Clinical significance of amphiregulin in patients with chronic kidney disease.

BACKGROUND: Amphiregulin (AREG) is a ligand of epidermal growth factor receptor (EGFR), which plays an important role in injury-induced kidney fibrosis. However, the clinical significance of serum soluble AREG in chronic kidney disease (CKD) is unclear. In this study, we elucidated the clinical significance of serum soluble AREG in CKD by analyzing the association of serum soluble AREG levels with renal function and other clinical parameters in patients with CKD. METHODS: In total, 418 Japanese patients with CKD were enrolled, and serum samples were collected for the determination of soluble AREG and creatinine (Cr) levels, and other clinical parameters. Additionally, these parameters were evaluated after 2 and 3 years. Moreover, immunohistochemical assay was performed ate AREG expression in the kidney tissues of patients with CKD. RESULTS: Soluble AREG levels were positively correlated with serum Cr (p < 0.0001). Notably, initial AREG levels were positively correlated with changes in renal function (ΔCr) after 2 (p < 0.0001) and 3 years (P = 0.048). Additionally, soluble AREG levels were significantly higher (p < 0.05) in patients with diabetic nephropathy or primary hypertension. Moreover, AREG was highly expressed in renal tubular cells in patients with advanced CKD, but only weakly expressed in patients with preserved renal function. CONCLUSION: Serum soluble AREG levels were significantly correlated with renal function, and changes in renal function after 2 and 3 years, indicating that serum soluble AREG levels might serve as a biomarker of renal function and renal prognosis in CKD.

Knockout of the interleukin-36 receptor protects against renal ischemia-reperfusion injury by reduction of proinflammatory cytokines.

IL-36, a newly named member of the IL-1 cytokine family, includes 3 isoforms, IL-36α, IL-36ß, and IL-36γ, all of which bind to a heterodimer containing the IL-36 receptor (IL-36R). Little is known about the role of the IL-36 axis in acute kidney injury (AKI) pathogenesis. Therefore, we evaluated IL-36 function in the bilateral renal ischemia-reperfusion injury model of AKI using IL-36R knockout and wild-type mice. IL-36R was found to be expressed in the kidney, mainly in proximal tubules. In IL-36R knockout mice, plasma creatinine, blood urea nitrogen, and IL-6 levels after ischemia-reperfusion injury were significantly lower than those in wild-type mice. Immunohistological analysis revealed mild tubular injury. IL-36α/ß/γ levels were increased after ischemia-reperfusion injury, and IL-36α was expressed in lymphocytes and proximal tubular cells, but post-ischemia-reperfusion injury mRNA levels of IL-6 and TNF-α were low in IL-36R knockout mice. In primary cultures of renal tubular epithelial cells, IL-36α treatment upregulated NF-κB activity and Erk phosphorylation. Notably, in patients with AKI, urine IL-36α levels were increased, and IL-36α staining in renal biopsy samples was enhanced. Thus, IL-36α/IL-36R blockage could serve as a potential therapeutic target in AKI.

Effects of topiroxostat in hyperuricemic patients with chronic kidney disease.

BACKGROUND: Hyperuricemia is associated with chronic kidney disease (CKD). Although topiroxostat, a novel, non-purine, selective xanthine oxidase inhibitor, has a strong effect against hyperuricemia, limited data are available on its renoprotective effect against CKD. METHODS: This study was conducted between October 2014 and May 2016. Thirty patients (20 male, 10 female) were administered 40 mg/day of topiroxostat twice daily. All patients were followed for a year. To elucidate the effects of topiroxostat, we evaluated the clinically documented primary indication of progression, viz. laboratory evidence of kidney function decline (reference indicator), uric acid, and hypertension in different patient groups, separated according to their baseline uProt levels and baseline eGFR. RESULTS: Topiroxostat treatment resulted in significant reduction in SUA (-1.53 mg/dL), systolic blood pressure (-8.9 mmHg), diastolic blood pressure (-5.0 mmHg), and urinary protein excretion (-795.5 mg/gCr) compared with baseline values. However, serum creatinine and urinary NAG levels, and estimated glomerular filtration rate did not change significantly. CONCLUSIONS: Topiroxostat reduced SUA levels effectively and may exhibit renoprotective effect in hyperuricemic patients with CKD. Further studies are required to clarify whether topiroxostat prevents the progression of renal disease and improves the prognosis of CKD patients.

Incidence of acute kidney injury among patients with chronic kidney disease: a single-center retrospective database analysis.

