Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats.

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

Prostaglandin E-major urinary metabolites as a new biomarker for acute mesenteric ischemia.

BACKGROUND: Acute mesenteric ischemia (AMI) is an emergent vascular disease caused by cessation of the blood supply to the small intestine. Despite advances in the diagnosis, intervention, and surgical procedures, AMI remains a life-threatening condition. Prostaglandin E2 major urinary metabolite (PGE-MUM), the urinary metabolite of prostaglandin E2, is known to be stable in urine and has been suggested to be a valuable biomarker for intestinal mucosal inflammation, such as ulcerative colitis. We therefore investigated whether or not PGE-MUM levels reflect the degree of ischemia in an intestinal ischemia-reperfusion model. METHODS: Male rats were used to establish a superior mesenteric artery occlusion (SMAO) group, in which the superior mesenteric artery was clamped, and a sham group. The clamping times in the SMAO group were either 30 minutes or 60 minutes, and reperfusion times were either 3 hours or 6 hours, after which PGE-MUM values were measured. RESULTS: The histological injury score of the SMAO (30-minute ischemia and 6-hour reperfusion group, 1.8 ± 0.4; 60-minute ischemia and 6-hour reperfusion group, 4.7 ± 0.5) and were significantly greater than that of the sham group (0.4 ± 0.7, p < 0.05). The PGE-MUM levels in the SMAO group (30-minutes ischemia and 6-hour reperfusion group, 483 ± 256; 60-minutes ischemia and 6-hour reperfusion group, 889 ± 402 ng/mL) were significantly higher than in the sham group (30-minute and 6-hour observation group, 51 ± 20; 60-minute and 6-hour observation group, 73 ± 32 ng/mL; p < 0.05). Furthermore, the PGE-MUM value was corrected by the concentration of urinary creatinine (Cr). The PGE-MUM/urinary Cr levels in the SMAO group were also significantly higher than in the sham group ( p < 0.05). CONCLUSION: We found that intestinal ischemia-reperfusion increased urinary PGE-MUM levels depending on the ischemic time. This suggests the potential utility of PGE-MUM as a noninvasive marker of intestinal ischemia.

In Vivo and In Vitro Evaluation of Urinary Biomarkers in Ischemia/Reperfusion-Induced Kidney Injury.

Oxidative stress plays an important role in the pathophysiology of acute kidney injury (AKI). Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study was aimed to determine whether urinary vanin-1 is a biomarker for the early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI, ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. On Day 1 after renal ischemia/reperfusion (I/R), serum creatinine (SCr) in I/R rats was higher than in sham-operated rats, but this did not reach significance. Urinary N-acetyl-ß-D-glucosaminidase (NAG) exhibited a significant increase but decreased on Day 2 in I/R rats. In contrast, urinary vanin-1 significantly increased on Day 1 and remained at a significant high level on Day 2 in I/R rats. Renal vanin-1 protein decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules of I/R rats. Our in vitro results confirmed that the supernatant from HK-2 cells under hypoxia/reoxygenation included significantly higher levels of vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker of AKI induced by I/R.

Hyperbaric Oxygen Preconditioning Upregulates Heme OxyGenase-1 and Anti-Apoptotic Bcl-2 Protein Expression in Spontaneously Hypertensive Rats with Induced Postischemic Acute Kidney Injury.

Renal ischemia and reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI). Pathogenesis of postischemic AKI involves hemodynamic changes, oxidative stress, inflammation process, calcium ion overloading, apoptosis and necrosis. Up to date, therapeutic approaches to treat AKI are extremely limited. Thus, the aim of this study was to evaluate the effects of hyperbaric oxygen (HBO) preconditioning on citoprotective enzyme, heme oxygenase-1 (HO-1), pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins expression, in postischemic AKI induced in normotensive Wistar and spontaneously hypertensive rats (SHR). The animals were randomly divided into six experimental groups: SHAM-operated Wistar rats (W-SHAM), Wistar rats with induced postischemic AKI (W-AKI) and Wistar group with HBO preconditioning before AKI induction (W-AKI + HBO). On the other hand, SHR rats were also divided into same three groups: SHR-SHAM, SHR-AKI and SHR-AKI + HBO. We demonstrated that HBO preconditioning upregulated HO-1 and anti-apoptotic Bcl-2 protein expression, in both Wistar and SH rats. In addition, HBO preconditioning improved glomerular filtration rate, supporting by significant increase in creatinine, urea and phosphate clearances in both rat strains. Considering our results, we can also say that even in hypertensive conditions, we can expect protective effects of HBO preconditioning in experimental model of AKI.

