Yoshifumi Hamasaki

Mild elevation of urinary biomarkers in prerenal acute kidney injury

Prerenal acute kidney injury (AKI) is thought to be a reversible loss of renal function without structural damage. Although prerenal and intrinsic AKI frequently coexist in clinical situations, serum creatinine and urine output provide no information to support their differentiation. Recently developed biomarkers reflect tubular epithelial injury; therefore, we evaluated urinary biomarker levels in an adult mixed intensive care unit (ICU) cohort of patients who had been clinically evaluated as having prerenal AKI. Urinary L-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), N-acetyl-β-D-glucosaminidase (NAG), and albumin in patients with prerenal AKI showed modest but significantly higher concentrations than in patients with non-AKI. We also conducted a proof-of-concept experiment to measure urinary biomarker excretion in prerenal AKI caused by volume depletion. Compared with cisplatinum and ischemia-reperfusion models in mice, volume depletion in mice caused a modest secretion of L-FABP and NGAL into urine with more sensitive response of L-FABP than that of NGAL. Although no histological evidence of structural damage was identified by light microscopy, partial kidney hypoxia was found by pimonidazole incorporation in the volume depletion model. Thus, our study suggests that new AKI biomarkers can detect mild renal tubular damage in prerenal acute kidney injury.

Urinary L-type fatty acid-binding protein as a new biomarker of sepsis complicated with acute kidney injury.

Objective:This study is aimed to examine whether urinary L-type fatty acid-binding protein can detect the severity of sepsis with animal sepsis models and septic shock patients complicated with established acute kidney injury. Design:Experimental animal models and a clinical, prospective observational study. Setting:University laboratory and tertiary hospital. Subjects and Patients:One hundred fourteen human L-type fatty acid-binding protein transgenic mice and 145 septic shock patients with established acute kidney injury. Interventions:Animals were challenged by abdominal (cecal ligation and puncture) and pulmonary (intratracheal lipopolysaccharide injection) sepsis models with different severities that were confirmed by survival analysis (n = 24) and bronchoalveolar lavage fluid analysis (n = 38). Measurements and Main Results:In animal experiments, significant increases of urinary L-type fatty acid-binding protein levels were induced by sepsis (severe cecal ligation and puncture 399.0 ± 226.8 &mgr;g/g creatinine [n = 12], less-severe cecal ligation and puncture 89.1 ± 25.3 [n = 11], sham 13.4 ± 3.4 [n = 10] at 6 hrs, p < .05 vs. sham; 200 &mgr;g of lipopolysaccharide 190.6 ± 77.4 &mgr;g/g creatinine [n = 6], 50 &mgr;g of lipopolysaccharide 145.4 ± 32.6 [n = 8], and saline 29.9 ± 14.9 [n = 5] at 6 hrs, p < .05 vs. saline). Urinary L-type fatty acid-binding protein predicted severity more accurately than blood urea nitrogen, serum creatinine, and urinary N-acetyl-d-glucosaminidase levels. In clinical evaluation, urinary L-type fatty acid-binding protein measured at admission was significantly higher in the nonsurvivors of septic shock with established acute kidney injury than in the survivors (4366 ± 192 &mgr;g/g creatinine [n = 68] vs. 483 ± 71 [n = 77], p < .05). Urinary L-type fatty acid-binding protein showed the higher value of area under the receiver operating characteristic curve for mortality compared with Acute Physiology and Chronic Health Evaluation (APACHE) II and Sepsis-related Organ Failure Assessment (SOFA) scores (L-type fatty acid-binding protein 0.994 [0.956–0.999], APACHE II 0.927 [0.873–0.959], and SOFA 0.813 [0.733–0.873], p < .05). Conclusions:Our results suggest that urinary L-type fatty acid-binding protein can be a useful biomarker for sepsis complicated with acute kidney injury for detecting its severity.

