Endothelial dysfunction is dampened by early administration of fresh frozen plasma in a rodent burn shock model.

BACKGROUND: Endothelial dysfunction has been implicated in the pathogenesis of burn shock affecting patients with large thermal injury. In response to injury, glycocalyx components like Syndecan-1 (SDC-1) are shed into circulation and have been used as markers of endothelial damage. Previous work in our lab has shown plasma inclusive resuscitation (PIR) with fresh frozen plasma (FFP) ameliorates endothelial damage. However, there remains a paucity of information regarding optimal timing and dosing of PIR as well as organ specific endothelial responses to shock. We aimed to examine the impact of PIR on endothelial dysfunction using clinically-translatable timing and dosing. METHODS: Spraque-Dawley rats were used to create thermal burns. Rats were subjected to 40% TBSA scald burns and were resuscitated with LR only, LR + albumin and LR + early 1 ml boluses of FFP at 0,2,4 and 8 hours post-injury. A late group also received LR + FFP starting at hour 10 post-injury. SDC-1 levels were quantified by ELISA and qRT-PCR analysis characterized transcription of glycocalyx components and inflammatory cytokines in the lung and spleen. Evan's blue dye (EBD) was used to quantify amount of vascular leakage. RESULTS: LR + early FFP reduced EBD extravasation when compared to LR only groups while late FFP did not. When comparing LR only vs LR + early FFP, SDC-1 levels were reduced in the LR + early FFP group at hour 8, 12 and 24 (5.23 vs. 2.07, p < 0.001, 4.49 vs. 2.05, p < 0.01, 3.82 vs 2.08, p < 0.05, respectively). LR only groups had upregulation of Exostosin-1 (EXT-1) and SDC-1 in the lung compared to LR + early FFP groups (p < 0.01 and p < 0.05) as well as upregulation of cytokines IL-10 and IFN-Ƴ (p < 0.001 and p < 0.001). CONCLUSIONS: Early administration of LR + FFP reduces the magnitude of SDC-1 shedding and dampens the cytokine response to injury. The upregulation of glycocalyx components as a response to endothelial injury is also decreased in the lung and spleen by LR + early FFP administration.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1ß appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Identification of prognostic biomarkers for antibiotic associated nephrotoxicity in cystic fibrosis.

BACKGROUND: Our objective was to discover novel urinary biomarkers of antibiotic-associated nephrotoxicity using an ex-vivo human microphysiological system (MPS) and to translate these findings to a prospectively enrolled cystic fibrosis (CF) population receiving aminoglycosides and/or polymyxin E (colistin) for a pulmonary exacerbation. METHODS: We populated the MPS with primary human kidney proximal tubule epithelial cells (PTECs) from three donors and modeled nephrotoxin injury through exposure to 50 µg/mL polymyxin E for 72 h. We analyzed gene transcriptional responses by RNAseq and tested MPS effluents. We translated candidate biomarkers to a CF cohort via analysis of urine collected prior to, during and two weeks after antibiotics and patients were followed for a median of 3 years after antibiotic use. RESULTS: Polymyxin E treatment resulted in a statistically significant increase in the pro-apoptotic Fas gene relative to control in RNAseq of MPS: fold-change = 1.63, FDR q-value = 7.29 × 10-5. Effluent analysis demonstrated an acute rise of soluble Fas (sFas) concentrations that correlated with cellular injury. In 16 patients with CF, urinary sFas concentrations were significantly elevated during antibiotic treatment, regardless of development of AKI. Over a median of three years of follow up, we identified seven cases of incident chronic kidney disease (CKD). Urinary sFas concentrations during antibiotic treatment were significantly associated with subsequent development of incident CKD (unadjusted relative risk = 2.02 per doubling of urinary sFas, 95 % CI = 1.40, 2.90, p < 0.001). CONCLUSIONS: Using an ex-vivo MPS, we identified a novel biomarker of proximal tubule epithelial cell injury, sFas, and translated these findings to a clinical cohort of patients with CF.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Endothelial damage occurs early after inhalation injury as measured by increased syndecan-1 levels.

