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Background: The dipstick urinalysis for proteinuria has been used for chronic kidney disease (CKD) screening at community-based health checkups; however, it has major drawbacks in that the result is only semi-quantitative and is influenced by urine concentration. Methods: We conducted urine protein/creatinine ratio (UPCR) measurements of 590 participants who showed a result of more than trace proteinuria on a dipstick analysis and evaluated the usefulness of UPCR measurements in community-based health checkups. Results: The UPCR values increased in accordance with the severity of the dipstick test findings, but statistical significance was only obtained between (±) and (1+), between (±) and (2+), and between (±) and (3+) groups. When the participants with (±) proteinuria were subjected to CGA classification (a classification of CKD by cause, glomerular filtration rate category, and albuminuria category) according to their UPCR data, a significant proportion of subjects (277, 77.0%) moved from the A2 category into A1, which is a less severe category. Conversely, 21 subjects (5.8%) were reclassified into a more severe category (A3). Thus, a dipstick test may produce a non-negligible number of false negatives as well as a large number of false positives. Similarly, the classifications of more than half of the subjects with (1+) or more severe proteinuria were changed based on their UPCR results. Conclusion: The dipstick urinalysis for proteinuria appears less reliable than expected, suggesting that the quantitative measurement of urine protein should be performed even during mass health checkups to ensure the early detection and prevention of CKD. [ABSTRACT FROM AUTHOR]

In this review, isolation and characterization of several kidney-derived molecules are described, namely carbonic anhydrase XIV, cysteine-rich protein 61, and kidney-liver-specific immunoglobulin-like protein. Features of neutrophil gelatinase-associated lipocalin (LCN2 or human neutrophil lipocalin) as a kidney differentiation inducer and renal injury biomarker and also as an iron-carrier protein are also summarized. Furthermore, the concepts of forest fire theory and the biology of siderophore-binding proteins are discussed. [ABSTRACT FROM AUTHOR]

CCNI (cysteine-rich protein 61; Cyr61) is an extracellular matrix-associated signaling molecule that functions in cell migration, adhesion, and differentiation. We previously reported that CCN1 is induced at podocytes in rat anti- Thy-I glomerulonephritis, a well-known model of reversible glomerular injury, but its expression and significance in the human kidney remain totally unknown (Sawai K, Mon K, Mukoyama M, Sugawara A, Suganami T, Koshikawa M, Yahata K, Makino H, Nagae T, Fujinaga Y, Yokoi H, Yoshioka T, Yoshimoto A, Tanaka I, Nakao K. JAm Soc Nephrol 14: 1154–1163,2003). Here we report that, in the human kidney, CCN1 expression was confined to podocytes in normal adult and embryonic glomeruli from the capillary loop stage. Podocyte CCN1 expression was decreased in IgA nephropathy, diabetic nephropathy, and membranous nephropathy, whereas it remained unchanged in minimal change disease and focal segmental glomeru- losclerosis. Downregulation of CCN1 was significantly greater in diseased kidneys with severe mesangial expansion. CCN1 protein was also localized in the thick ascending limb of Henle's loop, distal and proximal tubules, and collecting ducts, which was not altered in diseased kidneys. In vitro, recombinant CCN1 protein enhanced endothelial cell adhesion, whereas it prominently inhibited mesangial cell adhesion. CCN1 also completely suppressed mesangial cell migration, suggesting its role as a mesangial-repellent factor. In cultured podocytes, CCN 1 markedly induced the expression of cyclin-dependent kinase inhibitor p27KiP1 as well as synaptopodin in a dose-dependent manner and suppressed podocyte migration. These data indicate that CCN1 is expressed in podocytes, can act on glomerular cells to modulate glomerular remodeling, and is downregulated in diseased kidneys, suggesting that impairment of CCNI expression in podocytes may contribute to the progression of glomerular disease with mesangial expansion. [ABSTRACT FROM AUTHOR]

Analyzes the role of connective tissue growth factor (CTGF) in fibronectic expression and tubulointerstitial fibrosis. Properties of connective tissue growth factor; Effects of endogenous CTGF blockade on transforming growth factor-beta induced fibronectin expression; Discussion on fibronectin promoter activity.

Investigates the role of adrenomedullin (AM) in cultured endothelial and mesangial cells. Description about AM; Secretion of AM by endothelial cells (EC); Modulation of EC and mesangial cells.

Immunosuppressants and inhibitors of the renin angiotensin system are major reagents to treat nephrotic syndrome but their clinical effects are not necessarily satisfactory. Injection of doxorubicin in several strains of mice causes nephrotic syndrome-like disorder. Zhou et al. report that PPAR-α expression is downregulated in murine doxorubicin nephropathy and a PPAR-α agonist, fenofibrate, partially ameliorates the disorder induced likely through stabilization of nephrin expression and suppression of apoptosis in podocytes, providing a new preventive strategy. [ABSTRACT FROM AUTHOR]

Presents a study that examined whether leptin is effective in the prevention or treatment of renal injury in a mouse model of lipoatrophic diabetes (A-ZIP/F-1 mice). Characteristics of renal injury in A-ZIP/F-1 mice; Prevention of renal injury in A-ZIP/F-1 mice by crossing leptin transgenic mice; Reversal of renal injury in A-ZIP/F-1 mice by continuous leptin administration.

Background: We investigate whether Intensive uric acid (UA)-lowering therapy (ULT) provides increased renal protection compared with standard therapy in chronic kidney disease (CKD) patients. Methods: This was a multicenter randomized controlled trial. Only CKD patients with hyperuricemia were included in this study. The participants were randomly assigned to either the Intensive therapy group (target serum UA level ≥ 4.0 mg/dL and < 5.0 mg/dL) or the standard therapy group (serum UA level ≥ 6.0 mg/dL and < 7.0 mg/dL). ULT was performed using topiroxostat, a non-purine-type selective xanthine oxidase inhibitor. The primary endpoint was change in the logarithmic value of urine albumin to the creatinine ratio (ACR) between baseline and week 52 of the treatment. Results: Three hundred fifty-two patients were included in the full analysis set. In the Standard therapy group, mean serum UA was 8.23 mg/dL at baseline and 6.13 mg/dL at 52 weeks. In the Intensive therapy group, mean serum UA was 8.15 mg/dL at baseline and 5.25 mg/dL at 52 weeks. There was no significant difference in changes in log ACR at 52 weeks between the Intensive therapy and the Standard therapy groups. Conclusion: This study did not reveal the benefit of Intensive ULT to improve albuminuria levels. (UMIN000026741 and jRCTs051180146). [ABSTRACT FROM AUTHOR]

