Effect of roxadustat on red blood cell lifespan in patients with long-term haemodialysis: a single-centre, prospective, single-arm study.

Background: Reduced survival of red blood cells (RBCs) in patients with chronic kidney disease (CKD) is thought to contribute to renal anaemia. Although renal anaemia improved greatly because of the wide use of erythropoiesis-stimulating agents (ESAs) and the advancement of dialysis techniques, RBC longevity seems not to be obviously ameliorated. Methods: In this single-centre, single-arm trial, patients who had been undergoing haemodialysis and ESA therapy with epoetin alfa for at least 12 weeks changed their anti-anaemia drugs from epoetin alfa to oral roxadustat three times per week for 24 weeks. The primary endpoint was the change in RBC lifespan from baseline at week 24. The change in the circulating percentage of eryptotic RBCs, RBC deformability and RBC oxygen transport ability were also assessed. Results: A total of 27 patients were enrolled, with 26 completing the full course of intervention. At baseline, the average RBC lifespan was 60.1 days [standard deviation (SD) 14.4; n = 27]. At the end of the study period, 26 patients had an RBC lifespan measurement (83.9 days on average; SD 21.9). The RBC lifespan increased by 22.8 days on average [95% confidence interval (CI) 15.5-30.0, P < .001]. This equated to an average RBC lifespan increase of 39.2% (95% CI 27.8-50.6). The percentage of circulating eryptotic RBCs, erythrocyte filtration index and the pressure at which haemoglobin is 50% saturated decreased significantly from baseline to week 24 (1.39 ± 0.44% versus 0.89 ± 0.25%, P < .0001; 0.29 ± 0.12 versus 0.16 ± 0.08, P < .0001 and 32.54 ± 4.83 versus 28.40 ± 2.29, P < .001, respectively). Conclusion: Roxadustat prolonged RBC lifespan in patients with long-term haemodialysis.

Long Noncoding RNA ENSG00000254693 Promotes Diabetic Kidney Disease via Interacting with HuR.

Diabetic kidney disease (DKD) is one of the most common complications of diabetes mellitus (DM), without suitable therapies, causing end-stage renal diseases (ESRDs) ultimately. Moreover, there is increasing evidence demonstrating that long noncoding RNAs (lncRNAs) play crucial roles in the development of DKD. Our RNA sequencing data revealed a large group of differentially expressed lncRNAs in renal tissues of DKD, of which lncRNA ENSG00000254693 (lncRNA 254693 for short) changed drastically. In this study, we found that the expression of lncRNA 254693 was increased in both DKD patients and high-glucose-induced human podocytes. 5'/3'RACE and Northern blot assays were used to find the full length of lncRNA ENSG00000254693 which is 558 nucleotides and nonisoform that existed in human podocyte. Downregulation of lncRNA 254693 remarkably reversed the elevation of inflammation, apoptosis, and podocyte injury caused by high glucose. Then, we did bioinformatics analysis via RBPDB and found that lncRNA 254693 can combine with HuR, a RNA binding protein. Meanwhile, immunofluorescence and in situ hybridization double staining was used to prove the existence of colocalization between them. Intriguingly, lncRNA 254693 knockdown decreased HuR levels, while HuR knockdown also decreased the level of lncRNA 254693 and its stability. After this, RNA immunoprecipitation assay results confirmed the binding association between them again. In addition, we found that HuR was increased in high glucose-induced podocytes, and the silence of HuR could alleviate podocyte injury, inflammation, and apoptosis. These results together suggested a novel feedback regulation between lncRNA 254693 and HuR which could involve in podocyte injury and may serve as a predicted target for DKD therapies.

Inhibition of the lncRNA MIAT prevents podocyte injury and mitotic catastrophe in diabetic nephropathy.