MicroRNAs as Biomarkers and Therapeutic Targets in Inflammation- and Ischemia-Reperfusion-Related Acute Renal Injury.

Acute kidney injury (AKI), caused mainly by ischemia-reperfusion, sepsis, or nephrotoxins (such as contrast medium), is identified by an abrupt decline in kidney function and is associated with high morbidity and mortality. Despite decades of efforts, the pathogenesis of AKI remains poorly understood, and effective therapies are lacking. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level to control cell differentiation, development, and homeostasis. Additionally, extracellular miRNAs might mediate cell-cell communication during various physiological and pathological processes. Recently, mounting evidence indicates that miRNAs play a role in the pathogenesis of AKI. Moreover, emerging research suggests that because of their remarkable stability in body fluids, microRNAs can potentially serve as novel diagnostic biomarkers of AKI. Of note, our previous finding that miR-494 is rapidly elevated in urine but not in serum provides insight into the ultimate role of urine miRNAs in AKI. Additionally, exosomal miRNAs derived from stem cells, known as the stem cell secretome, might be a potential innovative therapeutic strategy for AKI. This review aims to provide new data obtained in this field of research. It is hoped that new studies on this topic will not only generate new insights into the pathophysiology of urine miRNAs in AKI but also might lead to the precise management of this fatal disease.

Impaired Tubular Reabsorption Is the Main Mechanism Explaining Increases in Urinary NGAL Excretion Following Acute Kidney Injury in Rats.

Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted low-molecular weight iron-siderophore-binding protein. NGAL overexpression in injured tubular epithelia partly explains its utility as a sensitive and early urinary biomarker of acute kidney injury (AKI). Herein, we extend mechanistic insights into the source and kinetics of urinary NGAL excretion in experimental AKI. Three models of experimental AKI were undertaken in adult male Wistar rats; renal ischemia-reperfusion injury (IRI) and gentamicin (G) and cisplatin (Cisp) nephrotoxicity. Alongside standard histological and biochemical assessment of AKI, urinary NGAL excretion rate, plasma NGAL concentration, and renal NGAL mRNA/protein expression were assessed. In situ renal perfusion studies were undertaken to discriminate direct shedding of NGAL to the urine from addition of NGAL to the urine secondary to alterations in the tubular handling of glomerular filtrate-derived protein. Renal NGAL expression and urinary excretion increased in experimental AKI. In acute studies in both the IRI and G models, direct renal perfusion with Kreb's buffer eliminated urinary NGAL excretion. Addition of exogenous NGAL to the Kreb's buffer circuit, reestablishment of perfusion with systemic blood or reperfusion with renal vein effluent restored high levels of urinary NGAL excretion. Urinary NGAL excretion in AKI arises in large proportion from reduced reabsorption from the glomerular filtrate. Hence, subclinical cellular dysfunction could increase urinary NGAL, particularly in concert with elevations in circulating prerenal NGAL and/or pharmacological inhibition of tubular reabsorption. More granular interpretation of urinary NGAL measurements could optimize the scope of its clinical utility as a biomarker of AKI.

Interleukin-24 is a novel diagnostic biomarker for the severity of acute kidney injury.

There is a clinical need for sensitive acute kidney injury (AKI) biomarkers that enable early therapeutic interventions and prediction of disease prognosis. In this study, we monitored interleukin (IL)-24 expressed in kidneys with severe AKI that progresses to atrophic kidney in a mouse model of ischemia-reperfusion injury (IRI). Therefore, we evaluated IL-24 as a potential biomarker not only for early diagnosis of AKI, but also for predicting progression to chronic kidney disease (CKD). Serum IL-24 was detected earlier than the elevation of serum creatinine levels and urinary IL-24 was detected as early as neutrophil gelatinase associated lipocalin (NGAL) in severe AKI (60 min of IRI). In addition, serum and urine IL-24 levels tended to increase in relation to ischemia duration. In such kidneys, vascular smooth muscle cells expressed IL-24 in response to the injury in the renal tubular epithelial cell and its target was the renal tubular epithelial cell itself. IL-24 may play a pivotal role in the communication between tubular epithelial cells and vascular smooth muscle cells and, in conclusion, IL-24 can be used as a sensitive biomarker for AKI.