Many large-scale studies show that exogenous erythropoietin, erythropoiesis-stimulating agents, lack any renoprotective effects. We investigated the effects of endogenous erythropoietin on renal function in kidney ischemic reperfusion injury (IRI) using the prolyl hydroxylase domain (PHD) inhibitor, Roxadustat (ROX). Four h of hypoxia (7% O2) and 4 h treatment by ROX prior to IRI did not improve renal function. In contrast, 24–72 h pretreatment by ROX significantly improved the decline of renal function caused by IRI. Hypoxia and 4 h ROX increased interstitial cells-derived Epo production by 75- and 6-fold, respectively, before IRI, and worked similarly to exogenous Epo. ROX treatment for 24–72 h increased Epo production during IRI by 9-fold. Immunohistochemistry revealed that 24 h ROX treatment induced Epo production in proximal and distal tubules and worked similarly to endogenous Epo. Our data show that tubular endogenous Epo production induced by 24–72 h ROX treatment results in renoprotection but peritubular exogenous Epo production by interstitial cells induced by hypoxia and 4 h ROX treatment did not. Stimulation of tubular, but not peritubular, Epo production may link to renoprotection. [ABSTRACT FROM AUTHOR]

In proteinuric renal diseases, the serine protease (SP) plasmin activates the epithelial sodium channel (ENaC) by cleaving its γ subunit. We previously demonstrated that a high-salt (HS) diet provoked hypertension and proteinuria in Dahl salt-sensitive (DS) rats, accompanied by γENaC activation, which were attenuated by camostat mesilate (CM), an SP inhibitor. However, the effects of CM on plasmin activity in DS rats remain unclear. In this study, we investigated the effects of CM on plasmin activity, ENaC activation, and podocyte injury in DS rats. The DS rats were divided into the control diet, HS diet (8.0% NaCl), and HS+CM diet (0.1% CM) groups. After weekly blood pressure measurement and 24-h urine collection, the rats were sacrificed at 5 weeks. The HS group exhibited hypertension, massive proteinuria, increased urinary plasmin, and γENaC activation; CM treatment suppressed these changes. CM prevented plasmin(ogen) attachment to podocytes and mitigated podocyte injury by reducing the number of apoptotic glomerular cells, inhibiting protease-activated receptor-1 activation, and suppressing inflammatory and fibrotic cytokine expression. Our findings highlight the detrimental role of urinary plasmin in the pathogenesis of salt-sensitive hypertension and glomerular injury. Targeting plasmin with SP inhibitors, such as CM, may be a promising therapeutic approach for these conditions. [ABSTRACT FROM AUTHOR]

Background Aldosterone has been assumed to be one of aggravating factors in diabetic kidney disease (DKD). Natriuretic peptides/guanylyl cyclase-A/cGMP signalling has been shown to ameliorate aldosterone-induced renal injury in mice. Sacubitril/valsartan (SAC/VAL) is used clinically for chronic heart failure and hypertension, in part by augmenting natriuretic peptide bioavailability. The effects of SAC/VAL on renal pathophysiology including in DKD, however, have remained unclarified. Methods Eight-week-old male db/db mice fed on a high-salt diet (HSD) were treated with vehicle or aldosterone (0.2 μg/kg/min), and divided into four groups: HSD control, ALDO (aldosterone), ALDO + VAL (valsartan), and ALDO + SAC/VAL group. After 4 weeks, they were analysed for plasma atrial natriuretic peptide (ANP) levels, renal histology, and haemodynamic parameters including glomerular filtration rate (GFR) by FITC-inulin and renal plasma flow (RPF) by para-amino hippuric acid. Results The ALDO + SAC/VAL group showed significantly increased plasma ANP concentration and creatinine clearance, and decreased tubulointerstitial fibrosis and neutrophil gelatinase-associated lipocalin expression compared to ALDO and ALDO + VAL groups. SAC/VAL treatment increased GFR and RPF, and suppressed expression of Tgfb1, Il1b, Ccl2 , and Lcn2 genes compared to the ALDO group. The percentage of tubulointerstitial fibrotic areas negatively correlated with the RPF and GFR. Conclusion In a mouse model of type 2 diabetes with aldosterone excess, SAC/VAL increased RPF and GFR, and ameliorated tubulointerstitial fibrosis. Furthermore, RPF negatively correlated well with tubulointerstitial injury, suggesting that the beneficial effects of SAC/VAL could be through increased renal plasma flow with enhanced natriuretic peptide bioavailability. [ABSTRACT FROM AUTHOR]

Renal fibrosis is the final manifestation of chronic kidney disease (CKD); its prevention is vital for controlling CKD progression. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, is produced in the liver via the enzyme sulfotransferase (SULT) 1A1 and accumulates significantly during CKD. We investigated the toxicopathological role of IS in renal fibrosis using Sult1a1-KO mice and the underlying mechanisms. The unilateral ureteral obstruction (UUO) model was created; kidney IS concentrations, inflammation, and renal fibrosis were assessed on day 14. After UUO treatment, inflammation and renal fibrosis were exacerbated in WT mice, with an accumulation of IS in the kidney. However, they were significantly suppressed in Sult1a1-KO mice. CD206+ expression was upregulated, and β-catenin expression was downregulated in Sult1a1-KO mice. To confirm the impact of erythropoietin (EPO) on renal fibrosis, we evaluated the time-dependent expression of EPO. In Sult1a1-KO mice, EPO mRNA expression was improved considerably; UUO-induced renal fibrosis was further attenuated by recombinant human erythropoietin (rhEPO). Thus, UUO-induced renal fibrosis was alleviated in Sult1a1-KO mice with a decreased accumulation of IS. Our findings confirmed the pathological role of IS in renal fibrosis and identified SULT1A1 as a new therapeutic target enzyme for preventing and attenuating renal fibrosis. [ABSTRACT FROM AUTHOR]

Introduction: Interdialytic weight gain (IDWG) is crucial in the association between long interdialytic intervals and mortality in hemodialysis patients. The impact of IDWG on changes in residual kidney function (RKF) has not been evaluated thoroughly. This study examined the associations of IDWG in the long intervals (IDWGL) with mortality and rapid RKF decline. Methods: This retrospective cohort study included patients who initiated hemodialysis in the United States dialysis centers from 2007 to 2011. IDWGL was defined as IDWG in the two‐day break between dialysis sessions. This study examined the associations of seven categories of IDWGL (0% to <1%, 1% to <2%, 2% to <3% [reference], 3% to <4%, 4% to <5%, 5% to <6%, and ≥6%) with mortality using Cox regression models and rapid decline of renal urea clearance (KRU) using logistic regression models. The continuous relationships between IDWGL and study outcomes were investigated using restricted cubic spline analyses. Findings: Mortality and rapid RKF decline were assessed in 35,225 and 6425 patients, respectively. Higher IDWGL categories were linked to increased risk of adverse outcomes. The multivariate adjusted hazard ratios (95% confidence intervals) of all‐cause mortality for 3% to <4%, 4% to <5%, 5% to <6%, and ≥6% IDWGL were 1.09 (1.02–1.16), 1.14 (1.06–1.22), 1.16 (1.06–1.28), and 1.25 (1.13–1.37), respectively. The multivariate adjusted odds ratios (95% confidence intervals) of rapid decline of KRU for 3% to <4%, 4% to <5%, 5% to <6%, and ≥6% IDWGL were 1.03 (0.90–1.19), 1.29 (1.08–1.55), 1.17 (0.92–1.49), and 1.48 (1.13–1.95), respectively. When IDWGL exceeded 2%, the hazard ratios of mortality and the odds ratios of rapid KRU decline continuously increased. Discussion: Higher IDWGL was incrementally associated with higher mortality risk and rapid KRU decline. IDWGL level over 2% was linked to higher risk of adverse outcomes. Therefore, IDWGL may be utilized as a risk parameter for mortality and RKF decline. [ABSTRACT FROM AUTHOR]

