Tubular Injury Markers Research Articles

Could KIM-1 and NGAL levels predict acute kidney injury after paracentesis? – preliminary study

Background Kidney dysfunction is a common complication in patients with severe liver cirrhosis. There is a need for discovery and validation of novel biomarkers for earlier AKI detection. The aim of this study was to determine if tubular injury markers: NGAL and KIM-1 could be helpful in the early diagnosis of AKI in patients undergoing therapeutic paracentesis. Methods This preliminary study included 24 adult patients diagnosed with liver cirrhosis who had been hospitalized due to massive ascites requiring paracentesis. Pre- and post-paracentesis plasma samples were taken from each patient and biomarkers were measured. Results Before paracentesis, the levels of serum and urinary NGAL were similar between patients and controls; while urinary KIM-1 was markedly increased in liver cirrhotic patients (0.76 vs. 0.24 ng/ml; respectively). Although urinary NGAL levels in AKI patients were 5-time greater than in non-AKI subgroup, the difference did not reach statistical significance (13.2 vs 1.5 pg/mL, p = 0.06). Serum NGAL level, post-procedure, was 3 times greater in AKI subgroup. Conclusion Kidney injury markers, especially serum NGAL, may be useful for the early detection of AKI. However, further research is required to determine if biomarkers of kidney injury may help identify patients with cirrhosis who would most likely benefit from early AKI prevention and treatment.

Biomarker evidence for distal tubular damage but cortical sparing in hospitalized diabetic patients with acute kidney injury (AKI) while on SGLT2 inhibitors

Background Inhibitors of sodium-glucose co-transporter-2 (SGLT2i) were found to improve renal outcome in diabetic patients in large prospective randomized trials. Yet, SGLT2i may acutely reduce kidney function through volume depletion, altered glomerular hemodynamics or intensified medullary hypoxia leading to acute tubular injury (ATI). The aim or this study was to prospectively assess the pathophysiology of acute kidney injury (AKI) in patients hospitalized while on SGLT2i, differing ATI from pre-renal causes using renal biomarkers. Methods Serum and urine Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Kidney Ischemia Molecule (KIM)-1, markers of distal and proximal tubular injury, respectively, were determined in 46 diabetic patients who were on SGLT2i upon hospitalization with an acute illness. Results Serum and urine NGAL, but not KIM-1, were significantly increased in 21 of the patients who presented with AKI upon admission, as compared with 25 patients that maintained kidney function. Both serum and urinary NGAL correlated with the degree of impaired renal function, which in many cases was likely the result of additional acute renal perturbations, such as sepsis. Conclusions Increased urinary and serum NGAL indicates that ATI, principally affecting distal tubular segments, may develop in some of the patients hospitalized with an acute illness and AKI while on SGLT2i. It is suggested that intensified medullary hypoxia by SGLT2i might be detrimental in this injury. By contrast, concomitantly unaltered KIM-1 might reflect improved cortical oxygenation by SGLT2i, and may explain an overall reduced risk of AKI with SGLT1i in large series. The independent potential of SGLT2i to inflict medullary hypoxic damage should be explored further.

Day-to-day variation of the kidney proximal tubular injury markers urinary cystatin C, KIM1, and NGAL in patients with chronic kidney disease

Background It is important to know the intraindividual variation of biomarkers to be able to distinguish a change of a biomarker due to the course of the disease from the normal biological variation of the marker. The purpose of this study was to investigate the day-to-day variability of urine markers in nephrology patients. Materials 23 nephrology patients were included in the study. First morning urine samples were collected daily for ten consecutive days and analyzed for U-cystatin C, U-KIM1, U-NGAL and U-creatinine. The day-to-day variation was calculated as concentrations of the markers and as creatinine ratios. Values deviating more than the 90th percentile of the normal intraindividual variation was used to define a disease/treatment specific change. Results The day-to-day coefficient of variation (CV) for individual patients varied between 9.6 and 100.3% for NGAL (mean 45.6%) and between 8.8 and 107.3% for the NGAL/creatinine ratio (mean 43.8%). The corresponding values for KIM1 were between 10.9 and 60.2% (mean 30.1%) and for the ratio between 8.7 and 59.8% (mean 23.4%) and for cystatin C 3.8–67.4% (mean 25.0%) and for the cystatin C/creatinine ratio 5.9–78.4% (mean 24.8%). Conclusions The similar intraindividual CV values between the renal tubules damage markers and their corresponding creatinine ratios speaks against using creatinine ratio. Using the 90th percentiles of the CV values as a limit for clinical change means that NGAL has to change by 83.3%, KIM1 by 45.5% and Cystatin C by 46.3% before the change can be considered clinically significant in patients with chronic kidney disease.