Remote Ischemic Perconditioning Modulates Apelin Expression After Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia/reperfusion injury (IRI) can result in impaired ability of urine concentration and increased sodium fractional excretion. Apelin, a (neuro) vasoactive peptide, enhances diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of arginine vasopressin on the tubules. However, changes in renal apelin expression in renal IRI rat model have not been elucidated. Remote ischemic perconditioning (RIPerC) improves renal sodium and water handling after IRI. Here, we investigated whether RIPerC prevents dysregulation of renal sodium and water handling in response to IRI by apelin signaling pathway in rats. MATERIALS AND METHODS: Renal IRI was induced by 45-min clamping of renal arteries followed by 24 h reperfusion. RIPerC was created by applying four cycles of 2-min ischemia of the left femoral artery followed by 3-min reperfusion at the start of renal ischemia. Rats were randomly divided into sham, ischemia/reperfusion, and RIPerC + ischemia/reperfusion groups. Urine and blood were sampled after reperfusion period. The kidney was harvested for mRNA isolation and histopathological study. RESULTS: IRI resulted in decreased clearance of creatinine, increased sodium fractional excretion, and reduced urine osmolality compared with sham animals. This occurred with an increase in mRNA expression levels of apelin and histological damages in both cortical and medullary regions of kidney tissues. RIPerC treatment ameliorated all these changes. CONCLUSIONS: This study showed that RIPerC has protective effects against dysregulation of renal sodium and water handling after renal IRI, which might be related with inhibition of apelin signaling pathway.

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.

Urine complement activation fragments are increased in patients with kidney injury after cardiac surgery.

Experiments in mouse models have shown that the complement cascade is activated within the kidney after ischemia-reperfusion and that complement activation contributes to tubular injury in this setting. Less is known, however, about complement activation in human kidneys after ischemia or whether complement activation in the tubulointerstitium can be detected by measurement of complement fragments in the urine. We hypothesized that urine biomarkers of complement activation would rapidly increase in patients who develop ischemic acute kidney injury, signaling complement activation within the kidney. We confirmed that the alternative pathway of complement is activated in the kidneys of mice after ischemia-reperfusion, and we found that levels of factor B fragments (generated during alternative pathway activation) rapidly increase in the urine. We next performed a case-control study in which we measured complement fragments in human urine samples from patients undergoing cardiac surgery using ELISAs. The level of Ba increased after cardiac surgery and was significantly higher in patients who developed acute kidney injury. The increase in Ba also correlated with magnitude of the subsequent rise in serum creatinine and with the need for hemodialysis during the hospitalization. These findings demonstrate that the alternative pathway of complement is activated in patients who develop acute kidney injury after cardiac surgery and that increases in the level of urine Ba may be a predictive and functional biomarker of severe kidney injury.

Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion.

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild-type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI-AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4-NF-κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR-4-NF-κB associated renal inflammation, including the expression of MCP-1, TNF-α, IL-1ß, IL-6, iNOS, CXCL15(IL-8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.

Glomerular filtrate proteins in acute cardiorenal syndrome.

Acute cardiorenal syndrome (CRS-1) is a morbid complication of acute cardiovascular disease. Heart-to-kidney signals transmitted by "cardiorenal connectors" have been postulated, but investigation into CRS-1 has been limited by technical limitations and a paucity of models. To address these limitations, we developed a translational model of CRS-1, cardiac arrest and cardiopulmonary resuscitation (CA/CPR), and now report findings from nanoscale mass spectrometry proteomic exploration of glomerular filtrate 2 hours after CA/CPR or sham procedure. Filtrate acquisition was confirmed by imaging, molecular weight and charge distribution, and exclusion of protein specific to surrounding cells. Filtration of proteins specific to the heart was detected following CA/CPR and confirmed with mass spectrometry performed using urine collections from mice with deficient tubular endocytosis. Cardiac LIM protein was a CA/CPR-specific filtrate component. Cardiac arrest induced plasma release of cardiac LIM protein in mice and critically ill human cardiac arrest survivors, and administration of recombinant cardiac LIM protein to mice altered renal function. These findings demonstrate that glomerular filtrate is accessible to nanoscale proteomics and elucidate the population of proteins filtered 2 hours after CA/CPR. The identification of cardiac-specific proteins in renal filtrate suggests a novel signaling mechanism in CRS-1. We expect these findings to advance understanding of CRS-1.