Podocyte damage is strongly associated with the progression of diabetic nephropathy. Mitotic catastrophe plays an essential role in accelerating podocyte loss and detachment from the glomerular basement membrane. In the current study, we observed that the long non-coding RNA (lncRNA) MIAT was noticeably upregulated in the plasma and kidney tissues of patients with diabetic nephropathy, and this upregulation was accompanied by higher albumin/creatinine ratios and serum creatinine levels. By generating CRISPR-Cas9 Miat-knockout (KO) mice in vivo and employing vectors in vitro, we found that the depletion of Miat expression significantly restored slit-diaphragm integrity, attenuated foot process effacement, prevented dedifferentiation, and suppressed mitotic catastrophe in podocytes during hyperglycemia. The mechanistic investigation revealed that Miat increased Sox4 expression and subsequently regulated p53 ubiquitination and acetylation, thereby inhibiting the downstream factors CyclinB/cdc2 by enhancing p21cip1/waf1 activity, and that Miat interacted with Sox4 by sponging miR-130b-3p. Additionally, the inhibition of miR-130b-3p with an antagomir in vivo effectively enhanced glomerular podocyte injury and mitotic dysfunction, eventually exacerbating proteinuria. Based on these findings, MIAT may represent a therapeutic target for diabetic nephropathy.

Preoperative Cardiac Index as a Predictor of Maturation and Primary Patency of Radiocephalic Arteriovenous Fistula in Hemodialysis.

BACKGROUND: Radiocephalic arteriovenous fistula (RCAVF) is the best access modality to be considered initially when planning arteriovenous fistula (AVF) for maintenance hemodialysis. Considering the higher incidence of RCAVF failed maturation (M), it is important to perform proper preoperative evaluation and identification of high-risk patients. There has been no study on the influence of preoperative cardiac function on the M and patency of AVFs. The purpose of this investigation is to determine whether preoperative cardiac index (CI) is a predictor of M and primary patency of RCAVF. METHOD: A total of 365 end-stage renal disease patients undergoing RCAVF surgery were consecutively enrolled with a median follow-up time of 20 months in this prospective cohort study. Demographics, vascular diameters measured by duplex ultrasound examination, and CI measured by echocardiography, were analyzed for effect on RCAVF primary functional M and primary patency. RESULT: Patients in the group achieving primary RCAVF functional M had a significantly larger mean CI than the group with early RCAVF failure (2.93 ± 0.77 vs. 3.57 ± 0.76 L/min/m2, p < 0.001). The receiver operating characteristic curve was plotted and demonstrated that preoperative vein diameter and CI can predict failure of RCAVF M. The AUC of CI was higher (0.745 vs. 0.666). Multivariate regression analysis, adjusted for age, sex, diabetes, preoperative dialysis status and vessel diameters, showed that decreased CI remained associated with increased risk of failure of M (FM) and worse primary unassisted patency. The Kaplan-Meier survival analysis suggested that patients with CI <3 L/min/m2 had a worse primary unassisted patency rate at all time points compared with patients with CI ≥3 L/min/m2. CONCLUSION: This study demonstrated that preoperative CI was associated with RCAVF M and long-term patency. A decreased CI may be a possible predictor of an increased risk of FM and a shorter primary patency time.

Red blood cell lifespan in long-term hemodialysis patients treated with roxadustat or recombinant human erythropoietin.

INTRODUCTION: A significant decrease in red blood cell (RBC) survival has been observed in patients with renal failure, which is supposed to contribute to renal anemia. The aim of this observational study was to determine RBC survival in hemodialysis (HD) patients treated with roxadustat or recombinant human erythropoietin (rhuEPO) compared with healthy persons. METHODS: RBC lifespan was measured by Levitt's CO breath test with newly developed automatic instrument ELS Tester. RESULTS: A total of 102 patients receiving long-term HD from two independent dialysis centers enrolled in the study, of whom 62 were treated with rhuEPO and 40 were on roxadustat therapy. A total of 25 healthy participants were recruited to match HD participants according to age and sex. Median RBC survival times in rhuEPO, roxadustat, and control groups were 65.0 (25th-75th percentile, 49.5-77.3), 75.5 (25th-75th percentile, 57.3-99.3), and 108.0 (25th-75th percentile, 89.0-141.5) d, respectively. Patients treated with roxadustat had significantly longer RBC survival time than patients treated with rhuEPO (p < .05). In multivariate analysis of factors affecting RBC lifespan in the whole HD patients, anemia treatment drugs (rhuEPO/roxadustat) and levels of hemoglobin were the significantly independent factors. RBC survival was not found to correlate with either weekly rhuEPO dosage (r = -0.087, p = .500) or weekly roxadustat dosage (r = -0.267, p = .110) in our cohort. CONCLUSIONS: HD patients treated with roxadustat had significantly longer RBC survival time than patients treated with rhuEPO, large prospective studies with long-term follow-up are warranted to verify the results in future. Abbreviations RBC: red blood cell; HD: hemodialysis; rhu EPO: recombinant human erythropoietin; ESRD: end-stage renal disease; EPO: erythropoietin; ROS: reactive oxygen species; CKD: chronic kideny disease; ESAs: erythropoiesis-stimulating agents; HIF-PHD: hypoxia-inducible factor prolyl hydroxylase; CO: carbon monoxide; Hb: hemoglobin.