Inhalation injury is a significant cause of morbidity and mortality in the burn patient population. However, the pathogenesis of inhalation injury and its potential involvement in burn shock is not well understood. Preclinical studies have shown endothelial injury, as measured by syndecan-1 (SDC-1) levels, to be involved in the increased vascular permeability seen in shock states. Furthermore, the lung has been identified as a site of significant SDC-1 shedding. Here we aim to characterize the contribution of endotheliopathy caused by inhalation alone in a swine model. When comparing injured animals, the fold change of circulating SDC-1 levels from preinjury was significantly higher at 2, 4, and 6 hours postinjury (P = .0045, P = .0017, and P < .001, respectively). When comparing control animals, the fold change of SDC-1 from preinjury was not significant at any timepoint. When comparing injured animals versus controls, the fold change of SDC-1 injured animals was significantly greater at 2, 4, 6, and 18 hours (P = .004, P = .03, P < .001, and P = .03, respectively). Histological sections showed higher lung injury severity compared to control uninjured lungs (0.56 vs 0.38, P < .001). This novel animal model shows significant increases in SDC-1 levels that provide evidence for the connection between smoke inhalation injury and endothelial injury. Further understanding of the mechanisms underlying inhalation injury and its contribution to shock physiology may aid in development of early, more targeted therapies.

Double trouble: a case of bilateral multiple epidermal inclusion cysts after reduction mammaplasty.

Epidermal inclusion cysts (EIC) of the breast are uncommon and can occur after previous surgery or trauma. Here we discuss a case of massive bilateral multiple EIC of the breast presenting seven years after reduction mammaplasty. This report highlights the importance of accurate diagnosis and management of this rare condition.

Feasibility and Acceptance of a Teletrauma Surgery Consult Service to Rural and Community Hospitals: A Pilot Study.

BACKGROUND: Many trauma patients currently transferred from rural and community hospitals (RCH) to Level I trauma centers (LITC) for trauma surgery evaluation may instead be appropriate for immediate discharge or admission to the local facility after evaluation by a trauma and acute care surgery (TACS) surgeon. Unnecessary use of resources occurs with current practice. We aimed to demonstrate the feasibility and acceptance of a teletrauma surgery consultation service between LITC and RCH. STUDY DESIGN: LITC TACS surgeons provided telehealth consults on trauma patients from 3 local RCHs. After consultation, appropriate patients were transferred to LITC; selected patients remained at or were discharged from RCH. Participating TACS surgeons and RCH physicians were surveyed. RESULTS: A total of 28 patients met inclusion criteria during the 5-month pilot phase, with 7 excluded due to workflow issues. The mean ± SD age was 63 ± 17 years. Of 21 patients, 7 had intracranial hemorrhage; 12 had rib fractures. The mean ± SD Injury Severity Score was 8.1 ± 4.0). A total of 6 patients were discharged from RCH, 4 admitted to RCH hospitalist service, 2 transferred to a LITC emergency room, and 9 transferred to LITC as direct admission. There was one 30-day readmission and no missed injuries or complications, or deaths. RCH providers were highly satisfied with the teletrauma surgery consultation service, TACS surgeons, and equipment used. Mental demand and effort of consulting TACS surgeons decreased significantly as the consult number increased. CONCLUSIONS: Teletrauma surgery consultation involving 3 RCH within our system is feasible and acceptable. A total of 10 transfers and 19 emergency department visits were avoided. There was favorable acceptance by RCH providers and TACS surgeons.

Optimal Timing of Cholecystectomy for Acute Cholecystitis: A Retrospective Cohort Study.

Background Laparoscopic cholecystectomy performed less than 72 hours from hospital admission for acute cholecystitis has shown to decrease hospital cost without an increase in length of stay (LOS). Very few studies have examined clinical and cost outcomes of performing cholecystectomy less than 24 hours from hospital admission. The aim of this study was to examine the cost and LOS of laparoscopic cholecystectomy performed on an early (less than 24 hours from admission) and late (more than 24 hours from hospital admission) basis. Methods We performed a retrospective observational study of 569 patients at Baystate Medical Center, Springfield, USA, who underwent urgent laparoscopic cholecystectomy for acute cholecystitis between January 1, 2018 and February 28, 2020. We evaluated preoperative/postoperative LOS, operative duration, hospital cost, and patient complications. Results 468 patients underwent urgent laparoscopic cholecystectomy for acute cholecystitis during our study period. Early cholecystectomy (less than 24 hours from admission) had an overall decreased LOS (43.6 hours versus 102.9 hours, p-value < 0.01) and decreased hospital cost ($23,736.70 versus $30,176.40, p-value < 0.01) compared to late cholecystectomy (more than 24 hours from admission). There was also a significantly higher rate of bile leak in patients who underwent surgery more than 24 hours from hospital admission compared to those who had surgery less than 24 hours from admission (5.9% versus 0.4%, p-value < 0.01). Additionally, those procedures performed greater than 24 hours from hospital admission were significantly more likely to be converted to an open procedure (6.9% versus 2.2%, p-value = 0.02).  Conclusion Urgent laparoscopic cholecystectomy performed within 24 hours of hospital admission for acute cholecystitis decreased hospital cost, LOS, and operative complications in our institution's patient population. Our data suggests that performing laparoscopic cholecystectomy within 24 hours of hospital admission would be beneficial from a patient and hospital standpoint.