Urine podoplanin heralds the onset of ischemia-reperfusion injury of the kidney.

Ischemia-reperfusion injury represents one of the most common causes of acute kidney injury, a serious and often deadly condition that affects up to 20% of all hospitalized patients in the United States. However, the current standard assay used universally for the diagnosis of acute kidney injury, serum creatinine, does not detect renal damage early in its course. Serendipitously, we found that the immunofluorescent signal of the constitutive podocyte marker podoplanin fades in the glomerulus and intensifies in the tubulointerstitial compartment of the kidney shortly after ischemia-reperfusion injury in 8- to 10-wk-old male C57Bl/6j mice. Therefore, we sought to define the appearance and course of the podoplanin-positive signal in the kidney after ischemia-reperfusion injury. The tubulointerstitial podoplanin-positive signal increased as early as 2 h but persisted for 7 days after ischemia-reperfusion injury. In addition, the strength of this tubulointerstitial signal was directly proportional to the severity of ischemia, and its location shifted from the tubules to interstitial cells over time. Finally, we detected podoplanin in the urine of mice after ischemia, and we observed that an increase in the urine podoplanin-to-creatinine ratio correlated strongly with the onset of renal ischemia-reperfusion injury. Our findings indicate that the measurement of urine podoplanin harbors promising potential for use as a novel biomarker for the early detection of ischemia-reperfusion injury of the kidney.

Early intraoperative iron-binding proteins are associated with acute kidney injury after cardiac surgery.

OBJECTIVES: Iron regulation is an important modifier of renal ischemia-reperfusion injury, but the role of iron-binding proteins during cardiopulmonary bypass remains unclear. The goal was to characterize iron-binding proteins throughout ischemia-reperfusion injury to determine their association with acute kidney injury development. METHODS: A prospective observational cohort of adult patients who underwent cardiac surgery (n = 301) was obtained, and acute kidney injury was defined by Kidney Disease Improving Global Outcomes. Serum ferritin, transferrin saturation, and urine hepcidin-25 were measured. RESULTS: Intraoperative serum ferritin was lower at the start of cardiopulmonary bypass (P = .005) and 1-hour cardiopulmonary bypass (P = .001) in patients with acute kidney injury versus patients without acute kidney injury. Lower serum ferritin and higher transferrin saturation at 1-hour cardiopulmonary bypass were independent predictors of acute kidney injury (serum ferritin odds ratio, 0.66; 95% confidence interval [CI], 0.48-0.91; transferrin saturation odds ratio, 1.26; 95% CI, 1.02-1.55) and improved model discrimination (area under the curve [AUC], 0.76; 95% CI, 0.67-0.85) compared with clinical prediction alone (AUC, 0.72; 95% CI, 0.62-0.81; ΔAUC and net reclassification index, P = .01). Lower ferritin, higher transferrin saturation at 1-hour cardiopulmonary bypass, and lower urine hepcidin-25 at postoperative day 1 were also independent predictors for acute kidney injury development, and this model demonstrated an AUC of 0.80 (0.72-0.87), which was superior to clinical prediction (ΔAUC P = .002, integrated discrimination improvement and net reclassification index P = .003). CONCLUSIONS: Our findings suggest that lower levels of intraoperative iron-binding proteins may reflect an impaired capacity to rapidly handle catalytic iron released during cardiopulmonary bypass, leading to kidney injury. These data highlight the importance of iron homeostasis in human ischemia-reperfusion injury and suggest it is a potentially modifiable risk during cardiac surgery. Intraoperative detection of incipient acute kidney injury may be feasible and could be used as an enrichment strategy for clinical trials.

The effect of remote ischemic preconditioning on serum creatinine in patients undergoing partial nephrectomy: a study protocol for a randomized controlled trial.

