Acute kidney injury (AKI) is a systemic catabolic condition that affects multiple organs. Clinically, AKI has been associated with muscle wasting, weakness, and delayed functional recovery. The underlying biochemical mechanisms driving these changes are not well understood. Ischemic AKI was induced in 8-10-week-old male C57BL/6 mice. Untargeted metabolomics on gastrocnemius samples were performed via ultra-high performance liquid chromatography-mass spectrometry at 24 h and 72 h after AKI. Of the 175 annotated analytes identified, 72 were significantly affected at 24 h with the majority of these metabolites being depleted in AKI compared to controls. Key depleted metabolites included multiple amino acids, glutathione and its precursors, and other energy-related substrates. Integration with our previously published metabolomics data in the kidney, liver, heart, and plasma highlights shared metabolic pathways across these organs, particularly reflected in arginine metabolism and the urea cycle, alanine/aspartate/glutamate metabolism, and glutathione/redox balance. This is the first study to characterize the metabolic profile of skeletal muscle after ischemic AKI in a murine model. Our data deepen the understanding of AKI as a systemic metabolic disease, underscoring the need to further understand the effects of AKI on skeletal muscle and opening potential avenues for therapeutic strategies to improve outcomes after AKI.

Prerenal azotemia (PRA) accounts for 10%-30% of hospitalized cases of acute kidney injury (AKI). In contrast to AKI from acute tubular injury (ATI), PRA is considered a benign condition, despite evidence that it is associated with adverse clinical consequences. In this study, we used untargeted metabolomics to study the potential systemic consequences of PRA as compared with ATI. We hypothesized that the PRA and ATI plasma metabolomes would share certain features, since both are marked by a decline in glomerular filtration rate (GFR), but that a unique PRA signature would be identified. Wild-type male C57BL/6 mice were used in established models of ischemic acute kidney injury (iAKI) or PRA induced by intraperitoneal furosemide followed by GFR restoration with normal saline resuscitation. Plasma from iAKI and PRA cohorts were analyzed by mass spectrometry metabolomics. The PRA plasma metabolome was defined by 40 significantly changed metabolites, including 7 uremic toxins, with significant enrichment of tryptophan and fatty acid oxidation metabolites. When comparing models, the PRA and iAKI metabolomes overlap at the individual metabolite level (50% of the PRA metabolome represented in the iAKI metabolome) and share three significantly enriched pathways involved in energy substrate and amino acid metabolism. Further analysis identified cystathionine, kynurenine, and indolepyruvate as possible PRA plasma biomarkers. Our results demonstrate that PRA causes systemic metabolic changes, some of which mirror changes found in ATI. Our results challenge the notion that PRA is inconsequential, and the metabolic pathways and uremic toxins identified herein warrant further study.NEW & NOTEWORTHY We completed the first untargeted plasma metabolomics analysis in a murine model of prerenal azotemia (PRA) and demonstrate that PRA has systemic consequences with numerous significantly altered metabolites and pathways. By comparing the metabolite signature to that of acute tubular injury (ATI), novel insights into the potential adverse consequences of PRA and potential methods to distinguish PRA from ATI were identified.

KEY POINTS:We identify a novel consequence of continuous KRT: clearance of thyroid-stimulating hormone and thyroid hormone. Severe non-thyroidal illness syndrome occurs in most of the continuous KRT patients and is persistent during continuous KRT. Notably, no patient achieved euthyroid status during continuous KRT therapy. BACKGROUND:AKI and ESKD requiring continuous KRT (CKRT) are associated with a high mortality rate. Solutes up to 40 kDa in size are amenable to CKRT clearance which includes thyroid-stimulating hormone (TSH; 28 kDa), free thyroxine (fT4; 0.78 kDa), and free triiodothyronine (fT3; 0.68 kDa). The effect of CKRT on TSH and thyroid hormone clearance, thyroid function, and the hypothalamic-pituitary-thyroid axis is unknown. METHODS:This prospective, single-center observational study enrolled 50 intensive care unit patients requiring CKRT and 50 control intensive care unit patients. Serum TSH, fT4, fT3, and reverse triiodothyronine were measured before and on days 1, 3, 8, and 14 after CKRT initiation and at the same time points relative to enrollment in control patients; effluent TSH, fT4, and fT3 were measured on days 1, 3, 8, and 14. Thyroid function status was adjudicated on days 1, 3, 8, and 14. Statistical analyses included employment of linear mixed modeling and time-dependent adjustments, and ANOVA with false discovery rate correction. RESULTS:Before and during CKRT, CKRT patients had lower fT4 and fT3 levels compared with controls, with a higher proportion of values below the normal reference range. TSH and fT4 were detected in the CKRT effluent, indicating clearance by CKRT. Severe and persistent non-thyroidal illness syndrome (NTIS) was seen in the CKRT cohort. No patient achieved euthyroid status while receiving CKRT. CONCLUSIONS:This is the first study to assess thyroid function and clearance of TSH and thyroid hormones during CKRT. We demonstrate that severe NTIS-characterized by low levels of fT4 and fT3-occurs in most of the CKRT patients. Furthermore, NTIS is persistent in patients receiving CKRT, which may be influenced by CKRT-mediated TSH and fT4 clearance. These novel complications may contribute to the high mortality rate observed in patients with either AKI or ESKD who receive CKRT.

