Clarifying expression patterns by renal lesion using transcriptome analysis and vanin-1 as a potential novel biomarker for renal injury in chickens.

Bird death is often caused by renal lesions induced by chemicals. The avian kidney has a renal portal system with significant blood flow that is sensitive to many chemicals. However, early avian biomarkers for kidney injury are yet to be identified. This study aimed to identify novel renal biomarkers. Acute kidney injury (AKI) can be divided into acute interstitial nephritis (AIN) and acute tubular necrosis (ATN). A chicken model of kidney damage was created by an injection of diclofenac or cisplatin, which caused either AIN or ATN, respectively. Microarray analysis was performed to profile the gene expression patterns in the chickens with nephropathy. A gene enrichment analysis suggested that the genes related to responses to external stimuli showed expression changes in both AIN and ATN. However, hierarchical clustering analyses suggested that gene expression patterns differed between AIN and ATN, and the number of biomarkers relating to renal damage was low. To identify early biomarkers for nephropathy, we focused on genes that were induced at various levels of renal damage. The gene, vanin-1 (VNN1) was highly induced in the early stages of renal damage. A quantitative real-time PCR analysis supported this finding. These results suggest VNN1 could be a useful early biomarker of kidney injury in avian species.

Lansoprazole promotes cisplatin-induced acute kidney injury via enhancing tubular necroptosis.

Acute kidney injury (AKI) is the main obstacle that limits the use of cisplatin in cancer treatment. Proton pump inhibitors (PPIs), the most commonly used class of medications for gastrointestinal complications in cancer patients, have been reported to cause adverse renal events. However, the effect of PPIs on cisplatin-induced AKI remains unclear. Herein, the effect and mechanism of lansoprazole (LPZ), one of the most frequently prescribed PPIs, on cisplatin-induced AKI were investigated in vivo and in vitro. C57BL/6 mice received a single intraperitoneal (i.p.) injection of cisplatin (18 mg/kg) to induce AKI, and LPZ (12.5 or 25 mg/kg) was administered 2 hours prior to cisplatin administration and then once daily for another 2 days via i.p. injection. The results showed that LPZ significantly aggravated the tubular damage and further increased the elevated levels of serum creatinine and blood urea nitrogen induced by cisplatin. However, LPZ did not enhance cisplatin-induced tubular apoptosis, as evidenced by a lack of significant change in mRNA and protein expression of Bax/Bcl-2 ratio and TUNEL staining. Notably, LPZ increased the number of necrotic renal tubular cells compared to that by cisplatin treatment alone, which was further confirmed by the elevated necroptosis-associated protein expression of RIPK1, p-RIPK3 and p-MLKL. Furthermore, LPZ deteriorated cisplatin-induced inflammation, as revealed by the increased mRNA expression of pro-inflammatory factors including, NLRP3, IL-1ß, TNF-α and caspase 1, as well as neutrophil infiltration. Consistently, in in vitro study, LPZ increased HK-2 cell death and enhanced inflammation, compared with cisplatin treatment alone. Collectively, our results demonstrate that LPZ aggravates cisplatin-induced AKI, and necroptosis may be involved in the exacerbation of kidney damage.

M2 macrophages predict worse long-term outcomes in human acute tubular necrosis.

Although macrophages are important players in the injury/repair processes in animal models of acute kidney injury (AKI), their roles in human AKI remains uncertain owing to a paucity of human biopsy studies. We investigated the role of macrophages in 72 cases of biopsy-proven acute tubular necrosis (ATN) and six cases of healthy kidney. Macrophages were identified by CD68 and CD163 immunohistochemistry and analyzed for their effect on renal outcomes. CD163+ M2 macrophages outnumbered CD68+ cells in the healthy kidneys, suggesting that CD163+ macrophages are resident macrophages. The infiltration of both subtypes of macrophages increased significantly in ATN. The density of the CD68+ macrophages was significantly higher in advanced-stage AKI, whereas CD163+ M2 macrophages was not. Eighty percent of patients exhibited renal functional recovery during follow-up. Older age and a higher density of CD163+ macrophages predicted non-recovery, whereas the AKI stage, tubular injury score, and density of CD68+ cells did not. The density of CD163+ M2 macrophages was an independent predictor of low eGFR at 3 months in advanced-stage AKI. This is the first human study demonstrating the possible role of macrophages in the injury and repair phases of AKI.