Background: Evaluating patients' risk for acute kidney injury (AKI) is crucial for positive outcomes following cardiac surgery. Our aims were first to select candidate risk factors from pre- or intra-operative real-world parameters collected from routine medical care and then evaluate potential associations between those parameters and risk of onset of post-operative cardiac surgery-associated AKI (CSA-AKI). Method: We conducted two cohort studies in Japan. The first was a single-center prospective cohort study (n = 145) to assess potential association between 115 clinical parameters collected from routine medical care and CSA-AKI (≥ Stage1) risk in the population of patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). To select candidate risk factors, we employed random forest analysis and applied survival analyses to evaluate association strength. In a second retrospective cohort study, we targeted patients undergoing cardiac surgery with CPB (n = 619) and evaluated potential positive associations between CSA-AKI incidence and risk factors suggested by the first cohort study. Results: Variable selection analysis revealed that parameters in clinical categories such as circulating inflammatory cells, CPB-related parameters, ventilation, or aging were potential CSA-AKI risk factors. Survival analyses revealed that increased counts of pre-operative circulating monocytes and neutrophils were associated with CSA-AKI incidence. Finally, in the second cohort study, we found that increased pre-operative circulating monocyte counts were associated with increased CSA-AKI incidence. Conclusions: Circulating monocyte counts in the pre-operative state are associated with increased risk of CSA-AKI development. This finding may be useful in stratifying patients for risk of developing CSA-AKI in routine clinical practice. [ABSTRACT FROM AUTHOR]

Background Practice facilitation program by multidisciplinary care for primary care physicians (PCPs) is expected to improve chronic kidney disease (CKD) outcomes, but there is no clear evidence of its long-term effectiveness. We have previously performed a cluster-randomized controlled trial for 3.5 years (the Frontier of Renal Outcome Modifications in Japan (FROM-J) study) with two arms—group A without the program and group B with the program. We aimed to assess the long-term effectiveness of the practice facilitation program on CKD outcomes via an extended 10-year follow-up of the FROM-J study. Methods We enrolled patients who were in the FROM-J study. The primary composite endpoint comprised cardiovascular disease (CVD), renal replacement therapy initiation and a 50% decrease in the estimated glomerular filtration rate (eGFR). The secondary endpoints were survival rate, eGFR decline rate and collaboration rate between PCPs and nephrologists. Results The occurrence of the primary composite endpoint tended to be lower in group B (group A: 27.1% versus group B: 22.1%, P = 0.051). Furthermore, CVD incidence was remarkably lower in group B (group A: 10.5% versus group B: 6.4%, P = 0.001). Although both mortality and the rate of eGFR decline were identical between both groups, the eGFR decline rate was significantly better in group B than in group A only in patients with stage G3a at enrollment (group A: 2.35 ± 3.87 mL/min/1.73 m2/year versus group B: 1.68 ± 2.98 mL/min/1.73 m2/year, P = 0.02). The collaboration rate was higher in group B. Conclusions The CKD practice facilitation program for PCPs reliably decreases CVD events and may reduce the progression of cases to end-stage kidney disease. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity. [ABSTRACT FROM AUTHOR]

Background: The prognosis of lupus nephritis (LN) has improved following the introduction of effective immunosuppressive therapy and progress in supportive care. This study examined recent renal and patient prognosis for adults with LN in Japan. Methods: We conducted a nationwide retrospective cohort study of LN patients who received a renal biopsy between 2007 and 2012 that were registered in the Japan Renal Biopsy Registry. Of 623 registered adults with LN from 25 institutions and their affiliated or community hospitals, 489 were eligible for this study. Results: The median age at renal biopsy was 39 years, and 82.2% of patients were female. Renal biopsies were performed in 348 patients with new-onset LN, 106 with relapse LN, and 35 with refractory LN. The distribution of ISN/RPS 2003 Classes was as follows: I 1.6%; II 5.3%; III (± V) 27.0%; IV (± V) 47.0%; V 18.4%; VI 0.6%. During the median observation period of 63.8 months, 36 patients (7.3%) reached a doubling of serum creatinine or end-stage kidney disease (ESKD), and 28 patients (5.7%) died. The 5 year renal and patient survival rates were 93.9% and 94.7%, respectively. Multivariate analysis revealed body mass index (BMI) and estimated glomerular filtration rate (eGFR) were independent risk factors for a doubling of serum creatinine in ESKD. Age and eGFR were independent risk factors for death. Conclusion: Recent prognosis for adults with LN are relatively good in Japan. Risk factors for impaired renal function are BMI and eGFR at renal biopsy, while age and eGFR are risk factors for death. [ABSTRACT FROM AUTHOR]

Dyslipidemia is an independent risk factor for the development and progression of diabetic nephropathy (DN). In this review, we summarize mouse models with both diabetes and dyslipidemia, and their associated complications. We then discuss molecules potentially involved in deterioration of DN by dyslipidemia. We focus especially upon toll-like receptor 4 (TLR4) and one of its endogenous ligands, myeloid-related protein 8 (MRP8 or S100A8), since we have found that their mRNA levels are commonly increased in glomeruli of type 1 (streptozotocin [STZ]-induced) and type 2 ( A-ZIP/F-1 lipoatrophic) diabetic mice. Gene expression of MRP8 and Tlr4 is further upregulated during worsening of STZ-induced DN by a high fat diet (HFD). Moreover, these HFD-induced changes are accompanied by enhanced gene expression of CCAAT element binding protein β and phosphorylation of c-Jun N-terminal kinase in the kidney, which have also been reported in pancreatic β cells under diabetic-hyperlipidemic conditions. Effects of a HFD upon DN are cancelled in Tlr4 knockout mice. Macrophages are the predominant source of MRP8 in glomeruli. In cultured macrophages, combinatorial treatment with high glucose and palmitate amplifies MRP8 expression in a Tlr4-dependent manner, and recombinant MRP8 protein markedly increases gene expression of the inflammatory cytokines interleukin-1β and tumor necrosis factor α. Here, we propose 'macrophage-mediated glucolipotoxicity' via activation of MRP8/TLR4 signaling as a novel mechanism of pathophysiology for DN. [ABSTRACT FROM AUTHOR]