Rationale & Objective Uremia is an indication for continuous kidney replacement therapy (CKRT) in patients with acute kidney injury (AKI). Existing studies of uremic toxins in AKI have several limitations, including modest cohort sizes and small numbers of uremic toxins assessed per study. We aimed to better understand the uremic milieu and its association with mortality in critically ill patients with AKI before and during CKRT. Study Design Single-center prospective observational study Setting & Participants We evaluated 21 uremic toxins in 89 critically ill adults with AKI enrolled before and during the first 3 days of CKRT and in 26 controls without kidney disease. Outcome(s) Thirty-day mortality. Analytical Approach The primary outcome was 30-day mortality which was assessed using logistic regression modeling of each individual uremic toxin before CKRT and development of a uremic toxin (UT) score that accounted for (1) all 21 uremic toxins, (2) levels before and during the first 3 days of CKRT, and (3) directionality (ie, adjustment for levels that were either higher or lower in nonsurvivors). Results Thirty-day mortality was 52%. In AKI before CKRT, 15 uremic toxins were increased, and 6 were similar or lower in AKI as compared with controls and 20 of the 21 uremic toxins were not associated with mortality. Before and during the first 3 days of CKRT we observed that 4 uremic toxins were significantly lower in nonsurvivors. Accounting for this, our UT score could differentiate survivors and nonsurvivors. Limitations Single center and lack of a validation cohort. Conclusions Uremic toxins should not be assumed to be elevated in AKI, and elevated levels are not universally associated with mortality. These data are in line with the growing appreciation that therapies beyond kidney replacement therapy will be necessary to improve outcomes in AKI. Plain-Language Summary Uremic toxins are harmful plasma solutes that accumulate in kidney failure and cause harm. We measured 21 plasma uremic toxins in critically ill patients with AKI for 4 days: just before and during 3 days of continuous kidney replacement therapy (CKRT). Before CKRT, not all uremic toxins were elevated compared with patients without kidney failure. During CKRT, 4 uremic toxins were lower in patients that died at 30 days. We developed a uremic toxin score to predict mortality at 30 days that accounted for (1) all 21 uremic toxin levels, (2) all 4 days, and (3) directionality (whether increased or decreased in survivors versus nonsurvivors). The UT score was able to distinguish 30-day survivors from nonsurvivors on all days.