Anti-renal fibrosis and anti-inflammation effect of urolithin B, ellagitannin-gut microbial-derived metabolites in unilateral ureteral obstruction rats

Abstract Urolithin B, gut microbiota metabolite of ellagitannins, contributes toward multiple health benefits attributed to ellagitannin-rich foods. The influence of urolithin B on unilateral ureteral obstruction (UUO) induced renal fibrosis and TGF-β1 induced HK-2 cells and the underlying mechanism was investigated. The results showed UUO-induced kidney injury was significantly ameliorated as shown by the improvement of renal function (BUN and Cr), kidney morphology and the suppression of renal tubular injury markers of TGF-β1, Ang II, alpha-SMA and Col IV mRNA levels. Meanwhile, the renal inflammatory induced by UUO were also strikingly attenuated in urolithin B-treated rats (TNF-α, IL-6 and MCP-1). In vitro, urolithin B treatment markedly inhibited TGF-β1-induced HK-2 cells fibrosis and proliferation, suppressed TGF-β1 and NF-κB activation. This works indicated that urolithin B contributes critically to renal inflammation in vivo and in vitro, involving the inhibition on TGF-β/Smad and TLR4/NF-кB signaling activation.

Protective Effects of Melatonin Against Aristolochic Acid-Induced Nephropathy in Mice.

Melatonin, a pineal hormone, is well known to regulate the sleep–wake cycle. Besides, the hormone has been shown to display pleiotropic effects arising from its powerful anti-oxidant and anti-inflammatory activities. Recent studies have reported that melatonin exerts protective effects in animal models of kidney disease. However, the potential effects of melatonin on aristolochic acid (AA)-induced nephropathy (AAN) have not yet been investigated. Here, we found that the administration of melatonin ameliorated AA-induced renal dysfunction, as evidenced by decreased plasma levels of blood urea nitrogen and creatinine and histopathological abnormalities such as tubular dilatation and cast formation. The upregulation of tubular injury markers after AA injection was reversed by melatonin. Melatonin also suppressed AA-induced oxidative stress, as evidenced by the downregulation of 4-hydroxynonenal and reduced level of malondialdehyde, and modulated expression of pro-oxidant and antioxidant enzymes. In addition, p53-dependent apoptosis of tubular epithelial cells, infiltration of macrophages and CD4+ T cells into damaged kidneys, and renal expression of cytokines and chemokines were inhibited by melatonin. Moreover, melatonin attenuated AA-induced tubulointerstitial fibrosis through suppression of the tumor growth factor-β/Smad signaling pathway. These results suggest that melatonin might be a potential therapeutic agent for AAN.

Association between 24-h urine sodium and proteinuria among hospitalized patients with type 2 diabetes

AimsThis study used estimated sodium intake from 24-h urine sodium (24hUNa) to explore the relationship of sodium intake with proteinuria among hospitalized patients with type 2 diabetes and with renal tubular injury markers [retinol-binding protein (RBP), beta 2-microglobulin (β2-MG), N-acetyl-beta-D-glucosaminidase (NAG)]. MethodsHospitalized patients with type 2 diabetes (N = 269) were divided into two groups according to the median (0.08 g/day) 24-h urinary protein (24hUpro) level. Logistic regression was used to analyze the association between 24hUNa and 24hUpro ≥ 0.08 g/L; scatter plots were used to analyze the association of RBP, β2-MG, and NAG with 24hUNa. ResultsOverall, 269 patients with type 2 diabetes mellitus were enrolled (average age, 56 ± 12 years; men, 61.3%). Multivariate logistic regression analysis revealed a positive correlation between 24hUNa and 24hUpro ≥ 0.08 g/L; every 10 mmol of 24hUNa had an increased risk of 24hUpro elevation [odds ratio (OR) (95% confidence interval [CI]: 1.06 (1.01–1.11)]. Compared with the lowest quartile of 24hUNa, the highest quartile had an increased risk of 24hUpro elevation [OR (95% CI): 2.76 (1.25–6.05)]; 24hUNa did not correlate with RBP, β2-MG, or NAG. ConclusionsIn hospitalized patients with type 2 diabetes, 24hUNa was independently related to 24hUpro ≥ 0.08 g/day. However, no correlation of 24hUNa with RBP, β2-MG, or NAG was found.