Background Osteocrin (OSTN), a bone-derived humoral factor, was reported to act on heart and bone by potentiating the natriuretic peptide (NP) system. Ostn gene polymorphisms have been associated with renal function decline, but its pathophysiological role in the kidney remains unclear. Methods The role of endogenous OSTN was investigated using systemic Ostn -knockout (KO) mice. As a model for OSTN administration, liver-specific Ostn -overexpressing mice crossed with KO (KO-Tg) were generated. These mice were subjected to unilateral ischemia–reperfusion injury (IRI) and renal lesions after 21 days of insult were evaluated. A comprehensive analysis of the Wnt/β-catenin pathway was performed using a polymerase chain reaction (PCR) array. Reporter plasmid-transfected proximal tubular cells (NRK52E) were used to investigate the mechanism by which OSTN affects the pathway. Results After injury, KO mice showed marginal worsening of renal fibrosis compared with wild-type mice, with comparable renal atrophy. KO-Tg mice showed significantly ameliorated renal atrophy, fibrosis and tubular injury, together with reduced expressions of fibrosis- and inflammation-related genes. The PCR array showed that the activation of the Wnt/β-catenin pathway was attenuated in KO-Tg mice. The downstream targets Mmp7, Myc and Axin2 showed similar results. MMP7 and Wnt2 were induced in corticomedullary proximal tubules after injury, but not in KO-Tg. In NRK52E, OSTN significantly potentiated the inhibitory effects of NP on transforming growth factor β1–induced activation of the Wnt/β-catenin pathway, which was reproduced by a cyclic guanosine monophosphate analog. Conclusions Ectopic Ostn overexpression ameliorated subsequent renal injury following ischemia–reperfusion. OSTN could represent possible renoprotection in acute to chronic kidney disease transition, thus serving as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Background. Podocytes play an important role in maintaining normal glomerular function. A podocyte-specific conditional knockout technology has been established by the use of transgenic mice expressing a podocyte-specific Cre recombinase to clarify the role of genes expressed in the podocytes. However, it may be difficult to examine the role of genes in certain pathologic conditions using conventional podocyte-specific knockout mice because they may be embryonically lethal or exhibit congenital renal abnormality. [ABSTRACT FROM PUBLISHER]

Urinary neutrophil gelatinase-associated lipocalin (Ngal or lipocalin 2) is a very early and sensitive biomarker of kidney injury. Here we determined the origin and time course of Ngal appearance in several experimental and clinically relevant renal diseases. Urinary Ngal levels were found to be markedly increased in lipoatrophic- and streptozotocin-induced mouse models of diabetic nephropathy. In the latter mice, the angiotensin receptor blocker candesartan dramatically decreased urinary Ngal excretion. The reabsorption of Ngal by the proximal tubule was severely reduced in streptozotocin-induced diabetic mice, but upregulation of its mRNA and protein in the kidney was negligible, compared to those of control mice, suggesting that increased urinary Ngal was mainly due to impaired renal reabsorption. In the mouse model of unilateral ureteral obstruction, Ngal protein synthesis was dramatically increased in the dilated thick ascending limb of Henle and N was found in the urine present in the swollen pelvis of the ligated kidney. Five patients with nephrotic syndrome or interstitial nephritis had markedly elevated urinary Ngal levels at presentation, but these decreased in response to treatment. Our study shows that the urinary Ngal level may be useful for monitoring the status and treatment of diverse renal diseases reflecting defects in glomerular filtration barrier, proximal tubule reabsorption, and distal nephrons.Kidney International (2009) 75, 285–294. doi:10.1038/ki.2008.499 [ABSTRACT FROM AUTHOR]

The klotho gene product and stanniocalcin (STC) 1 and 2 are recently identified molecules implicated in calcium and phosphorus homeostasis. In the present study, we investigated the regulation of STC1 and STC2 gene expression in the kidney by klotho gene expression. Mice deficient in klotho expression (klotho mice) have hypercalcemia and hyperphosphatemia, and increased renal gene expression of STC1 and STC2 compared with wild-type mice. Administration of vitamin D or CaCl2 to wild-type mice causes upregulation of STC1 but STC2 gene expression is not altered significantly. On the other hand, treatment of klotho mice with low phosphorus diet results in partial decrease in STC2 gene expression with normalization of hyperphosphatemia. These findings indicate that klotho gene expression plays a crucial role in the regulation of renal stanniocalcin gene expression in vivo, at least partly, through the control of circulating calcium and phosphate concentrations. [Copyright &y& Elsevier]

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury. [ABSTRACT FROM AUTHOR]

Background: Serial management of renal anemia using continuous erythropoietin receptor activator (CERA) throughout the peritoneal dialysis initiation period has rarely been reported. We investigated the efficacy and dosage of CERA treatment from pre- to post-peritoneal dialysis initiation for anemia management in patients with end-stage renal disease. Methods: Twenty-six patients (13 men; mean age 60.9 years) who started peritoneal dialysis between April 2012 and April 2018 were investigated. Serial changes in hemoglobin levels, transferrin saturation and ferritin levels, CERA dosage, and the erythropoietin resistance index (ERI) over a 48 week period were retrospectively examined. Results: Mean hemoglobin levels increased significantly from 10.5 g/dL at 24 weeks prior to the peritoneal dialysis initiation to 11.5 g/dL at 4 weeks post-initiation. The proportion of patients with hemoglobin levels ≥ 11 g/dL increased significantly after peritoneal dialysis initiation. The mean CERA dosage was 57.0 µg/month at 24 weeks prior to dialysis initiation, 86.5 µg/month at initiation, and 72.0 µg/month at 4 weeks post-initiation. Thus, the dosage tended to increase immediately before peritoneal dialysis initiation and then decreased thereafter. Hemoglobin levels were significantly lower, while the CERA dosage for maintaining hemoglobin levels and ERI tended to be higher at dialysis initiation in patients with diabetes than in those without diabetes. Conclusion: Treatment with CERA prior to and during the peritoneal dialysis initiation achieved fairly good anemia management in patients with and without diabetes. The CERA dosage could be reduced in patients without diabetes after dialysis initiation. [ABSTRACT FROM AUTHOR]

