Sepsis-associated acute kidney injury: recent advances in enrichment strategies, sub-phenotyping and clinical trials.

Acute kidney injury (AKI) often complicates sepsis and is associated with high morbidity and mortality. In recent years, several important clinical trials have improved our understanding of sepsis-associated AKI (SA-AKI) and impacted clinical care. Advances in sub-phenotyping of sepsis and AKI and clinical trial design offer unprecedented opportunities to fill gaps in knowledge and generate better evidence for improving the outcome of critically ill patients with SA-AKI. In this manuscript, we review the recent literature of clinical trials in sepsis with focus on studies that explore SA-AKI as a primary or secondary outcome. We discuss lessons learned and potential opportunities to improve the design of clinical trials and generate actionable evidence in future research. We specifically discuss the role of enrichment strategies to target populations that are most likely to derive benefit and the importance of patient-centered clinical trial endpoints and appropriate trial designs with the aim to provide guidance in designing future trials.

Overcoming barriers in the design and implementation of clinical trials for acute kidney injury: a report from the 2020 Kidney Disease Clinical Trialists meeting.

Acute kidney injury (AKI) is a growing epidemic and is independently associated with increased risk of death, chronic kidney disease (CKD) and cardiovascular events. Randomized-controlled trials (RCTs) in this domain are notoriously challenging and many clinical studies in AKI have yielded inconclusive findings. Underlying this conundrum is the inherent heterogeneity of AKI in its etiology, presentation and course. AKI is best understood as a syndrome and identification of AKI subphenotypes is needed to elucidate the disease's myriad etiologies and to tailor effective prevention and treatment strategies. Conventional RCTs are logistically cumbersome and often feature highly selected patient populations that limit external generalizability and thus alternative trial designs should be considered when appropriate. In this narrative review of recent developments in AKI trials based on the Kidney Disease Clinical Trialists (KDCT) 2020 meeting, we discuss barriers to and strategies for improved design and implementation of clinical trials for AKI patients, including predictive and prognostic enrichment techniques, the use of pragmatic trials and adaptive trials.

Optimizing the Design and Analysis of Future AKI Trials.

AKI is a complex clinical syndrome associated with an increased risk of morbidity and mortality, particularly in critically ill and perioperative patient populations. Most AKI clinical trials have been inconclusive, failing to detect clinically important treatment effects at predetermined statistical thresholds. Heterogeneity in the pathobiology, etiology, presentation, and clinical course of AKI remains a key challenge in successfully testing new approaches for AKI prevention and treatment. This article, derived from the "AKI" session of the "Kidney Disease Clinical Trialists" virtual workshop held in October 2021, reviews barriers to and strategies for improving the design and implementation of clinical trials in patients with, or at risk of, developing AKI. The novel approaches to trial design included in this review span adaptive trial designs that increase the knowledge gained from each trial participant; pragmatic trial designs that allow for the efficient enrollment of sufficiently large numbers of patients to detect small, but clinically significant, treatment effects; and platform trial designs that use one trial infrastructure to answer multiple clinical questions simultaneously. This review also covers novel approaches to clinical trial analysis, such as Bayesian analysis and assessing heterogeneity in the response to therapies among trial participants. We also propose a road map and actionable recommendations to facilitate the adoption of the reviewed approaches. We hope that the resulting road map will help guide future clinical trial planning, maximize learning from AKI trials, and reduce the risk of missing important signals of benefit (or harm) from trial interventions.

Subcellular expression of CYP2E1 in HepG2 cells impacts response to free oleic and palmitic acid.