Renal subcapsular transplantation of hepatocyte growth factor-producing mesothelial cell sheets improves ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a clinically important cause of acute kidney injury leading to chronic kidney disease. Furthermore, IRI in renal transplantation still remains a risk factor for delayed graft function. Previous studies on IRI have had some limitations, and few of the studied therapies have been clinically applicable. Therefore, a new method for treating renal IRI is needed. We examined the effects of human mesothelial cell (MC) sheets and hepatocyte growth factor (HGF)-transgenic MC (tg MC) sheets transplanted under the renal capsule in an IRI rat model and compared these two treatments with the intravenous administration of HGF protein and no treatment through serum, histological, and mRNA analyses over 28 days. MC sheets and HGF-tg MC sheets produced HGF protein and significantly improved acute renal dysfunction, acute tubular necrosis, and survival rate. The improvement in necrosis was likely due to the cell sheets promoting the migration and proliferation of renal tubular cells, as observed in vitro. Expression of α-smooth muscle actin at day 14 and renal fibrosis at day 28 after IRI were significantly suppressed in MC sheet and HGF-tg MC sheet treatment groups compared with the other groups, and these effects tended to be reinforced by the HGF-tg MC sheets. These results suggest that the cell sheets locally and continuously affect renal paracrine factors, such as HGF, and support recovery from acute tubular necrosis and improvement of renal fibrosis in chronic disease.

Translational Assessment of Drug-Induced Proximal Tubule Injury Using a Kidney Microphysiological System.

Drug-induced kidney injury, a major cause of acute kidney injury, results in progressive kidney disease and is linked to increased mortality in hospitalized patients. Primary injury sites of drug-induced kidney injury are proximal tubules. Clinically, kidney injury molecule-1, an established tubule-specific biomarker, is monitored to assess the presence and progression of injury. The ability to accurately predict drug-related nephrotoxicity preclinically would reduce patient burden and drug attrition rates, yet state-of-the-art in vitro and animal models fail to do so. In this study, we demonstrate the use of kidney injury molecule-1 measurement in the kidney microphysiological system as a preclinical model for drug toxicity assessment. To show clinical relevance, we use quantitative systems pharmacology computational models for in vitro-in vivo translation of the experimental results and to identify favorable dosing regimens for one of the tested drugs.

IL-17A improves the efficacy of mesenchymal stem cells in ischemic-reperfusion renal injury by increasing Treg percentages by the COX-2/PGE2 pathway.

Mesenchymal stem cells (MSCs) are effective for the management of experimental ischemia-reperfusion acute kidney injury (IRI-AKI). Immune modulation is one of the important mechanisms of MSCs treatment. Interleukin-17A (IL-17A) pretreated MSCs are more immunosuppressive with minimal changes in immunogenicity in vitro. Here, we demonstrated that administration of IL-17A-pretreated MSCs resulted in significantly lower acute tubular necrosis scores, serum creatinine, and BUN of mice with IRI-AKI, compared with the administration of MSCs. Of the co-cultured splenocytes, IL-17A-pretreated MSCs significantly increased the percentages of CD4+Foxp3+ Tregs and decreased concanavalin A-induced T cell proliferation. Furthermore, mice with IRI-AKI that underwent IL-17A-pretreated MSC therapy had significantly lower serum IL-6, TNF-α, and IFN-γ levels, a higher serum IL-10 level, and higher spleen and kidney Treg percentages than the mice that underwent MSCs treatment. Additionally, the depletion of Tregs by PC61 (anti-CD25 antibody) reversed the enhanced treatment efficacy of the IL-17A-pretreatedMSCs on mice with IRI-AKI. Additionally, IL-17A upregulated COX-2 expression and increased PGE2 production. The blockage of COX-2 by celecoxib reversed the benefit of IL-pretreated 17A-MSCs on the serum PGE2 concentration, spleen and kidney Tregs percentages, serum creatinine and BUN levels, renal acute tubular necrosis scores, and serum IL-6, TNF-α, IFN-γ, and IL-10 levels of IRI-pretreated mice with AKI, compared with MSCs. Thus, our results suggest that IL-17A pretreatment enhances the efficacy of MSCs on mice with IRI-AKI by increasing the Treg percentages through the COX-2/PGE2 pathway.

Tubular Peroxiredoxin 3 as a Predictor of Renal Recovery from Acute Tubular Necrosis in Patients with Chronic Kidney Disease.