Comparison of Postnatal Growth Charts of Singleton Preterm and Term Infants Using World Health Organization Standards at 40-160 Weeks Postmenstrual Age: A Chinese Single-Center Retrospective Cohort Study.

There remains controversy regarding whether the growth charts constructed from data of term infants, such as those produced by the World Health Organization (WHO) standards, can appropriately evaluate the postnatal growth of preterm infants. This retrospective cohort study, conducted in the First Affiliated Hospital of Shandong First Medical University in Jinan China, aimed to compare the postnatal growth charts of singleton preterm and term infants using WHO standards at 40-160 weeks postmenstrual age (PMA). A total of 5,459 and 15,185 sets of longitudinal measurements [length/height, weight, head circumference (HC), and body mass index (BMI)] from birth to 160 weeks PMA were used to construct growth charts for 559 singleton preterm (mean PMA at birth, 33.84 weeks) and 1,596 singleton term infants (born at 40 weeks PMA), respectively, using the Generalized Additive Models for Location, Scale, and Shape (GAMLSS) method. Z-scores (prematurity corrected) were calculated using WHO Anthro software. Compared to WHO standards, all parameters of preterm infants were increased, especially in terms of length/height and weight; the gap between the two almost spanned two adjacent centile curves. Compared to term controls, the length/height, weight, and BMI of preterm infants were higher at 40 weeks PMA, surpassed by term infants at 52-64 weeks PMA, and quite consistent thereafter. The HC of preterm infants at 40-160 weeks PMA was quite consistent with both term controls and the WHO standards. The Z-scores for length/height, weight, and BMI of preterm infants relative to the WHO standards gradually decreased from 1.20, 1.13, and 0.74 at 40-44 weeks PMA to 0.67, 0.42, and 0.03 at 132-160 weeks PMA, respectively; Z-scores for HC of preterm infants rapidly decreased from 0.73 to 0.29 at 40-50 weeks PMA, and then fluctuated in the range of 0.08-0.23 at 50-160 weeks PMA. Preterm infants had higher growth trajectories than the WHO standards and similar but not identical trajectories to term infants during the first 2 years of life. These findings reemphasize the necessity of constructing local growth charts for Chinese singleton preterm infants.

Podocyte-derived extracellular vesicles mediate renal proximal tubule cells dedifferentiation via microRNA-221 in diabetic nephropathy.

Podocyte injury is a key event in the initiation of Diabetic nephropathy (DN). Tubulointerstitium, especially the proximal tubule has been regarded as a target of injury. In the present study, we showed that podocytes induced dedifferentiation of proximal tubular epithelial cells(PTECs) in high-glucose conditions and extracellular vesicles (EVs) mediates the interaction. Then we extracted and identified these EVs derived from podocytes as exosome, further, the EVs induced PTECs dedifferentiation. Total microRNA(miRNA) expression of podocyte-derived EVs was extracted and miR-221 expression was remarkably increased. By making use of the miRNA gain- and loss-of-function approaches, we observed that miR-221 mediated PTECs dedifferentiation. In addition, a dual-luciferase reporter assay confirmed that miR-221 direct target DKK2, which was an inhibitor of Wnt signaling, and overexpression of miR-221 significantly resulted in ß-catenin nuclear accumulation. Moreover, we regulated the expression of ß-catenin and demonstrated that miR-221 in EVs mediated proximal tubule cells injury through Wnt/ß-catenin signaling. Furthermore, inhibition of miR-221 in diabetic mice reversed the abnormal expression of PTECs dedifferentiation related protein. These findings provide unique insights in the mechanisms of proximal tubule cell injury in diabetic nephropathy.