Phase-I Study of ETA Receptor Antagonist Ambrisentan in Sickle Cell Disease

Endothelin-1 (ET1) is a potent vasoconstrictor, mitogen, proinflammatory mediator and a mediator of nociception. Its synthesis is increased by hypoxia, ischemia, shear stress, oxidative stress and reduced nitric oxide (NO) bioavailability, all of which are well documented mechanisms in the pathophysiology of sickle cell disease. Two distinct receptors, ETA and ETB mediate opposing actions of ET1; while ETA mediates vasoconstriction, inflammation, cellular adhesion and vascular remodeling, ETB stimulation leads to increased NO production, vasodilatation, anticoagulation and anti-adhesion. We studied the role of ET1 in SCD in transgenic mouse models, and showed that selective ETA receptor blockade with ambrisentan provides renal protection by preventing the development of glomerular hyperfiltration, and preventing proteinuria (Kasztan et al, 2017; Taylor et al, 2019; Kasztan & Pollock, 2019).Additionaly, ETA receptor blockade was shown to decrease pulmonary inflammation in response to different stimuli, including hypoxia/reoxygenation and LPS (Meiler et al). Lutz et al (2018) showed that pharmacologic inhibition, or neuron specific knockdown of ETA receptor in primary sensory neurons of dorsal root ganglia in Berk mice alleviated basal and post-hypoxia evoked pain hypersensitivity. We then conducted a Phase 1, double blind, placebo controlled study to elucidate the role of ET-1 in 26 patients with SCD (SS or Sb0thal); patients were randomized to either ambrisentan 5 mg/day (N=13) or placebo (N=13) for 12 weeks. The primary end point was safety and tolerability. We also measured microalbuminuria, pressure pain threshold, transcranial doppler (TCD), echocardiograms (TR jet velocity), vascular endothelial function by flow mediated dilation (FMD), and inflammatory cytokines. Urine specimens were collected for measurement of markers of glomerular and tubular injury (NGAL, KIM-1, netrin and nephrin) at baseline and at the end of study (Day 85). Ambrisentan was well tolerated without any adverse events or fluid retention. No change in systolic and diastolic blood pressures were observed in either group (p=0.88, and p=049, respectively). No significant change in weight or serum creatinine was observed in the ambrisentan group (69.1→68.6 Kg, p=0.99, and 0.74→0.69 mg/dl, p=0.43). In the ambrisentan group, there was a tendency toward reduced microalbuminuria, (-36.6 mg/g Cr, vs +91.6), especially in the subgroup of patients who had been on a stable dose of ACE or ARB for 6 months (n=6, 286.1 mg/g Cr at baseline to 197.7 mg/g on day 85, p=0.06). FMD measurements showed increased arterial diameter, and improved microvascular function. Data collected for secondary end points including, TCD, TRV, inflammatory cytokines, markers of glomerular and tubular injury, quantitative sensory testing (QST) with pressure pain measurements, and von Frey monofilament readings, as well as quality of life measures (ASCQMe) are in the process of being analyzed. These data suggest that ETA receptor blockade is safe, well tolerated and has the potential to impact various aspects of disease pathophysiology in SCD. Disclosures Kutlar: Novartis: Consultancy; Global Blood Therapeutics, Inc. (GBT): Research Funding; Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Micelle Biopharma: Other: DSMB Chair. OffLabel Disclosure: Ambrisentan is an approved FDA drug used for treatment of another condition but it is expected that it will also help improve and/or prevent kidney damage that can be caused by sickle cell disease.

Autophagy-endoplasmic reticulum stress inhibition mechanism of superoxide dismutase in the formation of calcium oxalate kidney stones

BackgroundNephrolithiasis is a common disease in urology, and its pathogenesis is associated with various factors. Recent studies have shown that reactive oxygen species (ROS) can promote autophagy in the formation of kidney stones and exacerbate kidney injury. Endoplasmic reticulum stress (ERS), a key factor in regulating intracellular environmental homeostasis, is also directly related to ROS production. Therefore, this study aimed to investigate the regulatory effect of superoxide dismutase (SOD) on autophagy-ERS response during the formation of calcium oxalate (CaOx) kidney stones in rats. MethodsThirty-two rats were randomly divided into four groups (n = 8): normal control group, stone model group, stone model with atorvastatin group, and stone model with diethyldithiocarbamic acid (DETC) group. Rat models of CaOx kidney stones were established by intragastric administration of 0.75 % ethylene glycol for 4 weeks. Kidney/body weight was used to assess renal enlargement. Renal function was assessed by measuring serum SOD, creatinine (CRE), and blood urea nitrogen (BUN) levels. The expression of autophagy-related proteins LC3B and BECN1 was detected through immunohistochemical staining. Meanwhile, the expression of autophagy-ERS response-related proteins LC3B, BECN1, p62, GRP78, and CHOP was detected using Western blot and RT-PCR. Renal tubular injury markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (Kim-1) were determined through enzyme-linked immunosorbent assay. The apoptosis of renal tubular cells and the expression of their signature proteins cleaved Caspase-3, Bax and Bcl-2 were detected using Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Western blot assays, respectively. Crystal deposition and histological tissue injury were assessed through Von Kossa staining. ResultsCompared with the control group, the stone model group showed higher kidney/body weight ratio; evidently higher expression of autophagy-ERS response- and apoptosis-related proteins LC3B, BECN1, GRP78, CHOP, Bax and cleaved Caspase-3; and lower levels of p62, bcl-2 protein, and SOD. The stone model group also showed higher levels of apoptosis, serum CRE, BUN, NGAL, and Kim-1, as well as considerably greater crystal deposition and renal injury, than the control group. Atorvastatin reduced the levels of autophagy-ERS response, kidney injury, and crystal deposition, but they were increased by DETC. ConclusionEnhanced SOD activity can protect the kidneys by reducing autophagy-ERS response and CaOx kidney stone formation. Atorvastatin may be a new option for the prevention and treatment of nephrolithiasis.