Protection of Glucagon-Like Peptide-1 in Cisplatin-Induced Renal Injury Elucidates Gut-Kidney Connection

Accumulating evidence of the beyond-glucose lowering effects of a gut-released hormone, glucagon-like peptide-1 (GLP-1), has been reported in the context of remote organ connections of the cardiovascular system. Specifically, GLP-1 appears to prevent apoptosis, and inhibition of dipeptidyl peptidase-4 (DPP-4), which cleaves GLP-1, is renoprotective in rodent ischemia-reperfusion injury models. Whether this renoprotection involves enhanced GLP-1 signaling is unclear, however, because DPP-4 cleaves other molecules as well. Thus, we investigated whether modulation of GLP-1 signaling attenuates cisplatin (CP)-induced AKI. Mice injected with 15 mg/kg CP had increased BUN and serum creatinine and CP caused remarkable pathologic renal injury, including tubular necrosis. Apoptosis was also detected in the tubular epithelial cells of CP-treated mice using immunoassays for single-stranded DNA and activated caspase-3. Treatment with a DPP-4 inhibitor, alogliptin (AG), significantly reduced CP-induced renal injury and reduced the renal mRNA expression ratios of Bax/Bcl-2 and Bim/Bcl-2. AG treatment increased the blood levels of GLP-1, but reversed the CP-induced increase in the levels of other DPP-4 substrates such as stromal cell-derived factor-1 and neuropeptide Y. Furthermore, the GLP-1 receptor agonist exendin-4 reduced CP-induced renal injury and apoptosis, and suppression of renal GLP-1 receptor expression in vivo by small interfering RNA reversed the renoprotective effects of AG. These data suggest that enhancing GLP-1 signaling ameliorates CP-induced AKI via antiapoptotic effects and that this gut-kidney axis could be a new therapeutic target in AKI.

New biomarker panel of plasma neutrophil gelatinase–associated lipocalin and endotoxin activity assay for detecting sepsis in acute kidney injury

PURPOSE Septic acute kidney injury (AKI) shows an unacceptably high mortality rate. Detection of sepsis is important for the clinical management of AKI patients. This study was undertaken to evaluate 2 biomarkers of neutrophil gelatinase-associated lipocalin (NGAL) and endotoxin activity (EA) assay and their combination for detecting sepsis in AKI. MATERIALS AND METHODS Adult intensive care unit patients consisting of 40 non-AKI, 65 AKI without sepsis, 10 non-AKI with sepsis, and 24 septic AKI were examined in a cross-sectional manner. Plasma NGAL and EA values in whole blood were measured at recruitment. We evaluated whether combining 2 different biomarkers would improve the performance of each biomarker using receiver operating characteristic analysis. RESULTS Plasma NGAL was significantly higher in septic AKI patients than in the other AKI patients and non-AKI patients, whereas EA values were higher in septic patients than nonseptic patients irrespective of AKI complication. Combination of plasma NGAL and EA value increased the area under the curve of the receiver operating characteristic curve and showed better performance compared with a clinical model consisting of clinically available variables. CONCLUSION Combinations of plasma NGAL and EA, which are operating via different pathological pathways, significantly improved their detection performance in complicated conditions of septic AKI.

The high-mobility group protein B1–Toll-like receptor 4 pathway contributes to the acute lung injury induced by bilateral nephrectomy

Acute lung injury and acute kidney injury are severe complications in critically ill patients and synergistically increase mortality in intensive care units. Organ cross-talk between the kidney and the lung has been implicated recently as amplifying injury in each organ. Here we sought to identify a possible mechanism of acute kidney injury-induced acute lung injury using a mouse bilateral nephrectomy model. Toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice were more resistant to lung injury including neutrophil infiltration, increased neutrophil elastase activity, and vascular permeability caused by bilateral nephrectomy compared with TLR4-wild-type C3H/HeN mice 6 h after surgery. High-mobility group protein B1 (HMGB1) is one agonist for TLR4. Its blood concentrations were increased significantly by bilateral nephrectomy. Blockade of HMGB1 by neutralizing antibody reduced neutrophil infiltration in TLR4-wild-type C3H/HeN but not in TLR4-mutant C3H/HeJ mice. However, HMGB1 blockade in a renal ischemia reperfusion model reduced pulmonary neutrophil infiltration independent from TLR4. Thus, an enhanced HMGB1-TLR4 pathway contributes to lung injury induced by bilateral nephrectomy and the other HMGB1-dependent pathway exists in pulmonary neutrophil infiltration caused by renal ischemia reperfusion. Targeting the HMGB1-TLR4 pathway might enable development of a new therapeutic strategy to improve the outcomes of severely ill patients with both acute lung and acute kidney injury.