BACKGROUND: Acute kidney injury (AKI) may develop during partial nephrectomy due to ischemic reperfusion injury induced by renal artery clamping or surgical insult. The effect of remote ischemic preconditioning (RIPC) on reducing the renal injury after partial nephrectomy has not been evaluated in terms of urinary biomarkers. METHODS/DESIGN: We will conduct a randomized controlled trial enrolling the patients who will undergo partial nephrectomy. In the study group, RIPC which consisted of four 5-min cycles of limb ischemia and reperfusion will be conducted after induction of anesthesia. Postoperative serum creatinine values, the incidence of AKI, and urinary biomarkers, including urinary creatinine, microalbumin, ß-2 microglobulin, and N-acetyl-beta-D-glucosaminidase, will be compared between groups during the postoperative 2 weeks. Regional oxygen saturation on the skin of the contralateral kidney will be measured to evaluate the association between intraoperative regional oxygen saturation values and renal injury of the operating side. DISCUSSION: We expect that our trial may demonstrate the effect of RIPC on mitigating the immediate postoperative renal injury and improving patient outcomes after partial nephrectomy. Moreover, our patients will undergo 99mTc-DTPA radionuclide scintigraphy to calculate glomerular filtration rate 6 and 12 months after surgery. This data should show the long-term effect of RIPC. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03273751 . Registered on 6 September 2017.

Urinary angiotensinogen predicts progressive chronic kidney disease after an episode of experimental acute kidney injury.

One of the major obstacles to prevent AKI-CKD transition is the lack of effective methods to follow and predict the ongoing kidney injury after an AKI episode. In the present study, we test the utility of urinary angiotensinogen (UAGT) for dynamically evaluating renal structural changes and predicting AKI-CKD progression by using both mild and severe bilateral renal ischemia/reperfusion injury mice. UAGT returns to pre-ischemic levels 14 days after mild AKI followed by kidney architecture restoration, whereas sustained increase in UAGT accompanies by ongoing renal fibrosis after severe AKI. UAGT at day 14-42 correlates with renal fibrosis 84 days after AKI. For predicting fibrosis at day 84, the area under receiver operating characteristics curve of UAGT at day 14 is 0.81. Persistent elevation in UAGT correlates with sustained activation of intrarenal renin-angiotensin system (RAS) during AKI-CKD transition. Abrogating RAS activation post AKI markedly reduced renal fibrosis, with early RAS intervention (from 14 days after IRI) more beneficial than late intervention (from 42 days after IRI) in alleviating fibrosis. Importantly, UAGT decreases after RAS intervention, and its level at day 14-28 correlates with the extent of renal fibrosis at day 42 post RAS blockade. A pilot study conducted in patients with acute tubular necrosis finds that compared with those recovered, patients with AKI-CKD progression exhibits elevated UAGT during the 3-month follow-up after biopsy. Our study suggests that UAGT enables the dynamical monitoring of renal structural recovery after an AKI episode and may serve as an early predictor for AKI-CKD progression and treatment response.

Plasma and urinary p21: potential biomarkers of AKI and renal aging.

p21 is upregulated in renal tubules in response to acute kidney injury ( AKI). and localizes in the nucleus, where it induces cell cycle arrest (CCA). These events can mitigate early injury but can also facilitate the onset of the degenerative cell senescence/"aging" process. Hence, we asked the following: 1) can AKI-induced p21 upregulation be gauged by plasma and/or urinary p21 assay; 2) might p21 serve as an AKI/CCA biomarker; and 3) does p21 accumulate during normal renal aging, and might plasma p21 reflect this process? Mice were subjected to either ischemia-reperfusion (I/R) or nephotoxic (maleate) AKI. Renal cortical p21 expression (protein, mRNA) was assessed 2-18 h later and contrasted with plasma/urine p21 concentrations (ELISA). p21 mRNA/protein levels were also measured in aging mice (2, 12, 24 mo). AKI induced marked, progressive, increases in renal cortical p21 mRNA and protein levels. These changes were marked by acute (within 2-4 h) and profound increases (up to 200×) in both plasma and urine p21 concentrations. Renal I/R also activated p21 gene expression in extrarenal organs (heart, brain), consistent with so-called "organ cross talk". p21 efflux from damaged cells was confirmed with studies of hypoxia-injured, isolated proximal tubules. Aging was associated with progressive renal cortical p21 expression, which correlated ( r, 0.83) with rising plasma p21 concentrations. We concluded that 1) during AKI, renal p21 increases can be gauged by either plasma or urine p21 assay, serving as potentially useful AKI/CCA biomarkers; 2) AKI can activate p21 in extrarenal organs; and 3) plasma p21 levels may provide an index of the renal/systemic aging process.