Rationale: Pre-renal azotemia (PRA), defined as transient kidney dysfunction due to reduced kidney perfusion, is a common cause of acute kidney injury (AKI). PRA is associated with an increased risk of intensive care unit admission and need for mechanical ventilation, but mechanistic links underpinning these associations are unknown. Our PRA mouse model is characterized by increased plasma IL-6 and IL-6 dependent activation of the hepatic acute phase response. Our lab and others have shown that other experimental AKI mouse models demonstrate evidence of lung inflammation. It is unknown whether PRA causes lung inflammation. Methods: Baseline kidney function was assessed by transcutaneous glomerular filtration rate (tGFR) (MediBeacon) measurements in our C57BL/6J wildtype mice. Mice then received either 4 mg intraperitoneal (IP) furosemide injection (PRA) or 150 uL IP normal saline (Veh) injection at 0 and 3 hours and then had tGFR measured at 6 hours. Bronchoalveolar lavage fluid (BALF) was collected at 6 hours to assess total protein, flow cytometry (CD45, Siglec-F, and Ly6G), or IL-6 and CXCL1 ELISA. Whole long homogenates were assessed for myeloperoxidase (MPO) activity, flow cytometry (Liberase digestion and prior antibodies), and IL-6 and CXCL1 by ELISA. Two-tailed unpaired t-tests were completed to determine significance (p < 0.05). All groups were n = 5 unless specified otherwise. Results: PRA mice have decreased tGFR at 6 hours as compared to Veh mice (data not shown). PRA mice have increased whole lung MPO and CXCL1, but IL-6 is not different (Fig A-C). PRA mice have increased plasma CXCL1 and IL-6 (data not shown). PRA mice do not have increased BALF total protein or CXCL1 as compared to Veh (Fig D and F). BALF IL-6 was not detected in either group (Fig E). Flow cytometry of BALF and whole lung shows similar alveolar macrophage (AM) and neutrophil populations (Fig G-J). Conclusions: Our PRA model is characterized by increased lung MPO and CXCL1, suggesting inflammatory changes in the lung; however, flow analysis does not show a difference in the neutrophil population in PRA whole lung. Furthermore, BALF analysis of total protein, cytokine levels, and AM populations suggests that there is no disruption of the alveolar-capillary barrier. Overall, the lung inflammation demonstrated in this model is less severe than in the mouse ischemic AKI or nephrectomy models. Future directions will focus on upstream causes of these inflammatory changes and a more in-depth assessment of changes to lung myeloid populations in PRA.

Tertiary lymphoid tissues (TLTs) are ectopic lymphoid tissues that form de novo in nonlymphoid organs. In this study, we demonstrate that the kidneys of aged mice with a renal tubule-specific knockout of autophagy-related 7 (Atg7) contain numerous and large TLTs. p-S6 protein, a marker of mTORC1, was elevated in the tubules adjacent to the TLTs as well as within the TLTs themselves. In Atg7-/- kidneys, tubular injury and increased proinflammatory cytokines were observed, both of which are known to promote TLT formation and growth. In mice with either polycystic kidney disease (Pkd1RC/RC) or kidney ischemia, increased p-S6 was observed in tubules near TLTs and within the TLTs. Treatment with Torin2, an mTOR inhibitor, led to the virtual disappearance of TLTs in Pkd1RC/RC kidneys and a significant reduction in TLTs in ischemic kidneys. To assess whether p-S6 in the tubules was driving TLT formation, ischemia was induced in tubule-specific Atg7-/- Raptor (mTORC1)-/- mice. The tubule-specific Raptor knockout had little effect on the TLTs. In summary, Torin2, which inhibited p-S6 in both tubules and TLTs, resulted in a large decrease in TLTs in ischemic and Pkd1RC/RC kidneys. Tubule-specific knockout of mTORC1 (Raptor) had no effect on TLTs. In conclusion, p-S6 activity within the TLTs, rather than in the tubules, drives the proliferation of immune cells and the formation and growth of TLTs. These findings provide new insights into the role of mTOR in TLT development. The study has important therapeutic implications, as TLTs are involved in numerous disease processes and mTOR inhibitors are widely used in clinical practice.

Procalcitonin is a 14.5 kDa protein used clinically as a marker of sepsis and therapeutic response to antibiotic therapy. However, its utility in critically ill patients with either acute kidney injury (AKI) or end-stage kidney disease (ESKD) who require continuous kidney replacement therapy (CKRT) is unknown. The aim of this study was to determine if plasma levels of procalcitonin could reliably distinguish septic from nonseptic status in patients with AKI or ESKD prior to or during CKRT. Procalcitonin concentrations were measured in plasma of 41 critically ill septic or non-septic subjects with AKI or ESKD prior to CKRT (pre-CKRT) and on days 1, 2, and 3 of CKRT in this retrospective cohort study (n = 111 total plasma measurements). Continuous venovenous hemodialysis was the modality of CKRT in these patients. Sepsis status was stringently defined based on culture results. Effluent procalcitonin levels were ascertained on days 1, 2, and 3 of CKRT to assess the clearance of procalcitonin and effects on plasma levels. 92