Dietary phosphate restriction attenuates polycystic kidney disease in mice

Studies in rodents with reduced nephron mass have suggested a strong positive correlation between dietary phosphate consumption and CKD progression. Prior work by our group demonstrated that dietary phosphate restriction can prevent tubular injury and microcyst formation in rodents with glomerulonephritis. Tubular injury and cystic dilation of tubules are key contributors to kidney function decline in polycystic kidney disease (PKD). Here, we determined whether dietary phosphate restriction slows renal cyst growth and fibrosis in a mouse model of PKD. Pcy/pcy mice received a normal phosphate (0.54%) or a phosphate-restricted (0.02%) diet (n = 10/group) from 7 to 20 wk of age. All of the other major dietary constituents, including protein source and content, were comparable between the two diets. At 20 wk, body weight, kidney weight-to-body weight ratio (KW/BW), cystic area, cyst number, and kidney fibrosis were quantified. Pcy/pcy mice fed a phosphate-restricted diet had lower serum phosphate, fibroblast growth factor 23, and parathyroid hormone levels, along with elevated serum calcium levels and increased kidney Klotho gene expression compared with mice that consumed the control diet. Dietary phosphate restriction resulted in a 25% lower KW/BW ratio and reduced the cyst number, cystic index, and gene expression for the tubular injury markers neutrophil gelatinase-associated lipocalin and interleukin-18. Mice fed the phosphate-restricted diet exhibited lower kidney expression for pathways involved in collagen deposition and myofibroblast activation (collagen type I-α1, phosphorylated SMAD3, and α-smooth muscle actin); however, histological differences in kidney fibrosis were not appreciated. Dietary phosphate restriction slows cystogenesis and inhibits the activation of key pathways in the generation of kidney fibrosis in PKD mice.

Effects of Blood Transfusion on Hepatic Ischemia-Reperfusion Injury-Induced Renal Tubular Injury.

Hepatic ischemia-reperfusion injury and transfusion of red blood cells in liver surgery are wellknown risk factors to induce acute tubular injury. Transfusion of stored red blood cells may affect hepatic ischemia-reperfusion injury-induced acute tubular injury. Here, we hypothesized whether preischemic (due to increased severity of hepatic injury) and postischemic (due to renal uptake of free heme and iron) transfusion of stored red blood cells may potentiate acute tubular injury in rats subjected to hepatic ischemia-reperfusion injury. Sprague Dawley rats (n = 24) were divided into 4 groups: sham operation (sham group), hepatic ischemia-reperfusion injury only (injury-only group), red blood cell transfusion before hepatic ischemia-reperfusion injury (preinjury transfusion group), and red blood celltransfusion after hepatic ischemia-reperfusion injury (postinjury transfusion group). Partial hepatic ischemia was induced for 90 minutes, with reperfusion allowed for 12 hours. Hepatic and renal tubular injury markers, renal mRNA levels of oxidant stress markers, and inflammatory markers were assessed. Renal cortex samples were examined under hematoxylin and eosin staining for tubular histopathologic score and immunohistochemical staining forinflammatory cells. With regard to hepatic and renal tubular injury markers, serum alanine aminotransferase, serum urea nitrogen, and histopathologic scores were increased in the preinjury and postinjury transfusion groups versus injury-only group, with moderate to strong correlation between alanine aminotransferase and tubular injury markers. Renal oxidative stress markers (heme oxygenase-1 and neutrophil gelatinaseassociated lipocalin) were correlated with increased alanine aminotransferase, with upregulation of oxidant stress markers in the preinjury transfusion group versus sham group (all markers), as well as in the injury-only and postinjury transfusion groups (heme oxygenase-1 only). We observed no changes in renal inflammatory responses among the groups. Preischemic transfusion potentiated acute tubular injury without triggering renal inflammatory responses. Exacerbation of hepatic injury may induce acute tubular injury via renal oxidant stress.