Peroxiredoxin 3 (PRX3) is a mitochondrial antioxidant that regulates apoptosis in various cancers. However, whether tubular PRX3 predicts recovery of renal function following acute kidney injury (AKI) remains unknown. This retrospective cohort study included 54 hospitalized patients who had AKI with biopsy-proven acute tubular necrosis (ATN). The study endpoint was renal function recovery within 6 months. Of the 54 enrolled patients, 25 (46.3%) had pre-existing chronic kidney disease (CKD) and 33 (61%) recovered renal function. Tubular PRX3 expression was higher in patients with ATN than in those without renal function recovery. The level of tubular but not glomerular PRX3 expression predicted renal function recovery from AKI (AUROC = 0.76). In multivariate Cox regression analysis, high PRX3 expression was independently associated with a higher probability of renal function recovery (adjusted hazard ratio = 8.99; 95% CI 1.13-71.52, P = 0.04). Furthermore, the discriminative ability of the clinical model for AKI recovery was improved by adding tubular PRX3. High tubular PRX3 expression was associated with a higher probability of renal function recovery from ATN. Therefore, tubular PRX3 in combination with conventional predictors can further improve recovery prediction and may help with risk stratification in AKI patients with pre-existing CKD.

Bmi-1 plays a critical role in the protection from acute tubular necrosis by mobilizing renal stem/progenitor cells.

The regeneration of injured tubular cell occurs primarily from intrinsic renal stem/progenitor cells (RSCs) labeled with CD24 and CD133 after acute tubular necrosis (ATN). Bmi-1 plays a crucial role in regulating self-renewal, differentiation and aging of multiple adult stem cells and progenitor cells. Bmi-1 was rapidly elevated in the induction of adult kidney regeneration by renal injury. To determine whether Bmi-1 maintained mobilization of RSCs in the protection from ATN, glycerol-rhabdomyolysis-induced ATN were performed in wild type (WT) and Bmi-1-deficient (Bmi-1-/-) mice. Their ATN phenotypes were analyzed; CD24 and CD133 double positive (CD24+CD133+) cells were measured; and the levels of serum urea nitrogen (SUN) and serum creatinine (SCr) were detected. We found that CD24+CD133+ RSCs were mobilized in WT ATN mice with the increased expression of Bmi-1; Bmi-1 deficiency led to increased tubular cast formation and necrosis, elevated levels of SUN and SCr, decreased tubular proliferation, and immobilized ratio of RSCs in ATN. These findings indicated that Bmi-1 played a critical role in the protection from ATN by maintaining mobilization of RSCs and would be a novel therapeutic target for preventing the progression of ATN.

Transcription factor Nrf2 hyperactivation in early-phase renal ischemia-reperfusion injury prevents tubular damage progression.

Acute kidney injury is a devastating disease with high morbidity in hospitalized patients and contributes to the pathogenesis of chronic kidney disease. An underlying mechanism of acute kidney injury involves ischemia-reperfusion injury which, in turn, induces oxidative stress and provokes organ damage. Nrf2 is a master transcription factor that regulates the cellular response to oxidative stress. Here, we examined the role of Nrf2 in the progression of ischemia-reperfusion injury-induced kidney damage in mice using genetic and pharmacological approaches. Both global and tubular-specific Nrf2 activation enhanced gene expression of antioxidant and NADPH synthesis enzymes, including glucose-6-phosphate dehydrogenase, and ameliorated both the initiation of injury in the outer medulla and the progression of tubular damage in the cortex. Myeloid-specific Nrf2 activation was ineffective. Short-term administration of the Nrf2 inducer CDDO during the initial phase of injury ameliorated the late phase of tubular damage. This inducer effectively protected the human proximal tubular cell line HK-2 from oxidative stress-mediated cell death while glucose-6-phosphate dehydrogenase knockdown increased intracellular reactive oxygen species. These findings demonstrate that tubular hyperactivation of Nrf2 in the initial phase of injury prevents the progression of reactive oxygen species-mediated tubular damage by inducing antioxidant enzymes and NADPH synthesis. Thus, Nrf2 may be a promising therapeutic target for preventing acute kidney injury to chronic kidney disease transition.

Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps.

Acute kidney injury is often the result of ischemia reperfusion injury, which leads to activation of coagulation and inflammation, resulting in necrosis of renal tubular epithelial cells. Platelets play a central role in coagulation and inflammatory processes, and it has been shown that platelet activation exacerbates acute kidney injury. However, the mechanism of platelet activation during ischemia reperfusion injury and how platelet activation leads to tissue injury are largely unknown. Here we found that renal ischemia reperfusion injury in mice leads to increased platelet activation in immediate proximity of necrotic cell casts. Furthermore, platelet inhibition by clopidogrel decreased cell necrosis and inflammation, indicating a link between platelet activation and renal tissue damage. Necrotic tubular epithelial cells were found to release extracellular DNA, which, in turn, activated platelets, leading to platelet-granulocyte interaction and formation of neutrophil extracellular traps ex vivo. Renal ischemia reperfusion injury resulted in increased DNA-platelet and DNA-platelet-granulocyte colocalization in tissue and elevated levels of circulating extracellular DNA and platelet factor 4 in mice. After renal ischemia reperfusion injury, neutrophil extracellular traps were formed within renal tissue, which decreased when mice were treated with the platelet inhibitor clopidogrel. Thus, during renal ischemia reperfusion injury, necrotic cell-derived DNA leads to platelet activation, platelet-granulocyte interaction, and subsequent neutrophil extracellular trap formation, leading to renal inflammation and further increase in tissue injury.