Prophylactic Splenectomy and Hyposplenism in Spaceflight.

BACKGROUND: There is debate whether astronauts traveling to space should undergo a prophylactic splenectomy prior to long duration spaceflight. Risks to the spleen during flight include radiation and trauma. However, splenectomy also carries significant risks.METHODS: Systematic review of data published over the past 5 decades regarding risks associated with splenectomies and risks associated with irradiation to the spleen from long duration spaceflight were analyzed. A total of 41 articles were reviewed.RESULTS: Acute risks of splenectomy include intraoperative mortality rate (from hemorrhage) of 3-5%, mortality rate from postoperative complications of 6%, thromboembolic event rate of 10%, and portal vein thrombosis rate of 5-37%. Delayed risks of splenectomy include overwhelming postsplenectomy infection (OPSI) at 0.5% at 5 yr post splenectomy, mortality rate as high as 60% for pneumococcal infections, and development of malignancy with relative risk of 1.53. The risk of hematologic malignancy increases significantly when individuals reach 40 Gy of exposure, much higher than the 0.6 Gy of radiation experienced from a 12-mo round trip to Mars. Lower doses of radiation increase the risk of hyposplenism more so than hematologic malignancy.CONCLUSION:For protection against hematologic malignancy, the benefits of prophylactic splenectomy do not outweigh the risks. However, there is a possible risk of hyposplenism from long duration spaceflight. It would be beneficial to prophylactically provide vaccines against encapsulated organisms for long duration spaceflight to mitigate the risk of hyposplenism.Siu M, Levin D, Christiansen R, Kelly E, Alouidor R, Kamine TH. Prophylactic splenectomy and hyposplenism in spaceflight. Aerosp Med Hum Perform. 2022; 93(12):877-881.

Skin deep: Cutaneous manifestation of PIP implant rupture.

PIP (Poly Implant Prothèse, France) implants were readily employed for breast reconstruction until withdrawn from the market in 2010. These implants have an early and increased risk of rupture compared to non-PIP implants. This report outlines a significant cutaneous manifestation of PIP-implant rupture not previously described in the literature. This patient developed significant cutaneous xanthomatous inflammation with sinus tract formation that has yet to resolve despite explantation. Further investigation is warranted to elucidate the aetiology of this clinical sign and the optimal management of the cutaneous manifestation.

Pharmacokinetics, metabolism and off-target effects in the rat of 8-[(1H- benzotriazol-1-yl)amino]octanoic acid, a selective inhibitor of human cytochrome P450 4Z1: ß-oxidation as a potential augmenting pathway for inhibition.

8-[(1H-1,2,3-benzotriazol-1-yl)amino]octanoic acid (8-BOA) was recently identified as a selective and potent mechanism-based inactivator (MBI) of breast cancer-associated CYP4Z1 and exhibited favourable inhibitory activity in vitro, thus meriting in vivo characterization.The pharmacokinetics and metabolism of 8-BOA in rats was examined after a single IV bolus dose of 10 mg/kg. A biphasic time-concentration profile resulted in relatively low clearance and a prolonged elimination half-life.The major circulating metabolites identified in plasma were products of ß-oxidation; congeners losing two and four methylene groups accounted for >50% of metabolites by peak area. The -(CH2)2 product was characterized previously as a CYP4Z1 MBI and so represents an active metabolite that may contribute to the desired pharmacological effect.Ex vivo analysis of total CYP content in rat liver and kidney microsomes showed that off-target CYP inactivation was minimal; liver microsomal probe substrate metabolism also demonstrated low off-target inactivation. Standard clinical chemistries provided no indication of acute toxicity.In silico simulations using the free concentration of 8-BOA in plasma suggested that the in vivo dose used here may effectively inactivate CYP4Z1 in a xenografted tumour.

Microphysiological system modeling of ochratoxin A-associated nephrotoxicity.

Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic, and immunotoxic properties. Specifically, a major concern is severe nephrotoxicity, which is characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, the mechanism of OTA toxicity, as well as the genetic risk factors contributing to its toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate human kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern the renal disposition of OTA, and determine the role of metabolism in the bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that LC50 values of OTA in kidney MPS culture (0.375-1.21 µM) were in agreement with clinically relevant toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluent of the kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining. Instead, these biomarkers decreased in an OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), pan-inhibitors of P450 and glutathione S-transferase (GST) enzymes, respectively, on OTA-induced toxicity in kidney MPS was examined. These studies revealed significant enhancement of OTA-induced toxicity by NBDHEX (3 µM) treatment, whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting roles for GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Analysis of transcriptional changes using RNA-sequencing of kidney MPS treated with different concentrations of OTA revealed downregulation of several nuclear factor (erythroid derived-2)-like 2 (NRF2)-regulated genes by OTA treatment, including GSTs. The transcriptional repression of GSTs is likely playing a key role in OTA toxicity via attenuation of glutathione conjugation/detoxification. The sequential molecular events may explain the mechanism of toxicity associated with OTA. Additionally, OTA transport studies using kidney MPS in the presence and absence of probenecid (1 mM) suggested a role for organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a clearer understanding of the mechanism of OTA-induced kidney injury, which may support changes in risk assessment, regulatory agency policies on allowable exposure levels, and determination of the role of genetic factors in populations at risk for OTA nephrotoxicity.

Outcomes of a low-osmolar water-soluble contrast pathway in small bowel obstruction.

BACKGROUND: Adhesive small-bowel obstruction (SBO) is a common surgical condition accounting for a significant proportion of acute surgical admissions and surgeries. The implementation of a high-osmolar water-soluble contrast challenge has repeatedly been shown to reduce hospital length of stay and possibly the need for surgery in SBO patients. The effect of low-osmolar water-soluble contrast challenge however, is unclear. The aim of this study is to evaluate the outcomes of an SBO pathway including a low-osmolar water-soluble contrast challenge. METHODS: A prospective cohort of patients admitted for SBO were placed on an evidence-based SBO pathway including low-osmolar water-soluble contrast between January 2017 and October 2018 and were compared with a historical cohort of patients prior to the implementation of the pathway from September 2013 through December 2014. The primary outcome was length of stay less than 4 days with a secondary outcome of failure of nonoperative management. RESULTS: There were 140 patients enrolled in the SBO pathway during the study period and 101 historic controls. The SBO pathway was independently associated with a length of stay less than 4 days (odds ratio, 1.76; 95% confidence interval, 1.03-3.00). Median length of stay for patients that were successfully managed nonoperatively was lower in the SBO pathway cohort compared with controls (3 days vs. 4 days, p = 0.04). Rates of readmission, surgery, and bowel resection were not significantly different between the two cohorts. CONCLUSION: Implementation of an SBO pathway using a low-osmolarity contrast is associated with decreased hospital length of stay. Rates of readmission, surgery, and need for bowel resection for those undergoing surgery were unchanged. An SBO pathway utilizing low-osmolarity water-soluble contrast is safe and effective in reducing length of stay in the nonoperative management of adhesive small-bowel obstructions. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Emergency general surgery procedures in hematopoietic stem cell transplant recipients.

BACKGROUND: Outcomes of emergency general surgery (EGS) procedures on hematopoietic stem cell transplant (HST) recipients have not been defined in a large, national database. Whether EGS during HST engraftment admission, or in HST patients with graft versus host disease (GVHD) results in worse outcomes is unknown. METHODS: The National Inpatient Sample (NIS) was examined for patients with a history of BMT between 2001 and 2014. RESULTS: There were 520,000 HST admissions meeting inclusion criteria, of which, 14,143 (2.7%) required EGS. Of those requiring EGS, 378 (2.7%) were during engraftment admission and 13,765 (97.3%) on subsequent admission. For those requiring EGS during subsequent admission, 9,920 (72.1%) had a history of GVHD and 3,845 (27.9%) did not. On multivariate analysis, requirement of EGS was associated with mortality (OR: 1.71, 95%CI: 1.47-1.99, p < 0.001). For patients requiring EGS, engraftment admission or GVHD was not associated with mortality. CONCLUSIONS: While EGS results in worse survival for the HST population, patients in their engraftment admission do not appear to be at increased mortality risk. In addition, GVHD does not worsen survival.

Reconstructing the Human Renal Vascular-Tubular Unit In Vitro.