Abstract P123: Traditional Chinese Herbal Qian Yang Yu Yin Granules Treat Renal Damage in Spontaneously Hypertensive Rats by Inhibiting Renal Fibrosis

Background: Qian Yang Yu Yin Granules (QYYYG) is a Chinese herbal formula. It has been used in the clinical treatment of hypertensive renal impairment for more than 30 years. The pilot study curative effect is distinct. Methods: We elucidated the mechanisms underlying the therapeutic benefits of QYYYG, validate the drug-target predicted by the systems pharmacology analysis. WKY as control group. SHR were randomly divided into 3 groups: SHR, SHR+ Valsartan(0.03g/kg/d) and SHR +QYYY(5g/kg/d). WKY and SHR were gavage by distilled water. ELISA detected renal damage, tubular injury and fibrosis markers. HE and Masson staining to observe the general view and fibrosis degree. Results: After 4 weeks treatment, QYYYG reduced the levels of blood pressure and the expression of mALB, α1-MG, β2-MG, NGAL, KIM-1 and IL-18. HE staining of SHR kidneys showed classical pathological lesions including accumulation of extracellular matrix, infiltration of inflammatory cells and apparent interstitial fibrosis where renal fibrosis. These pathologic changes were reversed by QYYYG. The serum levels of LN, HA, pro-Col III and Col IV was also decreased. Similar results were found with Masson staining. RT-qPCR selected confirmed the reduced expression of TGF-β and the downstream fibrotic genes, closely associated with EMT in renal fibrosis. Overall, the therapeutic effects of QYYYG on renal fibrosis were consistent with those predicted by pathway analysis. Conclusions: Network-based analyses such as those described here may contribute to the holistic understanding and pharmacological evaluation of herbal remedies. QYYYG was validated as an effective treatment for hypertensive renal fibrosis.

Melatonin Attenuates Cisplatin-Induced Acute Kidney Injury through Dual Suppression of Apoptosis and Necroptosis.

Melatonin is well known to modulate the sleep–wake cycle. Accumulating evidence suggests that melatonin also has favorable effects such as anti-oxidant and anti-inflammatory properties in numerous disease models. It has been reported that melatonin has therapeutic effects against cisplatin-induced acute kidney injury (AKI). However, mechanisms underlying the therapeutic action of melatonin on the renal side-effects of cisplatin therapy remain poorly understood. In this study, we showed that melatonin treatment significantly ameliorates cisplatin-induced acute renal failure and histopathological alterations. Increased expression of tubular injury markers was largely reduced by melatonin. Melatonin treatment inhibited caspase-3 activation and apoptotic cell death. Moreover, protein levels of key components of the molecular machinery for necroptosis were decreased by melatonin. Melatonin also attenuated nuclear factor-κB activation and suppressed expression of pro-inflammatory cytokines. Consistent with in vivo findings, melatonin dose-dependently decreased apoptosis and necroptosis in cisplatin-treated mouse renal tubular epithelial cells. Collectively, our findings suggest that melatonin ameliorates cisplatin-induced acute renal failure and structural damages through dual suppression of apoptosis and necroptosis. These results reveal a novel mechanism underlying the therapeutic effect of melatonin against cisplatin-induced AKI and strengthen the idea that melatonin might be a promising therapeutic agent for the renal side-effects of cisplatin therapy.

Acute Kidney Injury and Fluid Overload in Pediatric Cardiac Surgery.

Acute kidney injury (AKI) and fluid overload affect a large number of children undergoing cardiac surgery, and confers an increased risk for adverse complications and outcomes including death. Survivors of AKI suffer long-term sequelae. The purpose of this narrative review is to discuss the short and long-term impact of cardiac surgery associated AKI and fluid overload, currently available tools for diagnosis and risk stratification, existing management strategies, and future management considerations. Improved risk stratification, diagnostic prediction tools and clinically available early markers of tubular injury have the ability to improve AKI-associated outcomes. One of the major challenges in diagnosing AKI is the diagnostic imprecision in serum creatinine, which is impacted by a variety of factors unrelated to renal disease. In addition, many of the pharmacologic interventions for either AKI prevention or treatment have failed to show any benefit, while peritoneal dialysis catheters, either for passive drainage or prophylactic dialysis may be able to mitigate the detrimental effects of fluid overload. Until novel risk stratification and diagnostics tools are integrated into routine practice, supportive care will continue to be the mainstay of therapy for those affected by AKI and fluid overload after pediatric cardiac surgery. A viable series of preventative measures can be taken to mitigate the risk and severity of AKI and fluid overload following cardiac surgery, and improve care.