Introduction: Patients with acute kidney injury (AKI) or end stage kidney disease (ESKD) may require continuous renal replacement therapy (CRRT) as a supportive intervention. While CRRT is effective at achieving solute control and fluid balance, the indiscriminate nature of this procedure raises the possibility that beneficial substances may similarly be removed. Hepcidin, an antimicrobial peptide with pivotal roles in iron homeostasis and pathogen clearance, has biochemical properties amenable to direct removal via CRRT. We hypothesized that serum hepcidin levels would significantly decrease after initiation of CRRT. Methods: In this prospective, observational trial, we enrolled 13 patients who required CRRT: 11 due to stage 3 AKI, and 2 due to critical illness in the setting of ESKD. Plasma was collected at the time of enrollment, and then plasma and effluent were collected at 10:00 a.m. on the following 3 days. Plasma samples were also collected from healthy controls, and we compared hepcidin concentrations in those with renal disease compared to normal controls, evaluated trends in hepcidin levels over time, and calculated the hepcidin sieving coefficient. Results: Plasma hepcidin levels were significantly higher in patients initiating CRRT than in normal controls (158 ± 60 vs. 17 ± 3 ng/mL respectively, p < 0.001). Hepcidin levels were highest prior to CRRT initiation (158 ± 60 ng/mL), and were significantly lower on day 1 (102 ± 24 ng/mL, p < 0.001) and day 2 (56 ± 14 ng/mL, p < 0.001) before leveling out on day 3 (51 ± 11 ng/mL). The median sieving coefficient was consistent at 0.82–0.83 for each of 3 days. Conclusions: CRRT initiation is associated with significant decreases in plasma hepcidin levels over the first 2 days of treatment regardless of indication for CRRT, or presence of underlying ESKD. Since reduced hepcidin levels are associated with increased mortality and our data implicate CRRT in hepcidin removal, larger clinical studies evaluating relevant clinical outcomes based on hepcidin trends in this population should be pursued.

AbstractAcute kidney injury (AKI) is a systemic disease that affects energy metabolism in various remote organs in murine models of ischemic AKI. However, AKI-mediated effects in the liver have not been comprehensively assessed. After inducing ischemic AKI in 8–10-week-old, male C57BL/6 mice, mass spectrometry metabolomics revealed that the liver had the most distinct phenotype 24 h after AKI versus 4 h and 7 days. Follow up studies with in vivo [13C6]-glucose tracing on liver and kidney 24 h after AKI revealed 4 major findings: (1) increased flux through glycolysis and the tricarboxylic (TCA) cycle in both kidney and liver; (2) depleted hepatic glutathione levels and its intermediates despite unchanged level of reactive oxygen species, suggesting glutathione consumption exceeds production due to systemic oxidative stress after AKI; (3) hepatic ATP depletion despite unchanged rate of mitochondrial respiration, suggesting increased ATP consumption relative to production; (4) increased hepatic and renal urea cycle intermediates suggesting hypercatabolism and upregulation of the urea cycle independent of impaired renal clearance of nitrogenous waste. Taken together, this is the first study to describe the hepatic metabolome after ischemic AKI in a murine model and demonstrates that there is significant liver-kidney crosstalk after AKI.

INTRODUCTION:Acute kidney injury (AKI) is associated with increased risk of heart failure (HF). Determining the type of HF experienced by AKI survivors (heart failure with preserved or reduced ejection fraction, HFpEF or HFrEF) could suggest potential mechanisms underlying the association and opportunities for improving post-AKI care. METHODS:In this retrospective study of adults within the Vanderbilt University health system with a diagnosis of HF, we tested whether AKI events in the two years preceding incident HF associated more with HFpEF or HFrEF while controlling for known predictors. HF outcomes were defined by administrative codes and classified as HFpEF or HFrEF by echocardiogram data. We used multivariable logistic regression models to estimate the effects of AKI on the odds of incident HFpEF versus HFrEF. RESULTS:AKI (all stages) trended towards a preferential association with HFpEF in adjusted analyses (adjusted OR 0.80, 95% CI 0.63 - 1.01). Stage 1 AKI was associated with higher odds of HFpEF that was statistically significant (adjusted OR 0.62, 95% CI 0.43 - 0.88), whereas stages 2-3 AKI showed a trend toward HFrEF that did not reach statistical significance (adjusted OR 1.11, 95% CI 0.76 - 1.63). CONCLUSIONS:AKI as a binary outcome trended towards a preferential association with HFpEF. Stage 1 AKI was associated with higher odds of HFpEF, whereas stage 2-3 trended towards an association with HFrEF that did not meet statistical significance. Different mechanisms may predominate in incident HF following mild versus more severe AKI. Close follow-up with particular attention to volume status and cardiac function after discharge is warranted after even mild AKI.