Aims: Cytochrome P450 2E1 (CYP2E1) is a mammalian monooxygenase expressed at high levels in the liver that metabolizes low molecular weight pollutants and drugs, as well as endogenous fatty acids and ketones. Although CYP2E1 has been mainly studied in the endoplasmic reticulum (ER, microsomal fraction), it also localizes in significant amounts to the mitochondria, where it has been far less studied. We investigated the effects of CYP2E1 expression in mitochondria, endoplasmic reticulum, or both organelles in transgenic HepG2 cells exposed to free oleic and palmitic acid, including effects on cytotoxicity, lipid storage, respiration, and gene expression. Results: We found that HepG2 cells expressing CYP2E1 in both the ER and mitochondria have exacerbated levels of palmitic acid cytotoxicity and inhibited respiration. CYP2E1 expression did not impact lipid accumulation from fatty acid exposures, but mitochondrial CYP2E1 expression promoted lipid droplet depletion during serum starvation. In contrast to HepG2 cells, differentiated HepaRG cells express abundant CYP2E1, but they are not sensitive to palmitic acid cytotoxicity. Oleic acid exposure prompted less cytotoxicity, and CYP2E1 expression in the ER prevented an oleic-acid-induced increase in respiration. HepG2 cells exposed to mixtures of palmitic and oleic acid are protected from palmitic acid cytotoxicity. Additionally, we identified that CYP2E1 was decreased at the gene and protein level in hepatocellular carcinoma. Moreover, patients with tumors that had higher CYP2E1 expression had a better prognosis compared to patients with lower CYP2E1 expression. Innovation: This study has demonstrated that transgenic CYP2E1 subcellular localization plays an important role in sensitivity to cytotoxicity, lipid storage, and respiration in the hepatoma cell line HepG2 exposed to palmitic and oleic acid. HepaRG cells, in contrast, were insensitive to palmitic acid. This work demonstrates the clear importance of CYP2E1 in dictating lipotoxicity and differential roles for the mitochondrial and ER forms of the enzyme. Additionally, our data supports a potentially unique role for CYP2E1 in cancer cells. Conclusion: There lies a role for CYP2E1 in altering lipotoxicity, and since CYP2E1 is known to be upregulated in both liver disease and hepatocellular carcinoma, it is important to better define how the role of CYP2E1 changes during disease progression.

Phenotypic anchoring of arsenic and cadmium toxicity in three hepatic-related cell systems reveals compound- and cell-specific selective up-regulation of stress protein expression: implications for fingerprint profiling of cytotoxicity.

Exposure of cells to toxic chemicals is known to up-regulate the expression of a number of stress proteins (SPs), including metallothionein (MT) and members of the heat shock protein (HSP) family, and this response may allow the development of a fingerprint profile to identify mechanisms of toxicity in an in vitro toxicology setting. To test this hypothesis, three hepatic-derived cell culture systems (rat hepatoma FGC4 cell line, rat hepatocytes, human hepatoma HepG2 cell line) were exposed to cadmium (as CdCl2) and arsenic (as NaAsO2), two compounds believed to exert their toxicity through an oxidative stress mechanism, under conditions of phenotypic anchoring defined as minimal and mild toxicity (approximately 5 and 25% reduction in neutral red uptake, respectively). The expression of six SPs--MT, HSP25/27, HSP40, HSP60, HSP70, and HSP90--was then determined by ELISA. Expression of four of these SPs--MT, HSP25/27, HSP40 and HSP70--was up-regulated in at least one experimental condition. However, the patterns of expression of these four SPs varied across the experimental conditions, according to differences in toxicant concentration and/or level of toxicity, cell-type and toxicant itself. This lack of uniformity in response of a focussed set of mechanistically defensible targets suggests that similar problems may emerge when using more global approaches based on genomics and proteomics, in which problems of redundancy in targets and uncertain mechanistic relevance will be greater.

CTL-promoting effects of CD40 stimulation outweigh B cell-stimulatory effects resulting in B cell elimination and disease improvement in a murine model of lupus.

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA-->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.

Boswellia carterii extract inhibits TH1 cytokines and promotes TH2 cytokines in vitro.

Traditional herbal formulas used to treat inflammatory arthritis in China and India include Boswellia carterii or Boswellia serrata. They both contain boswellic acids (BAs) which have been shown to exhibit anti-inflammatory and antiarthritic properties. This study tests the hypothesis that mixtures of BAs derived from B. carterii have immunomodulatory properties. B. carterii plant resin obtained from China was prepared as an ethanol extract, and the presence of seven BAs was confirmed by column chromatography, high-performance liquid chromatography, and UV laser desorption/ionization tandem mass spectroscopy. The extract was then tested for its ability to alter in vitro production of TH1 cytokines (interleukin-2 [IL-2] and gamma interferon) and TH2 cytokines (IL-4 and IL-10) by murine splenocytes. Delivery of the resin extract using ethanol as a solvent resulted in significant cellular toxicity not seen with the addition of ethanol alone. By contrast, delivery of the resin extract using a sesame oil solvent resulted in a dose-dependent inhibition of TH1 cytokines coupled with a dose-dependent potentiation of TH2 cytokines. These results indicate that a purified mixture of BAs from B. carterii plant resin exhibits carrier-dependent immunomodulatory properties in vitro.