Innovative Drugs to Target Renal Inflammation in Sepsis: Alkaline Phosphatase.

Sepsis-related mortality roughly doubles when acute kidney injury (AKI) occurs and end-stage renal disease is more common in sepsis-associated AKI survivors. So far, no licensed treatment for the prevention of AKI is available, however the data on alkaline phosphatase (AP) is promising and might change this. Sepsis-associated AKI is believed to be the result of inflammation and hypoxia combined. Systemic inflammation started by recognition of ‘pathogen-associated molecular patterns’ (PAMPs) such as lipopolysaccharide (LPS) which binds to Toll-like receptor 4 and leads to the production of inflammatory mediators. Due to this inflammatory process renal microcirculation gets impaired leading to hypoxia resulting in cell damage or cell death. In the process of cell damage so called ‘danger-associated molecular patterns’ (DAMPs) are released resulting in a sustained inflammatory effect. Apart from the systemic inflammation DAMPs and PAMPs also interact with receptors in the proximal tubule of the kidney causing a local inflammatory response leading to leukocyte infiltration and tubular lesions, combined with renal cell apoptosis and ultimately to AKI. In the longer-term, inflammation-mediated inadequate repair mechanism may lead to fibrosis and development of chronic kidney disease. AP is an endogenous enzyme that dephosphorylates and thereby detoxifies several compounds, including LPS. A small phase 2 clinical trial in sepsis patients showed that urinary excretion of tubular injury markers was attenuated and creatinine clearance improved in sepsis patients treated with AP. This renal protective effect was confirmed in a second small clinical phase 2 trial in sepsis patients with AKI. Subsequently, a large trial in sepsis patients with AKI was conducted using a human recombinant AP. In 301 patients no improvement of kidney function within 7 days after enrolment was observed, but kidney function was significantly better on day 21 and day 28 and all-cause 28-day mortality was significantly lower (14.4% in AP group versus 26.7% in the placebo group). Possible explanations of this lack of short-term kidney function improvement are discussed and potential effects of AP on renal repair mechanisms, including inflammation-mediated induction of fibrosis, that may explain the beneficial longer-term effects of AP are proposed.

Molecular nephrology: types of acute tubular injury.

The acute loss of kidney function has been diagnosed for many decades using the serum concentration of creatinine - a muscle metabolite that is an insensitive and non-specific marker of kidney function, but is now used for the very definition of acute kidney injury (AKI). Fortunately, myriad new tools have now been developed to better understand the relationship between acute tubular injury and elevation in serum creatinine (SCr). These tools include unbiased gene and protein expression analyses in kidney, urine and blood, the localization of specific gene transcripts in pathological biopsy samples by rapid in-situ RNA technology and single-cell RNA-sequencing analyses. However, this molecular approach to AKI has produced a series of unexpected problems, because the expression of specific kidney-derived molecules that are indicative of injury often do not correlate with SCr levels. This discrepancy between kidney injury markers and SCr level can be reconciled by the recognition that many separate subtypes of AKI exist, each with distinct patterning of molecular markers of tubular injury and SCr data. In this Review, we describe the weaknesses of isolated SCr-based diagnoses, the clinical and molecular subtyping of acute tubular injury, and the role of non-invasive biomarkers in clinical phenotyping. We propose a conceptual model that synthesizes molecular and physiological data along a time course spanning from acute cellular injury to organ failure.

Pharmacological Activation of Sirt1 Ameliorates Cisplatin-Induced Acute Kidney Injury by Suppressing Apoptosis, Oxidative Stress, and Inflammation in Mice.

Sirtuin 1 (Sirt1) is an essential modulator of cellular metabolism and has pleiotropic effects. It was recently reported that Sirt1 overexpression in kidney tubule ameliorates cisplatin-induced acute kidney injury (AKI). However, whether pharmacological activation of Sirt1 also has a beneficial effect against the disease remains unclear. In this study, we aimed to evaluate whether SRT1720, a potent and specific activator of Sirt1, could ameliorate cisplatin-induced AKI. We found that SRT1720 treatment ameliorated cisplatin-induced acute renal failure and histopathological alterations. Increased levels of tubular injury markers in kidneys were significantly attenuated by SRT1720. SRT1720 treatment also suppressed caspase-3 activation and apoptotic cell death. Increased expression of 4-hydroxynonenal, elevated malondialdehyde level, and decreased ratio of reduced glutathione/oxidized glutathione after cisplatin injection were significantly reversed by SRT1720. In addition, SRT1720 treatment decreased renal expression of pro-inflammatory cytokines and prevented macrophage infiltration into damaged kidneys. We also showed that the therapeutic effects of SRT1720 were associated with reduced acetylation of p53 and nuclear factor kappa-B p65 and preservation of peroxisome function, as evidenced by recovered expression of markers for number and function of peroxisome. These results suggest that Sirt1 activation by SRT1720 would be a useful therapeutic option for cisplatin-induced AKI.