Hydrogen Gas Does Not Ameliorate Renal Ischemia Reperfusion Injury in a Preclinical Model.

In renal transplantation, ischemia reperfusion injury impairs early graft function and can reduce long term graft survival. Hydrogen has antioxidant and anti-inflammatory properties that can reduce the effects of ischemic injury. The aim of this study was to examine the effects of hydrogen gas administered during reperfusion in a preclinical model of kidney ischemia reperfusion injury. Porcine kidneys underwent 15 min of warm ischemia followed by 22 h of cold ischemia. They were then reperfused for 6 h with whole autologous blood on an ex vivo reperfusion circuit. Paired kidneys were randomized to control (n = 6) (25% oxygen, 5% carbon dioxide, 70% nitrogen) or hydrogen (n = 6) (2% hydrogen, 25% oxygen, 5% carbon dioxide, 68% nitrogen) groups. Tissue, urine, and blood samples were collected at baseline and hourly throughout the reperfusion period. Baseline measurements were similar across groups. Following perfusion, there was no significant difference between control and hydrogen groups in urine output (693 mL vs. 608 mL, P = 0.86), renal blood flow (105.9 vs. 108 mL/min/100g, P = 0.89), acid-base homeostasis, or creatinine clearance. There was a significant increase in cytokine levels from baseline to 6 h in both groups (IL-1ß P = 0.002; IL-6 P = 0.004; IL-8 P = 0.002). However, there were no significant differences in levels of inflammatory cytokines (IL1ß, IL-6, and IL-8) between the groups. The administration of hydrogen gas did not improve renal function, reduce oxidative damage, or inflammation during the reperfusion of ischemically damaged kidneys.

Identification of Urinary Activin A as a Novel Biomarker Reflecting the Severity of Acute Kidney Injury.

Acute kidney injury (AKI) is a common but complex condition that is associated with increased morbidity and mortality. In the present study, we examined whether urinary activin A, a member of the TGF-beta superfamily, is present in mice with ischemia-reperfusion injury and in humans with AKI, as well as its potential as a biomarker for AKI. Expression of activin A was markedly increased in ischemic mouse kidneys. In situ hybridization demonstrated that activin mRNA was expressed in tubular cells of ischemic kidneys but not of normal kidneys. Immunoreactive activin A, which was absent in normal kidneys, was detected in the cytoplasm of proximal tubular cells in ischemic kidneys. Activin A was undetectable in the urine of normal mice. In contrast, activin A was significantly increased in the urine of ischemic mice at 3 h after reperfusion. Urinary activin A levels increased according to the period of ischemia. In humans, urinary activin A was almost undetectable in healthy volunteers and in patients with pre-renal AKI, but was significantly increased in patients with renal AKI. There was no significant correlation between urinary activin A and serum activin A. Collectively, urinary activin A might be a useful biomarker reflecting the severity of AKI.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats.

Acute kidney injury (AKI) is an important risk factor for the development of chronic kidney disease (CKD), and an alteration in renal water handling has been observed during the transition of AKI to CKD. Urinary exosomal release of aquaporin-1 (AQP1) and AQP2, important proteins for renal water handling, has recently been reported to predict their levels of renal expression. Therefore, we examined the patterns of urinary exosomal release of AQP1 and AQP2, and the exosomal marker proteins tumor susceptibility 101 protein (TSG101) and ALG-2 interacting protein X (Alix), in the acute and chronic phases following induction of AKI by renal bilateral ischemia/reperfusion (I/R) in rats. Blood tests and histological examinations indicated that AKI occurred before at 7 days after renal I/R ( day 7) and that renal fibrosis developed progressively thereafter. Immunoblotting demonstrated significant decreases in the urinary exosomal release of AQP1 and AQP2 during severe AKI. Urinary exosomal release of Alix and TSG101 was significantly increased on day 7. These data were also confirmed in rats with unilateral renal I/R causing more serious AKI. Urinary exosomal release of either the Ser-256- or Ser-269-phosphorylated form of AQP2, both of which are involved in apical trafficking of AQP2, was positively correlated with that of total AQP2. These results suggest that urinary exosomal release of AQP1 and AQP2 is reduced in I/R-induced AKI, whereas that of Alix and TSG101 is increased in the initial phase of renal fibrosis. Furthermore, apical trafficking of AQP2 appears to be related to urinary exosomal release of AQP2.