Role of Oxidative Stress in Drug-Induced Kidney Injury.

The kidney plays a primary role in maintaining homeostasis and detoxification of numerous hydrophilic xenobiotics as well as endogenous compounds. Because the kidney is exposed to a larger proportion and higher concentration of drugs and toxins than other organs through the secretion of ionic drugs by tubular organic ion transporters across the luminal membranes of renal tubular epithelial cells, and through the reabsorption of filtered toxins into the lumen of the tubule, these cells are at greater risk for injury. In fact, drug-induced kidney injury is a serious problem in clinical practice and accounts for roughly 20% of cases of acute kidney injury (AKI) among hospitalized patients. Therefore, its early detection is becoming more important. Usually, drug-induced AKI consists of two patterns of renal injury: acute tubular necrosis (ATN) and acute interstitial nephritis (AIN). Whereas AIN develops from medications that incite an allergic reaction, ATN develops from direct toxicity on tubular epithelial cells. Among several cellular mechanisms underlying ATN, oxidative stress plays an important role in progression to ATN by activation of inflammatory response via proinflammatory cytokine release and inflammatory cell accumulation in tissues. This review provides an overview of drugs associated with AKI, the role of oxidative stress in drug-induced AKI, and a biomarker for drug-induced AKI focusing on oxidative stress.

Overexpression of Intrarenal Renin-Angiotensin System in Human Acute Tubular Necrosis.

BACKGROUND/AIMS: Acute tubular necrosis (ATN), a leading cause of acute kidney injury (AKI), is associated with decreased survival and increased progression of chronic kidney disease. A barrier to improving the clinical outcomes is the incomplete understanding of the pathogenesis of AKI. Our objective is to test the hypothesis that intrarenal renin-angiotensin system (RAS) is overexpressed in patients with ATN and could be an indicator of ATN severity. METHODS: A transversal study was conducted in patients with biopsy-proven ATN. Intrarenal expression of angiotensinogen and angiotensin II, and urinary angiotensinogen were measured. RESULTS: Patients with ATN demonstrated upregulation of intrarenal RAS, evidenced by upregulation of intrarenal angiotensinogen and angiotensin II. Patients with ATN also have elevated urinary angiotensinogen level that correlated with the overexpressed intrarenal RAS. Moreover, this increase in intrarenal RAS expression and urinary angiotensinogen was associated with the extent of acute tubular injury and urinary albumin excretion, respectively. CONCLUSIONS: We demonstrate that the intrarenal RAS is upregulated in patients with ATN and is associated with the severity of ATN. Urinary angiotensinogen reflects intrarenal RAS status, and is of value to assess the severity of ATN.

Reduction in Renal Ischemia-Reperfusion Injury in Mice by a Phosphoinositide 3-Kinase p110gamma-Specific Inhibitor.

BACKGROUND: Although renal ischemia-reperfusion injury (IRI) can cause delayed graft function, a targeted therapy is not yet available. Because phosphoinositide 3-kinases (PI3K) p110γ and p110δ play important roles in immune cell migration and function, we investigated the effects of PI3K p110γ- and p110δ-specific inhibitors in a murine renal IRI model. METHODS: Renal function was assessed by serum creatine and hematoxylin-eosin staining. Immune cell migration was assessed by flow cytometry and an in vitro cell migration assay using Transwell plates. Gene expression analysis and a multiplex cytokine/chemokine assay were performed to find cytokines/chemokines whose expression was upregulated in renal IRI and affected by p110γ-specific inhibitor. RESULTS: The PI3K p110γ-specific inhibitor, but not p110δ-specific inhibitor, significantly reduced serum creatine levels and acute tubular necrosis. These were accompanied by reduced infiltration of B cells and reduced expression of CXCL9, a CXCR3 ligand, suggesting that p110γ plays an important role in B-cell migration toward injured kidneys. An in vitro cell migration assay revealed for the first time that B-cell migration to injured kidney cells and to CXCL9 requires p110γ. CONCLUSIONS: p110γ-specific inhibitor ameliorates renal IRI by reducing necrosis and immune cell migration. This inhibitor may have the potential to reduce renal graft failure caused by renal IRI.