Chronic low-grade inflammation is receiving much attention as a critical pathology that induces various aging phenotypes, a concept known as " inflammaging ". Uremic patients undergoing hemodialysis therapy show vascular aging phenotypes characterized by greater arterial stiffness and calcification compared to healthy controls of the same generation. In the current study, we investigated whether levels of inflammaging markers in the circulation were associated with vascular aging phenotypes in hemodialysis patients, as estimated by the cardio-ankle vascular index (CAVI). We conducted a multicenter cross-sectional study of 412 patients receiving hemodialysis and evaluated the relationship between circulating hs-CRP or ANGPTL2 levels, as markers of inflammaging, and CAVI. Of 412 patients, 376 were analyzed statistically. While circulating hs-CRP levels had no significant association with CAVI, generalized linear models revealed that high circulating ANGPTL2 levels were significantly associated with increasing CAVI after adjustment for classical metabolic factors and hemodialysis-related parameters [β 0.63 (95%CI 0.07–1.18)]. Exploratory analysis revealed that high circulating ANGPTL2 levels were also strongly associated with increased CAVI, particularly in patients with conditions of increased vascular mechanical stress, such elevated blood pressure [β 1.00 (95%CI 0.23–1.76)], elevated pulse pressure [β 0.75 (95%CI 0.52–0.98)], or excess body fluid [β 1.25 (95%CI 0.65–1.84)]. We conclude that circulating levels of ANGPTL2 rather than hs-CRP are positively associated with CAVI in the uremic population and that ANGPTL2 could be a unique marker of progression of vascular aging in patients receiving hemodialysis. Image 1 • Hs-CRP and ANGPTL2 are both inflammaging-related factors. • In patients receiving hemodialysis, we evaluated the relationship between cardio-ankle vascular index (CAVI) and circulating levels of hs-CRP or ANGPTL2. • High circulating ANGPTL2 levels were significantly associated with CAVI, and no significant association for hs-CRP levels was found. • ANGPTL2 could be a useful surrogate marker in evaluating the degree of vascular aging in patients receiving hemodialysis. [ABSTRACT FROM AUTHOR]

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross‐communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single‐nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER‐associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD‐related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)‐GFP knock‐in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD. [ABSTRACT FROM AUTHOR]

The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy. [ABSTRACT FROM AUTHOR]

To examine the cell-protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes, we analyzed the renal phenotype of tamoxifen (TM)-inducible podocyte-specific Atg5-deficient (iPodo-Atg5−/−) mice with experimental endothelial dysfunction. In both control and iPodo-Atg5−/− mice, high fat diet (HFD) feeding induced glomerular endothelial damage characterized by decreased urinary nitric oxide (NO) excretion, collapsed endothelial fenestrae, and reduced endothelial glycocalyx. HFD-fed control mice showed slight albuminuria and nearly normal podocyte morphology. In contrast, HFD-fed iPodo-Atg5−/− mice developed massive albuminuria accompanied by severe podocyte injury that was observed predominantly in podocytes adjacent to damaged endothelial cells by scanning electron microscopy. Although podocyte-specific autophagy deficiency did not affect endothelial NO synthase deficiency-associated albuminuria, it markedly exacerbated albuminuria and severe podocyte morphological damage when the damage was induced by intravenous neuraminidase injection to remove glycocalyx from the endothelial surface. Furthermore, endoplasmic reticulum stress was accelerated in podocytes of iPodo-Atg5−/− mice stimulated with neuraminidase, and treatment with molecular chaperone tauroursodeoxycholic acid improved neuraminidase-induced severe albuminuria and podocyte injury. In conclusion, podocyte autophagy plays a renoprotective role against diabetes-related structural endothelial damage, providing an additional insight into the pathogenesis of massive proteinuria in diabetic nephropathy. • Diabetes causes functional and structural glomerular endothelial damage. • Autophagy protects podocytes against structural glomerular endothelial damage. • Enhanced ER stress is involved in autophagy deficiency-mediated podocyte damage. [ABSTRACT FROM AUTHOR]

Aim: Metabolic acidosis occurs due to insufficient urinary ammonium excretion as chronic kidney disease (CKD) advances. Because obese subjects tend to have excessive consumption of protein and sodium chloride, they are prone to chronic acid loading and may therefore be predisposed to acid‐induced kidney injury. We investigated the involvement of obesity in ammoniagenesis within damaged kidneys. Methods: In the clinical study, urinary ammonium excretion was compared between 13 normal‐weight and 15 overweight/obese CKD outpatients whose creatinine clearance was higher than 25 mL/min. For animal experiments, NH4Cl was loaded to KKAy/TaJcl (KKAy), a metabolic syndrome model, and control BALB/c mice for 20 weeks. Kidney injury was evaluated through histological analysis and the expression of proinflammatory markers. Results: Urinary ammonium excretion was lower in overweight/obese patients than in normal‐weight patients, while intakes of protein and sodium chloride were higher in overweight/obese patients, implying that subclinical metabolic acidosis occurs in overweight/obese patients. The increase in urinary ammonium excretion induced by NH4Cl loading was attenuated in KKAy mice after 16 weeks, whereas the increase was maintained in BALB/c mice throughout the study period. Histological study and real‐time polymerase chain reaction analysis showed proximal tubular injury and enhanced expression levels of neutrophil gelatinase‐associated lipocalin (NGAL) protein and messenger RNA, respectively, in KKAy mice but not in BALB/c mice. Finally, urinary NGAL concentration was higher in overweight/obese patients than in normal‐weight patients in the early stage of CKD. Conclusion: Obesity could facilitate the induction of subclinical metabolic acidosis and acid accumulation in the kidney, which may potentially exacerbate kidney injury in CKD patients. SUMMARY AT A GLANCE: This study investigated the involvement of obesity in ammoniagenesis within damaged kidneys and found that obesity could facilitate the induction of subclinical metabolic acidosis and acid accumulation in the kidney. [ABSTRACT FROM AUTHOR]

Abstract Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34–45 kDa and kidney erythropoietin at 36–40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition. [ABSTRACT FROM AUTHOR]

What We Already Know About This Topic: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Natriuretic peptides are used, based on empirical observations, in intensive care units as antioliguric treatments. We hypothesized that natriuretic peptides prevent lipopolysaccharide-induced oliguria by activating guanylyl cyclase A, a receptor for natriuretic peptides, in proximal tubules and endothelial cells.Methods: Normal Sprague-Dawley rats and mice lacking guanylyl cyclase A in either endothelial cells or proximal tubular cells were challenged with lipopolysaccharide and assessed for oliguria and intratubular flow rate by intravital imaging with multiphoton microscopy.Results: Recombinant atrial natriuretic peptide efficiently improved urine volume without changing blood pressure after lipopolysaccharide challenge in rats (urine volume at 4 h, lipopolysaccharide: 0.6 ± 0.3 ml · kg · h; lipopolysaccharide + fluid resuscitation: 4.6 ± 2.0 ml · kg · h; lipopolysaccharide + fluid resuscitation + atrial natriuretic peptide: 9.0 ± 4.8 ml · kg · h; mean ± SD; n = 5 per group). Lipopolysaccharide decreased glomerular filtration rate and slowed intraproximal tubular flow rate, as measured by in vivo imaging. Fluid resuscitation restored glomerular filtration rate but not tubular flow rate. Adding atrial natriuretic peptide to fluid resuscitation improved both glomerular filtration rate and tubular flow rate. Mice lacking guanylyl cyclase A in either proximal tubules or endothelium demonstrated less improvement of tubular flow rate when treated with atrial natriuretic peptide, compared with control mice. Deletion of endothelial, but not proximal tubular, guanylyl cyclase A augmented the reduction of glomerular filtration rate by lipopolysaccharide.Conclusions: Both endogenous and exogenous natriuretic peptides prevent lipopolysaccharide-induced oliguria by activating guanylyl cyclase A in proximal tubules and endothelial cells. [ABSTRACT FROM AUTHOR]