Evaluation of urinary tissue inhibitor of metalloproteinase-2 in acute kidney injury: a prospective observational study

IntroductionTissue inhibitor of metalloproteinase-2 (TIMP-2) is an emerging acute kidney injury (AKI) biomarker. We evaluated the performance of urinary TIMP-2 in an adult mixed ICU by comparison with other biomarkers that reflect several different pathways of AKI.MethodsIn this study, we prospectively enrolled 98 adult critically ill patients who had been admitted to the adult mixed ICU. Urinary TIMP-2 and N-acetyl-β-d-glucosaminidase (NAG) and plasma neutrophil gelatinase-associated lipocalin (NGAL), interleukin-6 (IL-6) and erythropoietin (EPO) were measured on ICU admission. We evaluated these biomarkers’ capability of detecting AKI and its severity as determined by using the Kidney Disease Improving Global Outcomes serum creatinine criteria, as well as its capacity to predict in-hospital mortality. The impact of sepsis, the leading cause of AKI in ICUs, was also evaluated.ResultsWe found AKI in 42 patients (42.9%). All biomarkers were significantly higher in AKI than in non-AKI. In total, 27 patients (27.6%) developed severe AKI. Urinary TIMP-2 was able to distinguish severe AKI from non-severe AKI with an area under the receiver operating characteristic curve (AUC-ROC) of 0.80 (95% confidence interval, 0.66 to 0.90). A total of 41 cases (41.8%) were complicated with sepsis. Although plasma NGAL and IL-6 were increased by sepsis, urinary TIMP-2 and NAG were increased not by sepsis, but by the presence of severe AKI. Plasma EPO was increased only by septic AKI. In-hospital mortality was 15.3% in this cohort. Urinary TIMP-2 and NAG, and plasma NGAL, were significantly higher in non-survivors than in survivors, although plasma IL-6 and EPO were not. Among the biomarkers, only urinary TIMP-2 was able to predict in-hospital mortality significantly better than serum creatinine.ConclusionUrinary TIMP-2 can detect severe AKI with performance equivalent to plasma NGAL and urinary NAG, with an AUC-ROC value higher than 0.80. Furthermore, urinary TIMP-2 was associated with mortality. Sepsis appeared to have only a limited impact on urinary TIMP-2, in contrast to plasma NGAL.

Preserved Na/HCO3 cotransporter sensitivity to insulin may promote hypertension in metabolic syndrome

Hyperinsulinemia can contribute to hypertension through effects on sodium transport. To test whether the stimulatory effect of insulin on renal proximal tubule sodium transport is preserved in insulin resistance, we compared the effects of insulin on abdominal adipocytes and proximal tubules in rats and humans. Insulin markedly stimulated the sodium-bicarbonate cotransporter (NBCe1) activity in isolated proximal tubules through the phosphoinositide 3-kinase (PI3-K) pathway. Gene silencing in rats showed that while insulin receptor substrate (IRS)1 mediates the insulin effect on glucose uptake into adipocytes, IRS2 mediates the insulin effect on proximal tubule transport. The stimulatory effect of insulin on glucose uptake into adipocytes was severely reduced, but its stimulatory effect on NBCe1 activity was completely preserved in insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats and patients with insulin resistance. Despite widespread reduction of IRS1 and IRS2 expression in insulin-sensitive tissues, IRS2 expression in the kidney cortex was exceptionally preserved in both OLETF rats and patients with insulin resistance. Unlike liver, acute insulin injection failed to change the expression levels of IRS2 and sterol regulatory element-binding protein 1 in rat kidney cortex, indicating that regulatory mechanisms of IRS2 expression are distinct in liver and kidney. Thus, preserved stimulation of proximal tubule transport through the insulin/IRS2/PI3-K pathway may play an important role in the pathogenesis of hypertension associated with metabolic syndrome.

