohort study. SETTING & PARTICIPANTS: Optum Clinformatics, a national claims database, was used to identify patients hospitalized with and without an AKI discharge diagnosis between January 2007 and September 2020. EXPOSURE: Among patients with prior continuous enrollment for at least 2years without AKI hospitalization, 471,176 patients hospitalized with AKI were identified and PS-matched to 471,176 patients hospitalized without AKI. OUTCOME(S): All-cause and selected-cause rehospitalizations and mortality 90 and 365 days after index hospitalization. ANALYTICAL APPROACH: After PS matching, rehospitalization and death incidences were estimated using the cumulative incidence function method and compared using Gray's test. The association of AKI hospitalization with each outcome was tested using Cox models for all-cause mortality and, with mortality as competing risk, cause-specific hazard modeling for all-cause and selected-cause rehospitalization. Overall and stratified analyses were performed to evaluate for interaction between an AKI hospitalization and preexisting chronic kidney disease (CKD). RESULTS: After PS matching, AKI was associated with higher rates of rehospitalization for any cause (hazard ratio [HR], 1.62; 95% CI, 1.60-1.65), end-stage renal disease (HR, 6.21; 95% CI, 1.04-36.92), heart failure (HR, 2.81; 95% CI, 2.66, 2.97), sepsis (HR, 2.62; 95% CI, 2.49-2.75), pneumonia (HR, 1.47; 95% CI, 1.37-1.57), myocardial infarction (HR, 1.48; 95% CI, 1.33-1.65), and volume depletion (HR, 1.64; 95% CI, 1.37-1.96) at 90 days after discharge compared with the group without AKI, with similar findings at 365 days. Mortality rate was higher in the group with AKI than in the group without AKI at 90 (HR, 2.66; 95% CI, 2.61-2.72) and 365 days (HR, 2.11; 95% CI, 2.08-2.14). The higher risk of outcomes persisted when participants were stratified by CKD status (P<0.01). LIMITATIONS: Causal associations between AKI and the reported outcomes cannot be inferred. CONCLUSIONS: AKI during hospitalization in patients with and without CKD is associated with increased risk of 90- and 365-day all-cause/selected-cause rehospitalization and death.

The Application of Guanidinium to Improve Biomolecule Quality in Fixed, Paraffin-embedded Tissue.

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes.

BAM15 treats mouse sepsis and kidney injury, linking mortality, mitochondrial DNA, tubule damage, and neutrophils.

Sepsis pathogenesis is complex and heterogeneous; hence, a precision-medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial ROS (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP), and these mice were treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased after CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis and mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug-efficacy pair for clinical sepsis, are mechanistically linked via mtROS.

The effect of continuous intravenous norepinephrine infusion on systemic hemodynamics in a telemetrically-monitored mouse model of sepsis.

Sepsis, a life-threatening organ dysfunction, results from dysregulated host responses to infection and still has a high incidence and mortality. Although administration of vasopressors to treat septic shock is standard of care, the benefits are not well established. We evaluated the effect of continuous intravenous norepinephrine infusion in a septic cecal ligation and puncture (CLP) mouse model, evaluating systemic hemodynamics and body temperature post-hoc. CLP surgery significantly decreased mean arterial blood pressure (MAP), heart rate, and body temperature within six hours. Continuous norepinephrine infusion (NE+, n = 12) started at the time of CLP surgery significantly increased MAP at 24 and 30 hours and heart rate at 6, 18, 24, and 30 hours after CLP vs CLP alone (NE-, n = 12). However, addition of norepinephrine did not improve survival rate (NE+ n = 34, NE- n = 31). Early (6 hours or earlier, when the animal became visibly sick) MAP did not predict 7-day mortality. However, heart rates at 3 and at 6 hours after CLP/norepinephrine (NE+) were highly predictive of mortality, as also been found in one clinical study. We conclude that limited hemodynamic support can be provided in a mouse sepsis model. We propose that heart rate can be used to stratify severity of illness in rodent preclinical studies of sepsis therapeutics.

Class B Scavenger Receptors BI and BII Protect against LPS-Induced Acute Lung Injury in Mice by Mediating LPS.

Recent studies suggest an anti-inflammatory protective role for class B scavenger receptor BI (SR-BI) in endotoxin-induced inflammation and sepsis. Other data, including ours, provide evidence for an alternative role of SR-BI, facilitating bacterial and endotoxin uptake and contributing to inflammation and bacterial infection. Enhanced endotoxin susceptibility of SR-BI-deficient mice due to their anti-inflammatory glucocorticoid deficiency complicates the understanding of SR-BI's role in endotoxemia/sepsis, calling for the use of alternative models. In this study, using human SR-BI (hSR-BI) and hSR-BII transgenic mice, we found that SR-BI and, to a lesser extent, its splicing variant SR-BII protect against LPS-induced lung damage. At 20 h after intratracheal LPS instillation, the extent of pulmonary inflammation and vascular leakage was significantly lower in hSR-BI and hSR-BII transgenic mice than in wild-type mice. Higher bronchoalveolar lavage fluid (BALF) inflammatory cell count and protein content and lung tissue neutrophil infiltration found in wild-type mice were associated with markedly (2 to 3 times) increased proinflammatory cytokine production compared to these parameters in transgenic mice following LPS administration. The markedly lower endotoxin levels detected in BALF of transgenic versus wild-type mice and the significantly increased BODIPY-LPS uptake observed in lungs of hSR-BI and hSR-BII mice 20 h after the i.t. LPS injection suggest that hSR-BI- and hSR-BII-mediated enhanced LPS clearance in the airways could represent the mechanism of their protective role against LPS-induced acute lung injury.