BACKGROUND: Acute kidney injury (AKI) is a serious complication among hospitalized individuals and is closely associated with chronic kidney disease (CKD). METHODS: This retrospective cohort study evaluated the incidences of AKI according to CKD stage at Kochi Medical School hospital during 1981-2011. AKI was defined and staged according to the kidney disease improving global outcomes criteria, using serum creatinine levels. RESULTS: We analyzed data from 122,653 Japanese patients (57,105 men, 46.6 %). The incidence of AKI was 7.8 % (95 % confidence interval 7.7-8.0 %). Compared to non-AKI patients, patients with stage 1-2 AKI were more likely to be men. Patients with stage 1-2 AKI were significantly older than non-AKI or stage 3 AKI patients. The incidences of AKI were 6.7, 5.9, 10.4, 18.4, 30.0, and 48.8 % among individuals with estimated glomerular filtration rates of ≥90, 60-89, 45-59, 30-44, 15-29, and <15 mL/min/1.73 m2, respectively; these were significantly different from the incidence for the baseline eGFR. The proportions of inpatients with AKI exhibited step-wise increases with more severe pre-existing reduced kidney function, and the proportions among outpatients exhibited step-wise increases with milder pre-existing reduced kidney function. CONCLUSIONS: CKD was a risk factor for AKI, and the incidence of AKI was positively associated with pre-existing reduced kidney function (CKD stage). We also found that the prevalence of AKI at early-stage CKD among outpatients was higher than expected. We suggest that outpatients should be monitored for AKI, given its unexpected incidence in that population.

Serum uric acid level as a risk factor for acute kidney injury in hospitalized patients: a retrospective database analysis using the integrated medical information system at Kochi Medical School hospital.

BACKGROUND AND OBJECTIVES: Recent studies have shown that both low and high levels of serum uric acid (SUA) before cardiovascular surgery are independent risk factors for postoperative acute kidney injury (AKI). However, these studies were limited by their small sample sizes. Here, we investigated the association between SUA levels and AKI by performing a retrospective database analysis of almost 30 years of data from 81,770 hospitalized patients. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENT: Hospitalized patients aged ≥18 years were retrospectively enrolled. AKI was diagnosed according to the Kidney Disease: Improving Global Outcomes 2012 Clinical Practice Guideline (KDIGO) criteria. Multivariate logistic regression analyses were performed to investigate the independent association between SUA levels and the incidence of AKI. SUA levels were treated as categorical variables because the relationship between SUA and the incidence of AKI has been suggested to be J-shaped or U-shaped. In addition to stratified SUA levels, we considered kidney function and related comorbidities, medications, and procedures performed prior to AKI onset as possible confounding risk factors. RESULTS: The final study cohort included 59,219 adult patients. Adjusted odds ratios of AKI incidence were higher in both the high- and low-SUA strata. Odds ratios tended to become larger in the higher range of SUA levels in women than in men. Additionally, this study showed that AKI risk was elevated in patients with SUA levels ≤7 mg/dL. An SUA level >7 mg/dL is considered the point of initiation of uric acid crystallization. CONCLUSIONS: SUA level could be an independent risk factor for AKI development in hospitalized patients. Additionally, our results might suggest that intervention to lower SUA levels is necessary, even in cases of moderate elevation that does not warrant hyperuricemia treatment. Results also showed that SUA levels that require attention are lower for women than for men.

Possible roles of tumor necrosis factor-α and angiotensin II type 1 receptor on high glucose-induced damage in renal proximal tubular cells.

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22(phox) protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22(phox) protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22(phox) protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

Sestrin-2 and BNIP3 regulate autophagy and mitophagy in renal tubular cells in acute kidney injury.

Autophagy is a cellular recycling process induced in response to many types of stress. However, little is known of the signaling pathways that regulate autophagy during acute kidney injury (AKI). Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP)3 and sestrin-2 are the target proteins of hypoxia-inducible factor (HIF)-1α and p53, respectively. The aim of this study was to investigate the roles of BNIP3 and sestrin-2 in oxidative stress-induced autophagy during AKI. We used rat ischemia-reperfusion injury and cultured renal tubular (NRK-52E) cells as in vivo and in vitro models of AKI, respectively. Renal ischemia-reperfusion injury upregulated the expression of BNIP3 and sestrin-2 in the proximal tubules, as measured by immunohistochemical staining and Western blot analysis. In vitro, NRK-52E cells exposed to hypoxia showed increased expression of BNIP3 mRNA and protein in a HIF-1α-dependent manner. In contrast, sestrin-2 mRNA and protein expression were upregulated in a p53-dependent manner after exposure to oxidative stress (exogenous H2O2). NRK-52E cells stably transfected with a fusion protein between green fluorescent protein and light chain 3 were used to investigate autophagy. Overexpression of BNIP3 or sestrin-2 in these cells induced light chain 3 expression and formation of autophagosomes. Interestingly, BNIP3-induced autophagosomes were mainly localized to the mitochondria, suggesting that this protein selectively induces mitophagy. These observations demonstrate that autophagy is induced in renal tubules by at least two independent pathways involving p53-sestrin-2 and HIF-1α-BNIP3, which may be activated by different types of stress to protect the renal tubules during AKI.