Engineered human kidney-on-a-chip platforms show tremendous promise for disease modeling and drug screening. Outstanding challenges exist, however, in reconstructing the complex architecture, cellular make-up, and matrix composition necessary for the proper modeling of kidney function. Herein, the first fully tunable human kidney-on-a-chip platform is reported that allows the reconstruction of the native architecture of the renal endothelial-epithelial exchange interface using entirely cell-remodelable matrix and patient-derived kidney cells. This platform consists of a double-layer human renal vascular-tubular unit (hRVTU) enabled by a thin collagen membrane that replicates the kidney exchange interface. It is shown that endothelial and epithelial cells lining their respective lumens remodel the membrane in culture into a ≈1 µm thick exchange interface composed of native basement membrane proteins. This interface displays sufficient mechanical integrity for media flow and blood perfusion. As a proof of principle, it is demonstrated that the hRVTU performs kidney-specific functions including reabsorption of albumin and glucose from the epithelial channel. By incorporating multiple cell populations from single donors, it is demonstrated that the hRVTU may have utility for future precision medicine applications. The success of the system provides new opportunities for the next generation of organ-on-a-chip models.

A High-Throughput Screen Identifies DYRK1A Inhibitor ID-8 that Stimulates Human Kidney Tubular Epithelial Cell Proliferation.

BACKGROUND: The death of epithelial cells in the proximal tubules is thought to be the primary cause of AKI, but epithelial cells that survive kidney injury have a remarkable ability to proliferate. Because proximal tubular epithelial cells play a predominant role in kidney regeneration after damage, a potential approach to treat AKI is to discover regenerative therapeutics capable of stimulating proliferation of these cells. METHODS: We conducted a high-throughput phenotypic screen using 1902 biologically active compounds to identify new molecules that promote proliferation of primary human proximal tubular epithelial cells in vitro. RESULTS: The primary screen identified 129 compounds that stimulated tubular epithelial cell proliferation. A secondary screen against these compounds over a range of four doses confirmed that eight resulted in a significant increase in cell number and incorporation of the modified thymidine analog EdU (indicating actively proliferating cells), compared with control conditions. These eight compounds also stimulated tubular cell proliferation in vitro after damage induced by hypoxia, cadmium chloride, cyclosporin A, or polymyxin B. ID-8, an inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), was the top candidate identified as having a robust proproliferative effect in two-dimensional culture models as well as a microphysiologic, three-dimensional cell culture system. Target engagement and genetic knockdown studies and RNA sequencing confirmed binding of ID-8 to DYRK1A and upregulation of cyclins and other cell cycle regulators, leading to epithelial cell proliferation. CONCLUSIONS: We have identified a potential first-in-class compound that stimulates human kidney tubular epithelial cell proliferation after acute damage in vitro.

Risk factors for prolonged need for percutaneous endoscopic gastrostomy (PEG) tubes in adult trauma patients: Experience of a level 1 trauma.

BACKGROUND: Percutaneous endoscopic gastrostomy tubes are a means of providing an alternative enteric route of nutrition. This study sought to identify risk factors for the prolonged need of a percutaneous endoscopic gastronomy tube (≥90 days) in adult trauma patients. METHODS: The trauma database of a level 1 trauma center was queried retrospectively to identify patients who had percutaneous endoscopic gastronomy tubes placed. RESULTS: A total of 9,772 charts were reviewed with 282 patients (2.9%) undergoing successful percutaneous endoscopic gastronomy tube placement. On review of discharged living patients, 195 had adequate clinical documentation to allow for analysis. The mean age was 57.5 years, admission serum albumin was 3.7 g/dL, and Charlson Comorbidity Index score was 1.1. The first recorded mean Glasgow Coma Scale was 10.7, and their Injury Severity Score was 23.2. The mean duration of total hospital stay was 23.8 days, intensive care unit stay was 16.5 days, and in-hospital ventilator days was 11.5. Of the 272 patients, 77 (41.4%) required percutaneous endoscopic gastronomy tubes for >90 days. Statistically significant characteristics on univariate analysis included increasing age, a greater Charlson Comorbidity Index score, and a greater number of in-hospital ventilator days. On logistic regression, a Charlson Comorbidity Index score >1 (odds ratio 1.27, 95% confidence interval 1.03-1.56, P = .02) and greater in-hospital ventilator days (odds ratio 1.05, 95% confidence interval 1.02-1.09, P < .01) were predictive of the need for prolonged percutaneous endoscopic gastronomy tube placement. CONCLUSION: A Charlson Comorbidity Index score >1 and prolonged in-hospital ventilator days were risk factors for the necessity of a percutaneous endoscopic gastronomy tube for ≥90 days after placement. This observation may assist patients/surrogates in decision-making when needing alternative routes for nutrition.

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysio­logical systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, n = 8) and adjacent noncancerous (NC, n = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, n = 36) and HCV-cirrhotic (n = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues (n = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity.

Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.