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury.

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.

A Furosemide Excretion Stress Test Predicts Mortality in Mice After Sepsis and Outperforms the Furosemide Stress Test During Vasopressin Administration.

OBJECTIVES: The furosemide stress test measures the volume of urine produced after a furosemide challenge. Furosemide stress test has previously demonstrated sensitive and specific prediction of progression to Kidney Disease: Improving Global Outcomes guideline defined acute kidney injury stage III in the ICU. Furosemide is actively excreted into the nephron lumen where it inhibits the sodium-potassium-chloride cotransporter, causing diuresis. We hypothesize that furosemide excretion is a more direct measure of tubule health than diuresis. DESIGN: We developed a furosemide excretion stress test to evaluate this hypothesis in a murine model of septic-acute kidney injury. SETTING: Basic science laboratory. SUBJECTS: Male and female 8-week old CD-1 mice. INTERVENTIONS: Sepsis was induced by cecal ligation and puncture in male and female mice. Furosemide stress test/furosemide excretion stress test started 42 hours post-cecal ligation and puncture with a 1 mg/kg furosemide bolus and urine was collected for 12 hours. The mice were then euthanized or monitored until 7 days post-cecal ligation and puncture. In another cohort, mice were treated with vasopressin, which decreases urine volume. Furosemide concentration was determined by high performance liquid chromatography. MEASUREMENTS AND MAIN RESULTS: Urine production during the 12-hour collection varied from 0.08 to 2.62 mL. Both urine production (furosemide stress test) and furosemide excretion (furosemide excretion stress test) predicted mortality (area under the receiver operating characteristic curve = 0.925 and 0.916) and time of death (R 2 = 0.26 and 0.74). Male and female mice demonstrated consistent results. Following vasopressin treatment, furosemide stress test specificity fell to 33% (p = 0.016) but furosemide excretion stress test specificity was maintained. CONCLUSIONS: The furosemide stress test and furosemide excretion stress test performed similarly in predicting mortality; however, furosemide excretion stress test was superior in predicting time to death and maintained performance when challenged with vasopressin treatment in a mouse sepsis model.

Sex and the kidneys: current understanding and research opportunities.

Concerns regarding sex differences are increasingly pertinent in scientific and societal arenas. Although biological sex and socio-cultural gender are increasingly recognized as important modulators of renal function under physiological and pathophysiological conditions, gaps remain in our understanding of the mechanisms underlying sex differences in renal pathophysiology, disease development, progression and management. In this Perspectives article, we discuss specific opportunities for future research aimed at addressing these knowledge gaps. Such opportunities include the development of standardized core data elements and outcomes related to sex for use in clinical studies to establish a connection between sex hormones and renal disease development or progression, development of a knowledge portal to promote fundamental understanding of physiological differences between male and female kidneys in animal models and in humans, and the creation of new or the development of existing resources and datasets to make them more readily available for interrogation of sex differences. These ideas are intended to stimulate thought and interest among the renal research community as they consider sex as a biological variable in future research projects.

Chronic kidney diseases in agricultural communities: report from a workshop.

In June 2018, the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Environmental Health Sciences sponsored a workshop to identify research gaps in an increasingly common form of chronic kidney disease in agricultural communities, often termed "CKDu." The organizers invited a broad range of experts who provided diverse expertise and perspectives, many of whom had never addressed this particular epidemic. Discussion was focused around selected topics, including identifying and mitigating barriers to research in CKDu, creating a case definition, and defining common data elements. All hypotheses regarding etiology were entertained, and meeting participants discussed potential research strategies, choices in study design, and novel tools that may prove useful in this disease. Achievements of the workshop included robust cross-disciplinary discussion and preliminary planning of research goals and design. Specific challenges in implementing basic and clinical research and interventions in low- and middle-income countries were recognized. A balanced approach to leveraging local resources and capacity building without overreaching was emphasized.

Circadian variation in the release of small extracellular vesicles can be normalized by vesicle number or TSG101.