Nrf2 Plays a Protective Role Against Intravascular Hemolysis-Mediated Acute Kidney Injury.

Massive intravascular hemolysis is associated with acute kidney injury (AKI). Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a central role in the defense against oxidative stress by activating the expression of antioxidant proteins. We investigated the role of Nrf2 in intravascular hemolysis and whether Nrf2 activation protected against hemoglobin (Hb)/heme-mediated renal damage in vivo and in vitro. We observed renal Nrf2 activation in human hemolysis and in an experimental model of intravascular hemolysis promoted by phenylhydrazine intraperitoneal injection. In wild-type mice, Hb/heme released from intravascular hemolysis promoted AKI, resulting in decreased renal function, enhanced expression of tubular injury markers (KIM-1 and NGAL), oxidative and endoplasmic reticulum stress (ER), and cell death. These features were more severe in Nrf2-deficient mice, which showed decreased expression of Nrf2-related antioxidant enzymes, including heme oxygenase 1 (HO-1) and ferritin. Nrf2 activation with sulforaphane protected against Hb toxicity in mice and cultured tubular epithelial cells, ameliorating renal function and kidney injury and reducing cell stress and death. Nrf2 genotype or sulforaphane treatment did not influence the severity of hemolysis. In conclusion, our study identifies Nrf2 as a key molecule involved in protection against renal damage associated with hemolysis and opens novel therapeutic approaches to prevent renal damage in patients with severe hemolytic crisis. These findings provide new insights into novel aspects of Hb-mediated renal toxicity and may have important therapeutic implications for intravascular hemolysis-related diseases.

SAT-190 COMPREHENSIVE ANALYSIS OF URINARY AMINOPEPTIDASE ACTIVITIES FOR INVESTIGATING PROGNOSTIC MARKERS TO PREDICT THE PROGRESSION OF CHRONIC KIDNEY DISEASE

Renal prognosis is better correlated with the degree of tubulointerstitial damage than with that of glomerular injury (J Am Soc Nephrol. 2006: 17: 17–25), therefore a biomarker for tubular damage is important to predict chronic kidney disease (CKD) progression although a gold standard of tubular injury marker is yet to be established. Past reports showed that urinary GGT (γ-glutamyl transpeptidase) was one of renal prognostic factors (Transplant Proc. 2014; 46: 1812-8, Ren Fail. 2014; 36: 390-2). Urinary GGT activity is a tubular damage marker, considering that GGT is expressed in apical side of proximal tubule. In this viewpoint, other aminopeptidases are also expressed abundantly in proximal tubules, therefore it is possible that a certain aminopeptidase activity better reflects tubular damage than GGT. In addition, urinary aminopeptidase activity measurement has an advantage in quickness and cost-effectiveness once established. In this report, we examined comprehensive analysis of aminopeptidase activities in the urine of patients with CKD in order to search novel biomarkers to predict CKD progression, with a comparison to GGT activity. This study was approved by the ethical committee of University of Tokyo Graduate School of Medicine (11783, 11799). We collected the urine of adult patients with CKD G3b or G4. We measured urinary aminopeptidase activities by using our HMRG (hydroxymethyl rhodamine green)-based probe library, 384 kinds of fluorogenic probes for aminopeptidases. Urine was incubated with each probe for one hour and measured a fluorescent level, which was regarded as a target aminopeptidase activity. Each fluorescent probe activity was standardized by urinary creatinine concentration. We examined the association between urinary aminopeptidase activities and declines in eGFR for the past three years. We defined those whose eGFR decline rates per year, which was calculated by dividing eGFR, were over 5% as a ‘rapid’ group and the others as a ‘stable’ group. We also studied their clinical parameters obtained from medical records. A two-tailed paired Students’ t-test for continuous valuables and Fisher’s exact test for categorical valuables were used for the comparison of two groups. All statistical analysis was done with JMP Pro ver13.2.1. A p value < 0.5 was considered statistically significant. Forty patients were included in this study (70.2±13.2 years old: 19 women: estimated glomerular filtration rate (eGFR) 31.6±7.9 ml/min/1.73m2). Overall eGFR decline per year was 2.2±2.7 ml/min/1.73m2 and the decline rate was 8.3±10.7%. Twenty patients (50.0%) were in a rapid group. We analyzed 120 probes (31.3%), since the other 264 probes showed no fluorescence signals in almost all of the patients. We found several probes including ɤ-glutamyl-HMRG, a fluorogenic probe for detecting GGT, were significantly associated with CKD progression (Figure). Comparison of baseline characteristics between rapid and stable group was shown in Table.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We confirmed it was possible to measure urinary aminopeptidase activities by using our HMRG-based probes. Urinary GGT was significantly associated with eGFR decline, which was compatible with past reports. Other urinary aminopeptidase activity of cutting Lysine (Lys)-, LysLys- or Leucine-HMRG which are associated with eGFR decline may also become a novel biomarker for CKD progression.