Background. Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. Methods. To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Results. Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of aSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (aSMA), Pecam1 (CD31) and Vegfa. Conclusions. These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis. [ABSTRACT FROM AUTHOR]

Background: The clinical presentation and prognosis of adult and elderly patients with IgA vasculitis (Henoch-Schönlein purpura) accompanied by nephritis (IgAV-N) have not been investigated in detail. We therefore surveyed the features and outcomes of IgAV-N based on nationwide data derived from the Japan Renal Biopsy Registry (J-RBR). Methods: This multi-center cohort study compared the clinicopathological parameters at diagnosis, initial therapies and outcomes between 106 adult (age 19–64 years) and 46 elderly (≥65 years) patients with IgAV-N who were registered in the J-RBR between 2007 and 2012. The primary end-points comprised a 50% increase in serum creatinine (sCr) values or end-stage kidney disease. Factors affecting a decrease in renal function were assessed using Cox proportional hazards models. Results: Rates of hypertension, impaired renal function, hypoalbuminemia and crescentic glomerulonephritis were significantly higher among the elderly, than the adult patients. About 80% and 60% of the patients in both groups were respectively treated with corticosteroid and a renin-angiotensin system (RAS) blockade. Both groups had favorable renal survival rates for nine years (93.6% and 91.4% of the adult and elderly patients, respectively). Significantly more elderly than adult patients developed a 50% increase in sCr during a mean observation period of 3.9 years (21.7% vs. 4.7%, p = 0.012), and significantly fewer elderly, than adult patients achieved clinical remission (23.9% vs. 46.2%, p = 0.016). Multivariate analysis selected advanced age (≥65 years) and lower serum albumin values as independent prognostic factors for a decline in renal function, whereas steroid pulse therapy helped to preserve renal function. Conclusions: The renal prognosis of adult and elderly patients with IgAV-N was favorable when treated aggressively with corticosteroid and RAS blockade. However, the course of renal function should be carefully monitored in patients aged over 65 years and those with hypoalbuminemia. [ABSTRACT FROM AUTHOR]

Purpose: Angiopoietin-like proteins (ANGPTLs) 3, 4, and 8 reportedly contribute to progression of metabolic disease, a risk factor for cardiovascular disease (CVD). The purpose of this study was to investigate whether circulating ANGPTL levels are associated with CVD risk after adjustment for potential confounding factors. Methods: We conducted a single center, cross-sectional study of 988 Japanese subjects undergoing routine health checks. Serum ANGPTL3, 4, and 8 levels were measured using an enzyme-linked immunosorbent assay. Using multiple regression analysis we evaluated potential association of circulating ANGPTL3, 4, and 8 levels with general medical status including age, sex, smoking, drinking, obesity, hypertension, impaired glycometabolism, dyslipidemia, hyperuricemia, hepatic impairment, chronic kidney disease, anemia, cardiac abnormality, and inflammation. Results: Circulating ANGPTL3 levels were relatively high in health-related categories of hepatic impairment and inflammation. Circulating ANGPTL4 levels were also significantly high in impaired glycometabolism or hepatic impairment but decreased in inflammation. Finally, increased ANGPTL8 levels were observed in obesity, impaired glycometabolism and dyslipidemia. Particularly, increased levels of circulating ANGPTL8 were positively correlated with circulating triglycerides and LDL-cholesterol levels and inversely correlated with circulating HDL-cholesterol levels. Conclusions: Circulating ANGPTL3, 4, and 8 levels reflect some risk factors for CVD development. [ABSTRACT FROM AUTHOR]

Metabolic acidosis often results from chronic kidney disease; in turn, metabolic acidosis accelerates the progression of kidney injury. The mechanisms for how acidosis facilitates kidney injury are not fully understood. To investigate whether low pH directly affects the expression of genes controlling local homeostasis in renal tubules, we performed transcription start site sequencing (TSS-Seq) using IN-IC cells, a cell line derived from rat renal collecting duct intercalated cells, with acid loading for 24 h. Peak calling identified 651 up-regulated and 128 down-regulated TSSs at pH 7.0 compared with those at pH 7.4. Among them, 424 and 38 TSSs were ≥ 1.0 and ≤ -1.0 in Log2 fold change, which were annotated to 193 up-regulated and 34 down-regulated genes, respectively. We used gene ontology analysis and manual curation to profile the up-regulated genes. The analysis revealed that many up-regulated genes are involved in renal fibrosis, implying potential molecular mechanisms induced by metabolic acidosis. To verify the activity of the ubiquitin-proteasome system (UPS), a candidate pathway activated by acidosis, we examined the expression of proteins from cells treated with a proteasome inhibitor, MG132. The expression of ubiquitinated proteins was greater at pH 7.0 than at pH 7.4, suggesting that low pH activates the UPS. The in vivo study demonstrated that acid loading increased the expression of ubiquitin proteins in the collecting duct cells in mouse kidneys. Motif analysis revealed Egr1, the mRNA expression of which was increased at low pH, as a candidate factor that possibly stimulates gene expression in response to low pH. In conclusion, metabolic acidosis can facilitate renal injury and fibrosis during kidney disease by locally activating various pathways in the renal tubules. [ABSTRACT FROM AUTHOR]