Podocyte-specific Rac1 deficiency ameliorates podocyte damage and proteinuria in STZ-induced diabetic nephropathy in mice.

Activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) has been implicated in diverse kidney diseases, yet its in vivo significance in diabetic nephropathy (DN) is largely unknown. In the present study, we demonstrated a podocyte-specific Rac1-deficient mouse strain and showed that specific inhibition of Rac1 was able to attenuate diabetic podocyte injury and proteinuria by the blockade of Rac1/PAK1/p38/ß-catenin signaling cascade, which reinstated the integrity of podocyte slit diaphragms (SD), rectified the effacement of foot processes (FPs), and prevented the dedifferentiation of podocytes. In vitro, we showed Rac1/PAK1 physically bound to ß-catenin and had a direct phosphorylation modification on its C-terminal Ser675, leading to less ubiquitylated ß-catenin, namely more stabilized ß-catenin, and its nuclear migration under high-glucose conditions; further, p38 activation might be responsible for ß-catenin nuclear accumulation via potentiating myocyte-specific enhancer factor 2C (MEF2c) phosphorylation. These findings provided evidence for a potential renoprotective and therapeutic strategy of cell-specific Rac1 deficiency for DN and other proteinuric diseases.

LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucose-induced podocyte injury via its interplay with ß-catenin.

Metastasis associated lung adenocarcinoma transcript 1(MALAT1) is a long non-coding RNA, broadly expressed in mammalian tissues including kidney and up-regulated in a variety of cancer cells. To date, its functions in podocytes are largely unknown. ß-catenin is a key mediator in the canonical and non-canonical Wnt signalling pathway; its aberrant expression promotes podocyte malfunction and albuminuria, and contributes to kidney fibrosis. In this study, we found that MALAT1 levels were increased in kidney cortices from C57BL/6 mice with streptozocin (STZ)-induced diabetic nephropathy, and dynamically regulated in cultured mouse podocytes stimulated with high glucose, which showed a trend from rise to decline. The decline of MALAT1 levels was accompanied with ß-catenin translocation to the nuclei and enhanced expression of serine/arginine splicing factor 1 (SRSF1), a MALAT1 RNA-binding protein. Further we showed early interference with MALAT1 siRNA partially restored podocytes function and prohibited ß-catenin nuclear accumulation and SRSF1 overexpression. Intriguingly, we showed that ß-catenin was involved in MALAT1 transcription by binding to the promotor region of MALAT1; ß-catenin knock-down also decreased MALAT1 levels, suggesting a novel feedback regulation between MALAT1 and ß-catenin. Notably, ß-catenin deletion had limited effects on SRSF1 expression, demonstrating ß-catenin might serve as a downstream signal of SRSF1. These findings provided evidence for a pivotal role of MALAT1 in diabetic nephropathy and high glucose-induced podocyte damage.

FAK contributes to proteinuria in hypercholesterolaemic rats and modulates podocyte F-actin re-organization via activating p38 in response to ox-LDL.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that regulates cell adhesion, proliferation and differentiation. In the present study, a rat model of high fat diet-induced hypercholesterolaemia was established to investigate the involvement of FAK in lipid disorder-related kidney diseases. We showed focal fusion of podocyte foot process that occurred at as early as 4 weeks in rats consuming high fat diet, preceding the onset of proteinuria when aberrant phosphorylation of FAK was found. These abnormalities were ameliorated by dietary intervention of TAE226, a reported inhibitor of FAK. FAK is also an adaptor protein initiating cascades of intracellular signals including c-Src, Rho GTPase and mitogen-activated protein kinase (MAPK). P38 MAPK belongs to the latter and is centrally involved in kidney diseases. Our cell culture data revealed oxidized low-density lipoprotein (ox-LDL) triggered hyper-phosphorylation of FAK and p38, ectopic expression of cellular markers (manifested as decreased WT1, podocin and NEPH1, and increased vimentin and mmp9), and re-arrangement of F-actin filaments with enhanced cell motility; these mutations were significantly rectified by FAK shRNA. Notably, pre-treatment of p38 inhibitor did not alter FAK activation, albeit its deletion of p38 hyper-activity and attenuation of cellular abnormalities, demonstrating that p38 acted as a downstream effector of FAK signalling and ox-LDL damaged podocytes in a FAK/p38-dependent manner. This was further identified by animal data that p38 activation was also abrogated by TAE226 treatment in hypercholesterolaemic rats, suggesting that FAK/p38 axis might also be involved in in vivo events. These findings provided a potential early mechanism of hypercholesterolaemia-related podocyte damage and proteinuria.

Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation.

Rac1/PAK1 signaling promotes epithelial-mesenchymal transition of podocytes in vitro via triggering ß-catenin transcriptional activity under high glucose conditions.

Ras-related C3 botulinum toxin substrate 1 (Rac1), together with its major downstream effector p21-activated kinase 1 (PAK1), has been identified a central role in cellular events such as cell cytoskeletal remodeling that contributed to cell migration and epithelial-mesenchymal transition (EMT). And there are data implicating that podocytes underwent EMT under pathological conditions. However, little is known about mechanisms of podocytes undergoing EMT. To address this, we assessed the cellular changes of podocytes after high glucose stimulation in vitro, detected the effects of Rac1/PAK1 signaling on podocytes in response to the stimuli, and investigated interactions of Rac1/PAK1 axis with ß-catenin and Snail under high glucose conditions. We found that in vitro high glucose treatment led to remarkable down-regulation of nephrin and P-cadherin, as well as significant up-regulation of α-SMA and FSP-1, suggesting that in the presence of high glucose, podocytes underwent EMT, during which Rac1/PAK1 signaling was activated. And these were notably ameliorated by Rac1 gene knockdown. Furthermore, ß-catenin and Snail nuclear translocation were triggered by Rac1/PAK1 axis, which were both markedly reversed via Rac1 gene knockdown or pretreatment of IPA-3, a PAK1 inhibitor. These findings elaborated that Rac1/PAK1 signaling contributed to high glucose-induced podocyte EMT via promoting ß-catenin and Snail transcriptional activities, which could be a potential mechanism involved in podocytes injury in response to stimuli under diabetic conditions.

The role of the p38 MAPK signaling pathway in high glucose-induced epithelial-mesenchymal transition of cultured human renal tubular epithelial cells.

BACKGROUND: Epithelial-mesenchymal transition of tubular epithelial cells, which is characterized by a loss of epithelial cell characteristics and a gain of ECM-producing myofibroblast characteristics, is an essential mechanism that is involved in tubulointerstitial fibrosis, an important component of the renal injury that is associated with diabetic nephropathy. Under diabetic conditions, p38 MAPK activation has been reported in glomeruli and mesangial cells; however, studies on p38 MAPK in TECs are lacking. In this study, the role of p38 MAPK in AP-1 activation and in the EMT in the human proximal tubular epithelial cell line (HK-2) under high glucose concentration conditions is investigated. METHODOLOGY/PRINCIPAL FINDINGS: A vector for small interfering RNA that targets p38 MAPK was constructed; the cells were then either transfected with p38 siRNA or pretreated with a chemical inhibitor of AP-1 and incubated with low glucose plus TGF-ß1 or high glucose for 48 h. Cells that were not transfected or pretreated and were exposed to low glucose with or without TGF-ß1 or high glucose for 48 h were considered to be the controls. We found that high glucose induced an increase in TGF-ß1. And high glucose-induced p38 MAPK activation was inhibited by p38 siRNA (P<0.05). A significant decline in E-cadherin and CK expression and a notable increase in vimentin and α-SMA were detected when exposed to low glucose with TGF-ß1 or high glucose, and a significant raise of secreted fibronectin were detected when exposed to high glucose; whereas these changes were reversed when the cells were treated with p38 siRNA or AP-1 inhibitor (P<0.05). AP-1 activity levels and Snail expression were up-regulated under high glucose conditions but were markedly down-regulated through knockdown of p38 MAPK with p38 siRNA or pretreatment with AP-1 inhibitor (P<0.05). CONCLUSION: This study suggests that p38 MAPK may play an important role in the high glucose-induced EMT by activating AP-1 in tubular epithelial cells.