Renal Klotho expression in patients with acute kidney injury is associated with the severity of the injury.

BACKGROUND/AIMS: The potential physiologic roles of Klotho in acute kidney injury (AKI) have recently been demonstrated in animal models. However, to date, there have been no human studies investigating the expression of renal Klotho in AKI. METHODS: We retrospectively collected biopsy specimens and clinical data of AKI patients between January 2001 and December 2012. Klotho expression was determined by immunohistochemical staining, and the clinical-pathological correlation was examined. RESULTS: Among the 34 patients diagnosed with acute tubular necrosis or acute tubulointerstitial nephritis, 21 patients without chronic histological lesions were included. The mean age was 37.3 ± 18.5 years and the mean peak creatinine level was 8.2 ± 5.5 mg/dL. In total, 10 patients (47.6%) received temporary renal replacement therapy (RRT); however, 17 patients (81%) showed functional recovery with creatinine levels of < 1.3 mg/dL after 1 month. The intensity of Klotho expression was scored as a percentage of Klotho-positive area. The renal Klotho score showed a significant negative correlation with the initial or peak creatinine level. When the patients were divided into three groups according to the Klotho score (low, middle, high), the low group had a significantly higher peak creatinine level and a more frequent requirement for RRT. However, the Klotho score was not a significant predictor of renal recovery. CONCLUSIONS: The results demonstrated that renal Klotho expression in humans decreased significantly according to the severity of AKI, regardless of the etiology, and that low expression was associated with a poor short-term outcome.

Novel biomarkers indicating repair or progression after acute kidney injury.

PURPOSE OF REVIEW: Although recovery of kidney function following acute kidney injury (AKI) is not uncommon, it is often incomplete and associated with the development of chronic kidney disease (CKD). In order to improve AKI management, there is a critical need to develop a series of tests and biomarkers to detect renal function recovery and identify patients with progressive kidney disease. This article examines the current body of literature in the field. RECENT FINDINGS: The recently established consensus definition for AKI has resulted in significant advances in pathophysiologic understanding, patient identification, and disease prognostication. Unfortunately, the definition for renal recovery following AKI remains inconsistent. Proteinuria and microalbuminuria - classical markers of CKD progression - have been used and validated for the progression of AKI to CKD. Data on the performance of other biomarkers of kidney repair and the progression toward CKD are very limited. Specifically, the role of novel biomarkers including neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and nephronectin (NPNT) in the recovery process has been studied, but it has not reached the point of widespread clinical implementation. SUMMARY: There is a critical need for translational and clinical investigations to verify the performance of potential kidney injury repair and progression biomarker candidates in the clinical setting.

Acute tubular necrosis (ATN) presenting with an unusually prolonged period of marked polyuria heralded by an abrupt oliguric phase.

A 50-year-old African-American man presented with acute tubular necrosis (ATN) secondary to hypotension from non-typhoid Salmonella gastroenteritis and bacteraemia. The oliguric phase lasted only 24 h followed by prolonged polyuria for 20 days, with urine output in excess of 16 L/day at maximum. As indexed in PubMed this is only the second published case of this nature since 1974, in which an abrupt oliguric phase of 24 h or less heralded prolonged polyuria in ATN. The diagnosis is challenging as fractional excretion of sodium early in the clinical course and rapid normalisation of serum creatinine with intravenous fluids (IVF) may point towards prerenal azotaemia resulting in a premature discharge from hospital. Patients with an abrupt oliguric phase may suffer a secondary renal insult from the profound fluid loss that is to follow and may need inpatient monitoring with supplemental IVF to prevent deleterious outcomes.

Molecular mediators of favism-induced acute kidney injury.

Intolerance to fava beans in subjects with glucose-6-phosphate-dehydrogenase deficiency (favism) may lead to severe hemolytic crises and decreased renal function. Renal biopsy findings exploring the molecular mechanisms of renal damage in favism have not been previously reported. We report a case of favism-associated acute kidney injury in which renal biopsy showed acute tubular necrosis and massive iron deposits in tubular cells. Interestingly, iron deposit areas were characterized by the presence of oxidative stress markers (NADPH-p22 phox and heme-oxigenase-1) and macrophages expressing the hemoglobin scavenger receptor CD163. In addition, iron deposits, NADPH-p22 phox, hemeoxigenase- 1 and CD163 positive cells were observed in some glomeruli. These results identify both glomerular and tubular involvement in favism-associated acute kidney injury and suggest novel therapeutic targets to prevent or accelerate recovery from acute kidney injury.