Background: Neutrophil gelatinase-associated lipocalin (NGAL or LCN2) is an iron-transporting factor which possesses various activities such as amelioration of kidney injury and host defense against pathogens. Its circulating concentrations are elevated in acute and chronic kidney diseases and show a positive correlation with poor renal outcome and mortality, but its clinical significance in maintenance hemodialysis (HD) patients remains elusive. Methods: Serum NGAL levels were determined by enzyme-linked immunosorbent assay in out-patient, Japanese HD subjects. Their correlation to laboratory findings and morbidity (as development of severe infection or serum albumin reduction) was investigated using linear regression analysis and χ2 test. Results: Pre-dialysis serum NGAL levels in HD patients were elevated by 13-fold compared to healthy subjects (n=8, P<0.001). In a cross-sectional study of 139 cases, serum NGAL concentrations were determined independently by % creatinine generation rate (an indicator of muscle mass, standardized coefficient β=0.40, P<0.001), peripheral blood neutrophil count (β=0.38, P<0.001) and anion gap (which likely reflects dietary protein intake, β=0.16, P<0.05). Iron administration to anemic HD patients caused marked elevation of peripheral blood hemoglobin, serum ferritin and iron-regulatory hormone hepcidin-25 levels, but NGAL levels were not affected. In a prospective study of 87 cases, increase in serum albumin levels a year later was positively associated to baseline NGAL levels by univariate analysis (r=0.36, P<0.01). Furthermore, within a year, patients with the lowest NGAL tertile showed significantly increased risk for marked decline in serum albumin levels (≥0.4 g/dl; odds ratio 5.5, 95% confidence interval 1.5–20.3, P<0.05) and tendency of increased occurrence of severe infection requiring admission (odds ratio 3.1, not significant) compared to the middle and highest tertiles. Conclusion: Low serum NGAL levels appear to be associated with current malnutrition and also its progressive worsening in maintenance HD patients. [ABSTRACT FROM AUTHOR]

Background: Lipocalin 2 (LCN2 or neutrophil gelatinase-associated lipocalin) is a secretory protein discovered from neutrophils, which accumulates in the blood and urine during acute kidney injury (AKI) and in the blood by bacterial infection. Little is known about the tissue source and molecular forms of this protein under normal and pathophysiologic conditions. Methods: By sandwich ELISA, serum and urinary LCN2 levels were measured in 36 patients with hematologic malignancies who transiently became neutropenic by stem cell transplantation (SCT). To evaluate contribution of neutrophil-derived LCN2 in the physiologic blood LCN2 concentrations, we examined CCAAT/enhancer-binding protein ε (C/EBPε) knockout mice, which lack mature neutrophils. Results: In patients without AKI and bacterial infection, at 1 week after SCT, the median blood neutrophil counts became zero and serum LCN2 levels were decreased by 76 ± 6 % ( p < 0.01), but urinary LCN2 levels were not altered. During neutropenic conditions, bacterial infection caused only a modest rise of serum LCN2 but AKI produced a marked rise of serum and urinary LCN2 levels. Serum LCN2 concentrations in C/EBPε knockout mice were reduced by 66 ± 11 % compared to wild-type mice ( p < 0.05). Blood LCN2 existed predominantly in high molecular weight forms (>100 kDa), while urinary LCN2 was mainly in low molecular weight forms. Conclusion: Our findings suggest that neutrophils are the major source of circulating LCN2 in normal and infected conditions, whereas blood and urinary LCN2 mainly derive from the kidney during AKI, and that the molecular forms and regulation of blood and urinary LCN2 are clearly distinct. [ABSTRACT FROM AUTHOR]

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro . Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro , NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo . These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is not merely a disorder featuring renal dysfunction, but also accelerates the progression of cardiovascular disease. Recently, the prevalence of CKD has been increasing in parallel with that of metabolic syndrome, suggesting that these two disorders are closely related. Metabolic syndrome is generally characterized by several metabolic and cardiovascular features, including obesity, insulin resistance and hypertension, all of which are known to cause renal impairment. Hence, it is likely that metabolic syndrome could be a key factor in the development of renal disease. Among several factors associated with metabolic syndrome, lipid abnormality, in particular a high level of serum low-density lipoprotein, has been emerging as a cause of kidney injury. Accumulating evidence has demonstrated that statin therapy for treating hyperlipidemia could slow the progression of renal disease, indicating that a treatment for lipid abnormality might prevent the progression of renal disease. In other words, statin therapy might be renoprotective in addition to its beneficial effect on the cardiovascular system. We are carrying out a clinical trial, the ASUCA trial, in which we examine whether statins might slow the progression of CKD in the Japanese population. We organized the multi-center clinical trial to investigate the effect of atorvastatin on estimated glomerular filtration rate in patients with CKD and lipid abnormality. In this paper, we discuss the outline and significance of this trial. [ABSTRACT FROM AUTHOR]

Background and Objective: We have reported that toll-like receptor 4 (TLR4) and one of its endogenous ligands, myeloid-related protein 8 (MRP8 or S100A8), play an important role in the progression of diabetic nephropathy in mice. The aim of this study was to evaluate significance of kidney MRP8 expression in patients with obesity- or type 2 diabetes-associated kidney diseases. Methods: In diabetic, obese or control subjects, MRP8 mRNA and protein expression levels in renal biopsy samples were determined by real-time RT-PCR and immunohistochemistry (n = 28 and 65, respectively), and their associations with baseline and prognostic parameters were analyzed. Effects of MRP8 upon pro-inflammatory gene expressions were examined using macrophages. Results: Kidney MRP8 gene and protein expression levels were elevated in obese or diabetic groups compared to control group. Among all subjects, by univariate linear regression analysis, glomerular MRP8-positive cell count and tubulointerstitial MRP8-positive area at baseline were both, respectively, correlated not only with various known risk factors for diabetic nephropathy (such as systolic blood pressure, proteinuria and serum creatinine) but also with extent of glomerulosclerosis and tubulointerstitial fibrosis. Independent factors predicting urinary protein levels a year later were examined by multivariate analysis, and they included glomerular MRP8-positive cell count (β = 0.59, P<0.001), proteinuria (β = 0.37, P = 0.002) and systolic blood pressure (β = 0.21, P = 0.04) at baseline, after adjustment for known risk factors. MRP8 protein expression was observed in CD68-positive macrophages and atrophic tubules. In cultured mouse macrophages, MRP8 protein induced proinflammatory cytokine expressions and also triggered auto-induction of MRP8 in a TLR4-dependent manner. Conclusions: Glomerular MRP8 expression appears to be associated with progression of proteinuria in obese or type 2 diabetic patients, possibly by inducing inflammatory changes in macrophages through TLR4 signaling. [ABSTRACT FROM AUTHOR]