Abstract 304: Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity

Abstract Cisplatin is an anticancer drug widely used in the treatment of many cancers, including lung cancers. Although cisplatin causes various types of adverse events, the main dose-limiting toxicity of cisplatin is nephrotoxicity. Megalin is an endocytic receptor expressed at the apical membranes of proximal tubules. We previously demonstrated that cisplatin was reabsorbed through megalin and caused kidney injury. Cilastatin, an inhibitor of renal dehydropeptidase-I and used with imipenem, blocked the binding of cisplatin to megalin and reduced the nephrotoxicity induced by cisplatin. In the current study, we precisely evaluated the effect of cilastatin-mediated suppression of cisplatin nephrotoxicity to safely enhance the antitumor activity of cisplatin. BALB/c mice were administrated cisplatin with or without cilastatin. Tubular dilation or atrophy, brush border loss, tubular cell lysis and cast formation were observed in mice treated with cisplatin alone. However, these kidney injuries were decreased or disappeared in mice treated with cisplatin and cilastatin. Cilastatin also decreased the urinary levels of N-acetyl-β-D-glucosaminidase and neutrophil gelatinase-associated lipocalin, proximal tubular injury markers. Next, SCID mice were injected s.c. with A549, a human lung cancer cell line, and treated with cisplatin with or without cilastatin. Cilastatin did not affect the antitumor activity of cilastatin. Notably, A549 did not express megalin. Combined with cilastatin, the mice were successfully treated with 1.5 times dose of cisplatin with enhanced antitumor effects of cisplatin but without nephrotoxicity. In conclusion, cilastatin effectively suppressed nephrotoxicity of cisplatin by blocking the binding of cisplatin to megalin. These findings indicated that we could administer cisplatin into cancer patients without nephrotoxicity if we used cilastatin. Moreover, we might increase the dose of cisplatin and improve the outcome of cancer patients. Citation Format: Masashi Arita, Satoshi Watanabe, Nobumasa Aoki, Miho Takahashi, Satoshi Shoji, Koichiro Nozaki, Kosuke Ichikawa, Rie Kondo, Shoji Kuwahara, Junta Tanaka, Toshiyuki Koya, Akihiko Saito, Toshiaki Kikuchi. Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 304.

Spironolactone reduces oxidative stress in living donor kidney transplantation: a randomized controlled trial.

Mineralocorticoid receptor antagonism prevents acute kidney injury induced by ischemia-reperfusion in rodent and pig preclinical models. In a pilot study, we showed that spironolactone (25 mg) reduced oxidative stress after 5 days of kidney transplant (KT). In the present study, we investigated the effects of higher doses (50 and 100 mg) of spironolactone on kidney function, tubular injury markers, and oxidative stress in living donor KT recipients. We included KT recipients aged 18 yr or older who received immunosuppression therapy with IL-2 receptor antagonist, mycophenolate mofetil, corticosteroids, and tacrolimus with negative cross-match, and compatible blood group. Patients were randomized to receive placebo (n = 27), spironolactone (50 mg, n = 25), or spironolactone (100 mg, n = 25). Treatment was given from 3 days before and up to 5 days after KT. Serum creatinine, K+, urine neutrophil gelatinase-associated lipocalin-2, heat shock protein 72, and 8-hydroxy-2-deoxyguanosine levels were assessed. As expected, kidney function was improved after KT. Serum K+ remained in the normal range along the study. There was no significant effect of spironolactone on urinary neutrophil gelatinase-associated lipocalin-2 levels, whereas the increase in urinary heat shock protein 72 levels tended to be less intense in the 100 mg spironolactone-treated group (P = 0.054). In the placebo-treated group, urinary 8-hydroxylated-guanosine levels increased on days 3 and 5 after transplantation. This effect was prevented in patients that received spironolactone. In conclusion, spironolactone reduces the acute increase in urinary oxidative stress in living donor KT recipients.