Numerous candidate biomarkers in urine extracellular vesicles (EVs) have been described for kidney diseases, but none are yet in clinical use, possibly due to a lack of proper normalization. Proper normalization corrects for normal biological variation in urine flow rate or concentration, which can vary by over one order of magnitude. Here, we observed inter- and intra-animal variation in urine excretion rates of small EVs (<200 nm in diameter) in healthy rats as a series of six 4-h fractions. To visualize intra-animal variation, we normalized a small EV excretion rate to a peak excretion rate, revealing a circadian pattern for each rat. This circadian pattern was distinct from urine volume, urine albumin, urine creatinine, and urine albumin-to-creatinine ratio. Furthermore, urine small EV excretion was not significantly altered by sex, food/water deprivation, or ischemic acute kidney injury. Urine excretion of the exosomal/small EV marker protein tumor susceptibility gene 101 (TSG101) displayed a similar circadian pattern to urine small EV excretion; both measurements were highly correlated (R2 = 0.85), with an average stoichiometry of 10.0 molecules of TSG101/vesicle in healthy rats. The observed stoichiometry of TSG101/vesicle in rat urine translated to human spot urine samples (10.2 molecules/vesicle) and cultured kidney-derived cell lines (human embryonic kidney-293 and normal rat kidney 52E cells). Small EV number and its surrogate, TSG101 protein, can normalize for circadian variation when testing candidate biomarkers in small EVs. Just as creatinine has emerged as the customary normalization factor for liquid-phase urine biomarkers, vesicle number and its surrogate, molecules of exosome/small EV-associated TSG101, should be considered as viable, normalizing factors for small EV biomarkers.

Gut Leakage of Fungal-Derived Inflammatory Mediators: Part of a Gut-Liver-Kidney Axis in Bacterial Sepsis.

Sepsis is a life-threatening response to systemic infection. In addition to frank gastrointestinal (GI) rupture/puncture, sepsis can also be exacerbated by translocation of pathogen-associated molecular patterns (PAMPs) from the GI tract to the systemic circulation (gut origin of sepsis). In the human gut, Gram-negative bacteria and Candida albicans are abundant, along with their major PAMP components, endotoxin (LPS) and (1 → 3)-ß-D-glucan (BG). Whereas the influence of LPS in bacterial sepsis has been studied extensively, exploration of the role of BG in bacterial sepsis is limited. Post-translocation, PAMPs enter the circulation through lymphatics and the portal vein, and are detoxified and then excreted via the liver and the kidney. Sepsis-induced liver and kidney injury might therefore affect the kinetics and increase circulating PAMPs. In this article, we discuss the current knowledge of the impact of PAMPs from both gut mycobiota and microbiota, including epithelial barrier function and the "gut-liver-kidney axis," on bacterial sepsis severity.

Statistical methods for building better biomarkers of chronic kidney disease.

The last two decades have witnessed an explosion in research focused on the development and assessment of novel biomarkers for improved prognosis of diseases. As a result, best practice standards guiding biomarker research have undergone extensive development. Currently, there is great interest in the promise of biomarkers to enhance research efforts and clinical practice in the setting of chronic kidney disease, acute kidney injury, and glomerular disease. However, some have questioned whether biomarkers currently add value to the clinical practice of nephrology. The current state of the art pertaining to statistical analyses regarding the use of such measures is critical. In December 2014, the National Institute of Diabetes and Digestive and Kidney Diseases convened a meeting, "Toward Building Better Biomarker Statistical Methodology," with the goals of summarizing the current best practice recommendations and articulating new directions for methodological research. This report summarizes its conclusions and describes areas that need attention. Suggestions are made regarding metrics that should be commonly reported. We outline the methodological issues related to traditional metrics and considerations in prognostic modeling, including discrimination and case mix, calibration, validation, and cost-benefit analysis. We highlight the approach to improved risk communication and the value of graphical displays. Finally, we address some "new frontiers" in prognostic biomarker research, including the competing risk framework, the use of longitudinal biomarkers, and analyses in distributed research networks.

Mesenchymal stromal cell potency to treat acute kidney injury increased by ultrasound-activated interferon-γ/interleukin-10 axis.

Mesenchymal stromal cell (MSC) therapies combined with renal pulsed focused ultrasound (pFUS) pretreatment increase MSC homing and improve cisplatin-induced acute kidney injury (AKI) better than MSC alone. However, mechanisms underlying improved outcomes remain unknown. We hypothesize pFUS up-regulates renal interferon-γ (IFNγ) and stimulates MSC to produce interleukin-10 (IL-10) after migrating to kidneys. To demonstrate initially, MSC cultured with IFNγ up-regulated IL-10. More MSC-derived IL-10 was detected in kidneys when IFNγ-stimulated MSC were infused and they improved AKI better than unstimulated MSC. Next, IFNγ-knockout mice with AKI received pFUS+MSC, but MSC-derived IL-10 expression and AKI were similar to using MSC alone. AKI in wild-type mice receiving pFUS and IL-10-deficient MSC was also unimproved compared to administering IL-10-deficient MSC alone. Indoleamine 2,3-dioxygenase (IDO), an anti-inflammatory enzyme up-regulated in MSC by IFNγ, was up-regulated during AKI, but was not further elevated in MSC from pFUS-treated kidneys, suggesting that IDO is not involved in improved AKI healing by pFUS+MSC. These data suggest IFNγ is up-regulated by pFUS and after i.v.-infused MSC home to pFUS-treated kidneys, IFNγ stimulates additional IL-10 production by MSC to improve AKI. Analogous mechanisms of ultrasound-treated tissue microenvironments stimulating therapeutic MSC may exist in other pathologies where adjuvant ultrasound techniques are successful.