Regulation of Mitochondrial Dynamics by Dynamin-Related Protein-1 in Acute Cardiorenal Syndrome

Experimental evidence has clarified distant organ dysfunctions induced by AKI. Crosstalk between the kidney and heart, which has been recognized recently as cardiorenal syndrome, appears to have an important role in clinical settings, but the mechanisms by which AKI causes cardiac injury remain poorly understood. Both the kidney and heart are highly energy-demanding organs that are rich in mitochondria. Therefore, we investigated the role of mitochondrial dynamics in kidney-heart organ crosstalk. Renal ischemia reperfusion (IR) injury was induced by bilateral renal artery clamping for 30 min in 8-week-old male C57BL/6 mice. Electron microscopy showed a significant increase of mitochondrial fragmentation in the heart at 24 h. Cardiomyocyte apoptosis and cardiac dysfunction, evaluated by echocardiography, were observed at 72 h. Among the mitochondrial dynamics regulating molecules, dynamin-related protein 1 (Drp1), which regulates fission, and mitofusin 1, mitofusin 2, and optic atrophy 1, which regulate fusion, only Drp1 was increased in the mitochondrial fraction of the heart. A Drp1 inhibitor, mdivi-1, administered before IR decreased mitochondrial fragmentation and cardiomyocyte apoptosis significantly and improved cardiac dysfunction induced by renal IR. This study showed that renal IR injury induced fragmentation of mitochondria in a fission-dominant manner with Drp1 activation and subsequent cardiomyocyte apoptosis in the heart. Furthermore, cardiac dysfunction induced by renal IR was improved by Drp1 inhibition. These data suggest that mitochondrial fragmentation by fission machinery may be a new therapeutic target in cardiac dysfunction induced by AKI.

High-throughput screening identified disease-causing mutants and functional variants of α-galactosidase A gene in Japanese male hemodialysis patients

Fabry disease is a genetic disorder caused by deficient activity of lysosomal enzyme α-galactosidase A (GLA) and end-stage renal disease (ESRD) will be present after accumulation of glycosphingolipids within the kidney. Undiagnosed atypical variants of Fabry disease, which are limited to renal involvement, were found in several ESRD patient populations. On the other hand, unexpectedly high frequencies of male subjects having the c.196G>C nucleotide change (p.E66Q) showing low α-GLA activity have been reported on Japanese and Korean screening for Fabry disease. However, several evidences indicate the c.196G>C is not a pathogenic mutation but is a functional polymorphism. In the present study, high-throughput screening of serum GLA could successfully indentify two Fabry disease patients in a cohort consisted of 1080 male hemodialysis patients. Moreover, our serum assay was able to distinguish two patients with disease-causing genetic mutations (p.G195V and p.M296I) from eight functional variants that showed relatively decreased enzyme activity (p.E66Q). In conclusion, high-throughput serum enzyme assay distinctly identified disease-causing mutants and functional variants of GLA gene in Japanese male hemodialysis patients. In addition, our results underscore the high prevalence of not only undiagnosed Fabry patients but functional variants of p.E66Q among the ESRD population.

The effect of different apheresis modalities on coagulation factor XIII level during antibody removal in ABO-blood type incompatible living related renal transplantation

Apheresis therapy is used to remove pathogenic antibodies within the recipient blood during ABO-incompatible living related renal transplantation (LRRT). Factor XIII (FXIII) is a coagulating factor. Its deficiency reportedly engenders perioperative bleeding. This study compared apheresis modalities from the perspective of the FXIII level. Cases 1-3 were treated only with double-filtration plasmapheresis (DFPP) without (case 1) or with (cases 2 and 3) fresh frozen plasma (FFP) supplementation. Cases 4 and 5 were treated with simple plasma exchange (PEx) with FFP supplementation for the last session. Cases 1-3 showed a marked (case 1, 8.6%) or moderate (case 2, 26.2%; case 3, 28.4%) decrease in FXIII on the day before the procedure after the last apheresis session, although cases 4 (81.9%) and 5 (66.2%) did not. Case 1 experienced perioperative bleeding. The last session is usually performed the day before the surgical procedure. Therefore, FXIII elimination by DFPP might cause bleeding complications because of its slow recovery. The fact warrants that the last apheresis modality during the course might be PEx from the viewpoint of FXIII depletion.