Various bioactive peptides have been implicated in the homeostasis of organs and tissues. Adrenomedullin (AM) is a peptide with various bioactivities. AM-receptor, calcitonin-receptor-like receptor (CLR) associates with one of the subtypes of the accessory proteins, RAMPs. Among the RAMP subisoforms, only RAMP2 knockout mice (−/−) reproduce the phenotype of embryonic lethality of AM−/−, illustrating the importance of the AM-RAMP2-signaling system. Although AM and RAMP2 are abundantly expressed in kidney, their function there remains largely unknown. We used genetically modified mice to assess the pathophysiological functions of the AM-RAMP2 system. RAMP2+/− mice and their wild-type littermates were used in a streptozotocin (STZ)-induced renal injury model. The effect of STZ on glomeruli did not differ between the 2 types of mice. On the other hand, damage to the proximal urinary tubules was greater in RAMP2+/−. Tubular injury in RAMP2+/− was resistant to correction of blood glucose by insulin administration. We examined the effect of STZ on human renal proximal tubule epithelial cells (RPTECs), which express glucose transporter 2 (GLUT2), the glucose transporter that specifically takes up STZ. STZ activated the endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK). AM suppressed PERK activation, its downstream signaling, and CCAAT/enhancer-binding homologous protein (CHOP)-induced cell death. We confirmed that the tubular damage was caused by ER stress-induced cell death using tunicamycin (TUN), which directly evokes ER stress. In RAMP2+/− kidneys, TUN caused severe injury with enhanced ER stress. In wild-type mice, TUN-induced tubular damage was reversed by AM administration. On the other hand, in RAMP2+/−, the rescue effect of exogenous AM was lost. These results indicate that the AM-RAMP2 system suppresses ER stress-induced tubule cell death, thereby exerting a protective effect on kidney. The AM-RAMP2 system thus has the potential to serve as a therapeutic target in kidney disease. [ABSTRACT FROM AUTHOR]

Identifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD. Diabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx. Neuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD. [Display omitted] • Endothelial damage-related albuminuria elongates mitochondria in podocytes. • High glucose inhibits the cytoprotective response of mitochondrial elongation. • Drp1 deficiency prevents albumin-induced podocyte damage in diabetes • Inhibition of Drp1 function may be a novel therapy for proteinuria in diabetes. [ABSTRACT FROM AUTHOR]

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury. [ABSTRACT FROM AUTHOR]

OBJECTIVE--The aim of the current study was to evaluate the long-term effects of leptin on glucose metabolism diabetes complications, and life span in an insulin-dependent diabetes model, the Akita mouse. RESEARCH DESIGN AND METHODS--We cross-mated Akita mice with leptin-expressing transgenic (LepTg) mice to produce Akita mice with physiological hyperleptinemia (LepTg:Akita). Metabolic parameters were monitored for 10 months. Pair-fed studies and glucose and insulin tolerance tests were performed. The pancreata and kidneys were analyzed histologically. The plasma levels and pancreatic contents of insulin and glucagon, the plasma levels of lipids and a marker of oxidative stress, and urinary albumin excretion were measured. Survival rates were calculated. RESULTS--Akita mice began to exhibit severe hyperglycemia and hyperphagia as early as weaning. LepTg:Akita mice exhibited normoglycenfia after an extended fast even at 10 months of age. The 6-h fasting blood glucose levels in LepTg:Akita mice remained about half the level of Akita mice throughout the study. Food intake in LepTg:Akita mice was suppressed to a level comparable to that in WT mice, but pair feeding did not affect blood glucose levels in Akita mice. LepTg:Akita mice maintained insulin hypersensitivity and displayed better glucose tolerance than did Akita mice throughout the follow-up. LepTg:Akita mice had normal levels of plasma glucagon, a marker of oxidative stress, and urinary albumin excretion rates. All of the LepTg:Akita mice survived for >12 months, the median mortality time of Akita mice. CONCLUSIONS--These results indicate that leptin is therapeutically useful in the long-term treatment of insulin-deficient diabetes. [ABSTRACT FROM AUTHOR]

Kake T, Kitamura H, Adachi Y, Yoshioka T, Watanabe T, Matsushita H, Fujii T, Kondo E, Tachibe T, Kawase Y, Jishage K, Yasoda A, Mukoyama M, Nakao K. Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice. Am J Physiol Endocrinol Metab 297: El 339-El 348, 2009. First published October 6, 2009; doi: 10.1152/ajpendo.00272.2009.—C- type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia. [ABSTRACT FROM AUTHOR]

Since the 1980s, a number of bioactive molecules, now known as cardiovascular hormones, have been isolated from the heart and blood vessels, particularly from the subset of vascular endothelial cells. The natriuretic peptide family is the prototype of the cardiovascular hormones. Over the following decade, a variety of hormones and cytokines, now known as adipokines or adipocytokines, have also been isolated from adipose tissue. Leptin is the only adipokine demonstrated to cause an obese phenotype in both animals and humans upon deletion. Thus, the past two decades have seen the identification of two important classes of bioactive molecules secreted by newly recognized endocrine cells, both of which differentiate from mesenchymal stem cells. To assess the physiological and clinical implications of these novel hormones, we have investigated their functions using animal models. We have also developed and analyzed mice overexpressing transgenic forms of these proteins and knockout mice deficient in these and related genes. Here, we demonstrate the current state of the translational research of these novel hormones, the natriuretic peptide family and leptin, and discuss how lessons learned from excellent animal models and rare human diseases can provide a better understanding of common human diseases. [ABSTRACT FROM AUTHOR]

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys. [ABSTRACT FROM AUTHOR]

Glomerular injury plays a pivotal role in the development of diabetic nephropathy. To elucidate molecular mechanisms underlying diabetic glomerulopathy, we compared glomerular gene expression profiles of db/db mice with those of db/m control mice at a normoalbuminuric stage characterized by hyperglycemia and at an early stage of diabetic nephropathy with elevated albuminuria, using cDNA microarray. In db/db mice at the normoalbuminuric stage, hypoxia-inducible factor-1α (HIF-1α), ephrin B2, glomerular epithelial protein 1, and Pod-1, which play key roles in glomerulogenesis, were already upregulated in parallel with an alteration of genes related to glucose metabolism, lipid metabolism, and oxidative stress. Podocyte structure-related genes, actinin 4α and dystroglycan 1 (DG1), were also significantly upregulated at an early stage. The alteration in the expression of these genes was confirmed by quantitative RT-PCR. Through pioglitazone treatment, gene expression of ephrin B2, Pod-1, actinin 4α, and DG1, as well as that of oxidative stress and lipid metabolism, was restored concomitant with attenuation of albuminuria. In addition, HIF-1α protein expression was partially attenuated by pioglitazone. These results suggest that not only metabolic alteration and oxidative stress, but also the alteration of gene expression related to glomerulogenesis and podocyte structure, may be involved in the pathogenesis of early diabetic glomerulopathy in type 2 diabetes. Diabetes 55:2747-2756, 2006 [ABSTRACT FROM AUTHOR]

Neutrophil gelatinase-associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules. In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion injury, we show that a single dose of Ngal (10 microg), introduced during the initial phase of the disease, dramatically protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse renal diseases, and it provides a unique method for their treatment. [ABSTRACT FROM AUTHOR]

Studies the coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells. Effects of hydrogen peroxide on the expression of the endothelium-derived vasoactive peptides; Significance of oxiidative stress in the modulation of endothelial functions.