Serum level of soluble (pro)renin receptor is modulated in chronic kidney disease.

BACKGROUND: Prorenin, the precursor of renin, binds to the (pro)renin receptor [(P)RR] and triggers intracellular signaling. The ligand binding sites of (P)RR are disconnected and are present in the soluble form of the receptor in serum. Given that the clinical significance of serum prorenin and soluble (P)RR in chronic kidney disease (CKD) is unclear, we investigated the relationship between serum prorenin, soluble (P)RR, and various clinical parameters in patients with CKD. METHODS: A total of 374 patients with CKD were enrolled. Serum samples were collected, and the levels of soluble (P)RR and prorenin were measured using ELISA kits. Serum creatinine (Cr), blood urea nitrogen (BUN), uric acid (UA), hemoglobin (Hb), soluble secreted α-Klotho, and the urine protein/Cr ratio were also measured. Similarly, clinical parameters were also evaluated using serum and urine sample collected after 1 year (n = 204). RESULTS: Soluble (P)RR levels were positively associated with serum Cr (P < 0.0001, r = 0.263), BUN (P < 0.0001, r = 0.267), UA (P < 0.005, r = 0.168) levels, CKD stage (P < 0.0001, r = 0.311) and urine protein/Cr ratio (P < 0.01, r = 0.157), and inversely with estimated glomerular infiltration rate (eGFR) (P < 0.0001, r = -0.275) and Hb (P < 0.005, r = -0.156). Soluble (P)RR levels were inversely associated with α-Klotho levels (P < 0.001, r = -0.174) but did not correlate with prorenin levels. With respect to antihypertensive drugs, soluble (P)RR levels were significantly lower in patients treated with an angiotensin II receptor blocker (ARB) than in those without ARB therapy (P < 0.005). Soluble (P)RR levels were significantly lower in CKD patients with diabetes mellitus or primary hypertension than in those without these conditions (P < 0.05). In contrast, serum levels of prorenin did not correlate with parameters related to renal function. Serum prorenin levels were significantly higher in CKD patients with diabetes mellitus than in nondiabetic patients (P < 0.05), but not in CKD patients with hypertension (P = 0.09). Finally, with respect to the relationship between basal soluble (P)RR levels and the progression rates of renal function, soluble (P)RR levels were positively associated with ΔCr (P < 0.05, r = 0.159) and inversely associated with ΔeGFR (P < 0.05, r = -0.148). CONCLUSION: Serum levels of soluble (P)RR correlated with the stage of CKD. Our findings suggest that soluble (P)RR may be involved in renal injury and influence the progression of CKD.

Upregulation of HNF-1ß during experimental acute kidney injury plays a crucial role in renal tubule regeneration.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a transcription factor expressed in the kidney, liver, pancreas, and other organs. Mutations of HNF-1ß cause maturity-onset diabetes of the young type 5 (MODY5). The aims of this study were to investigate the functional roles of the HNF-1ß/suppressor of cytokine signaling-3 (SOCS-3) pathway in tubule damage after acute kidney injury (AKI) both in vivo and in vitro and to examine the effect of HNF-1ß on renal tubule formation. To clarify the significance of the HNF-1ß/SOCS-3 pathway in AKI, we used a rat ischemia/reperfusion (I/R) AKI model and cultured renal tubular cells (NRK-52E cells). Western blot analysis showed that HNF-1ß and polycystic kidney disease 2 (PKD2) expressions were increased at 3-12 h and 12-24 h after I/R, respectively. The expression level of SOCS-3 was decreased at 3-48 h. Immunohistological examination revealed that expression of HNF-1ß was increased in proximal tubules. Overexpression of HNF-1ß resulted in decreased SOCS-3 expression, activation of signal transducer and activator of transcription 3 (STAT3) and Erk, and increased [(3)H]thymidine uptake in the presence of hepatocyte growth factor. Furthermore, tubule formation in three-dimensional gels was inhibited by dominant-negative HNF-1ß. Our study shows that HNF-1ß is upregulated after AKI in proximal tubular cells and that HNF-1ß controls cellular proliferation and tubule formation by regulating SOCS-3 expression and STAT3/Erk activation. Therefore, the current study unravels the physiological and pathological significance of the HNF-1ß pathway in AKI.

Aldosterone stimulates nuclear factor-kappa B activity and transcription of intercellular adhesion molecule-1 and connective tissue growth factor in rat mesangial cells via serum- and glucocorticoid-inducible protein kinase-1.

Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. To determine the signaling pathway of aldosterone in relation to fibrosis and inflammation in mesangial cells, we investigated the effects of aldosterone on expression and activation of serum- and glucocorticoid-inducible protein kinase-1 (SGK1), the activation of nuclear factor-kappa B (NF-κB activation, and the expressions of intercellular adhesion molecule-1 (ICAM-1) and connective tissue growth factor (CTGF). Aldosterone stimulated SGK1 expression, phosphorylation (Ser-256), and kinase activity. The increments of phosphorylation and expression of SGK1 induced by aldosterone were inhibited by mineralocorticoid receptor (MR) inhibitor (eplerenone). Aldosterone stimulated NF-κB activity measured by NF-κB responsive elements, luciferase assay, and the levels of inhibitor of kappa B (IκB) phosphorylation. This aldosterone-induced activation of NF-κB was inhibited by the transfection of dominant-negative SGK1. Furthermore, aldosterone augmented the promoter activities and protein expressions of ICAM-1 and CTGF. The effects of aldosterone on ICAM-1 and CTGF promoter activities and protein expressions were inhibited by the transfection of dominant-negative SGK1 and dominant-negative IκBα. We also found that the MR antagonist significantly ameliorated the glomerular injury and enhancements in SGK1, ICAM-1, and CTGF expressions induced by 1% sodium chloride and aldosterone in vivo. In conclusion, our findings suggest that aldosterone stimulates ICAM-1 and CTGF transcription via activation of SGK1 and NF-κB, which may be involved in the progression of aldosterone-induced mesangial fibrosis and inflammation. MR antagonists may serve as useful therapeutic targets for the treatment of glomerular inflammatory disease.

Serum levels of soluble secreted α-Klotho are decreased in the early stages of chronic kidney disease, making it a probable novel biomarker for early diagnosis.

BACKGROUND: α-Klotho was first identified as an aging gene and was later shown to be a regulator of phosphate metabolism. Fibroblast growth factor 23 (FGF23) is the key regulator of phosphate metabolism. Serum levels of soluble α-Klotho in chronic kidney disease (CKD) patients have not previously been determined, especially in relation with FGF23 and creatinine levels. This study was designed to investigate whether serum soluble α-Klotho levels are modulated by renal function, age, and FGF23 level in CKD patients. This study is the first report on the utility of measuring soluble α-Klotho levels in human CKD. METHODS: A total of 292 CKD patients were enrolled. Serum samples were collected, and FGF23 and soluble α-Klotho levels were measured using enzyme-linked immunosorbent assay kits. In addition, serum creatinine, hemoglobin, albumin, calcium, and phosphate levels were measured. RESULTS: Serum soluble α-Klotho levels were associated positively with estimated glomerular filtration rate (eGFR) (P < 0.0001) and inversely with serum creatinine level (P < 0.01). Interestingly, α-Klotho levels were significantly decreased in stage 2 CKD compared with stage 1 (P = 0.0001). Serum FGF23 levels were associated positively with serum creatinine and negatively with eGFR. FGF23 levels were significantly increased in stage 5 compared with stage 1 CKD. Soluble α-Klotho was associated inversely with log-transformed FGF23 level (P < 0.01). CONCLUSION: Our data indicate that soluble α-Klotho levels are significantly decreased in stage 2 CKD compared to stage 1, and not only in the advanced stages of the disease. Soluble α-Klotho may thus represent a new biomarker for the diagnosis of CKD, especially in the early stage.

Cisplatin-induced macroautophagy occurs prior to apoptosis in proximal tubules in vivo.

BACKGROUND: Autophagy is an intracellular bulk degradation process induced by cell starvation. Autophagy was recently reported to be induced by various stresses such as hypoxia, ischemia/reperfusion, toxins, and denatured proteins, and to affect cell survival and death. Light chain 3-II (LC3-II) is specifically located on double membrane-bound autophagosomes that envelop disused proteins or organelles. METHOD: Transgenic mice in which green fluorescent protein (GFP) was fused to LC3 (LC3-GFP) were administered cisplatin (20 mg/kg). After euthanasia at times between 0 and 72 h, kidneys were excised for immunohistochemical analyses. Microscopic examinations of the generated NRK-52E cell lines stably transfected with LC3-GFP, and Western blot analyses of NRK-52E cells, were undertaken after cisplatin treatment with or without autophagy inhibitors and beclin 1 small interfering RNA (siRNA). RESULTS: Autophagosomes increased in the proximal tubular cells of transgenic mice from 12 h after cisplatin injection (20 mg/kg). The time course for this was faster than those for tubular necrosis and apoptosis. Autophagosomes also increased in NRK-52E cells after cisplatin treatment, with the time course for this being faster than that for apoptosis. When autophagy was suppressed by autophagy inhibitors or beclin 1 siRNA, the level of apoptosis was also suppressed. CONCLUSION: Autophagy occurs in proximal tubular cells after cisplatin treatment and is involved in cell death in renal tubular injury. Our data suggest that autophagy is a kind of cell damage index and that cells with activated autophagy will be scavenged by apoptosis.