BACKGROUND:Plasma cystatin C is a reliable marker to estimate kidney function; however, it is unknown whether this remains true in patients receiving continuous kidney replacement therapy (CKRT). Herein, we tested the hypothesis that lower concentrations of plasma cystatin C during the first three days of CKRT would predict kidney function recovery. METHODS:We performed a retrospective observational study of 72 patients from a 126-patient, single-center CKRT study. We studied two a priori defined cohorts of patients without advanced CKD who had acute kidney injury requiring CKRT (AKI-CKRT): 1) with early kidney function recovery defined as liberation from KRT within seven days of CKRT initiation versus 2) with delayed kidney function recovery defined as receipt of KRT for >21 days or death while on KRT. Subsequent analysis included patients with advanced CKD and intermediate kidney function recovery (liberation between 8 and 21 days). Cystatin C was then measured on stored plasma, urine, and dialysis effluent collected prior to CKRT initiation and on days 1, 2, and 3 of CKRT. RESULTS:Plasma cystatin C was significantly lower in patients with early kidney function recovery in comparison to patients with delayed kidney function recovery on days 1 (1.79 vs. 2.39mg/L), 2 (1.91 vs. 2.38mg/L) and 3 (2.04 vs. 2.67mg/L) of CKRT. Sieving coefficient and CKRT clearance of cystatin C were similar for patients with early and delayed kidney function recovery. The lowest plasma cystatin C concentration on days 1-3 of CKRT predicted early kidney function recovery with an area under the receiver operating curve of 0.77 (P = 0.002), positive likelihood ratio of 5.60 for plasma cystatin C <1.30mg/L, and negative likelihood ratio of 0.17 for plasma cystatin C ≥1.88mg/L. CONCLUSION:Lower plasma cystatin C concentrations during the first three days of CKRT are associated with early kidney function recovery.

Background Acute kidney injury (AKI) is common in sick neonates and associated with poor pulmonary outcomes, however, the mechanisms responsible remain unknown. We present two novel neonatal rodent models of AKI to investigate the pulmonary effects of AKI. Methods In rat pups, AKI was induced surgically via bilateral ischemia-reperfusion injury (bIRI) or pharmacologically using aristolochic acid (AA). AKI was confirmed with plasma blood urea nitrogen and creatinine measurements and kidney injury molecule-1 staining on renal immunohistochemistry. Lung morphometrics were quantified with radial alveolar count and mean linear intercept, and angiogenesis investigated by pulmonary vessel density (PVD) and vascular endothelial growth factor (VEGF) protein expression. For the surgical model, bIRI, sham, and non-surgical pups were compared. For the pharmacologic model, AA pups were compared to vehicle controls. Results AKI occurred in bIRI and AA pups, and they demonstrated decreased alveolarization, PVD, and VEGF protein expression compared controls. Sham pups did not experience AKI, however, demonstrated decreased alveolarization, PVD, and VEGF protein expression compared to controls. Conclusion Pharmacologic AKI and surgery in neonatal rat pups, with or without AKI, decreased alveolarization and angiogenesis, producing a bronchopulmonary dysplasia phenotype. These models provide a framework for elucidating the relationship between AKI and adverse pulmonary outcomes. Impact There are no published neonatal rodent models investigating the pulmonary effects after neonatal acute kidney injury, despite known clinical associations. We present two novel neonatal rodent models of acute kidney injury to study the impact of acute kidney injury on the developing lung. We demonstrate the pulmonary effects of both ischemia-reperfusion injury and nephrotoxin-induced AKI on the developing lung, with decreased alveolarization and angiogenesis, mimicking the lung phenotype of bronchopulmonary dysplasia. Neonatal rodent models of acute kidney injury provide opportunities to study mechanisms of kidney-lung crosstalk and novel therapeutics in the context of acute kidney injury in a premature infant.