Disrupting circadian control of autophagy induces podocyte injury and proteinuria.

The circadian clock influences a wide range of biological process and controls numerous aspects of physiology to adapt to the daily environmental changes caused by Earth's rotation. The kidney clock plays an important role in maintaining tubular function, but its effect on podocytes remains unclear. Here, we found that podocytes expressed CLOCK proteins, and that 2666 glomerular gene transcripts (13.4%), including autophagy related genes, had 24-hour circadian rhythms. Deletion of Clock in podocytes resulted in 1666 gene transcripts with the loss of circadian rhythm including autophagy genes. Podocyte-specific Clock knockout mice at age three and eight months showed deficient autophagy, loss of podocytes and increased albuminuria. Chromatin immunoprecipitation (ChIP) sequence analysis indicated autophagy related genes were targets of CLOCK in podocytes. ChIP-PCR further confirmed Clock binding to the promoter regions of Becn1 and Atg12, two autophagy related genes. Furthermore, the association of CLOCK regulated autophagy with chronic sleep fragmentation and diabetic kidney disease was analyzed. Chronic sleep fragmentation resulted in the loss of glomerular Clock rhythm, inhibition of podocyte autophagy, and proteinuria. Rhythmic oscillations of Clock also disappeared in high glucose treated podocytes and in glomeruli from diabetic mice. Finally, circadian differences in podocyte autophagy were also abolished in diabetic mice. Deletion Clock in podocytes aggravated podocyte injury and proteinuria in diabetic mice. Thus, our findings demonstrate that clock-dependent regulation of autophagy may be essential for podocyte survival. Hence. loss of circadian controlled autophagy may play an important role in podocyte injury and proteinuria.

Loss of UCP2 causes mitochondrial fragmentation by OMA1-dependent proteolytic processing of OPA1 in podocytes.

Mitochondrial dysfunction plays an important role in the onset and progression of podocyte injury and proteinuria. However, the process by which the change in the podocyte mitochondria occurs is not well understood. Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which is located in the mitochondrial inner membrane. Here, we reported that mice with podocyte-specific Ucp2 deficiency developed podocytopathy with proteinuria with aging. Furthermore, those mice exhibited increased proteinuria in experimental models evoked by Adriamycin. Our findings suggest that UCP2 mediates mitochondrial dysfunction by regulating mitochondrial dynamic balance. Ucp2-deleted podocytes exhibited increased mitochondrial fission and deficient in ATP production. Mechanistically, opacity protein 1 (OPA1), a key protein in fusion of mitochondrial inner membrane, was regulated by UCP2. Ucp2 deficiency promoted proteolysis of OPA1 by activation OMA1 which belongs to mitochondrial inner membrane zinc metalloprotease. Those finding demonstrate the role of UCP2 in mitochondrial dynamics in podocytes and provide new insights into pathogenesis associated with podocyte injury and proteinuria.

Pyruvate kinase M2 regulates mitochondrial homeostasis in cisplatin-induced acute kidney injury.

An important pathophysiological process of acute kidney injury (AKI) is mitochondrial fragmentation in renal tubular epithelial cells, which leads to cell death. Pyruvate kinase M2 (PKM2) is an active protein with various biological functions that participates in regulating glycolysis and plays a key role in regulating cell survival. However, the role and mechanism of PKM2 in regulating cell survival during AKI remain unclear. Here, we found that the phosphorylation of PKM2 contributed to the formation of the PKM2 dimer and translocation of PKM2 into the mitochondria after treatment with staurosporine or cisplatin. Mitochondrial PKM2 binds myosin heavy chain 9 (MYH9) to promote dynamin-related protein 1 (DRP1)-mediated mitochondrial fragmentation. Both in vivo and in vitro, PKM2-specific loss or regulation PKM2 activity partially limits mitochondrial fragmentation, alleviating renal tubular injury and cell death, including apoptosis, necroptosis, and ferroptosis. Moreover, staurosporine or cisplatin-induced mitochondrial fragmentation and cell death were reversed in cultured cells by inhibiting MYH9 activity. Taken together, our results indicate that the regulation of PKM2 abundance and activity to inhibit mitochondrial translocation may maintain mitochondrial integrity and provide a new therapeutic strategy for treating AKI.

Serum PTH Associated with Malnutrition Determined by Bioelectrical Impedance Technology in Chronic Kidney Disease Patients.

Purpose: Chronic malnutrition and cachexia are common in chronic kidney disease (CKD), and importance should be given to these complications because they affect the patient's quality of life and prognosis. This study analyzed the correlation between the serum PTH level, nutritional status, and body composition of patients with CKD. Methods: CKD patients were enrolled in Center for Kidney Disease, Second Affiliated Hospital of Nanjing Medical University, from December 1, 2016, to November 30, 2020. Bioelectrical impedance technology was applied to estimate the body composition. The characteristics of the body composition were compared among different stages of CKD patients, and then the correlation between PTH and body composition was analyzed. Results: 205 CKD patients were enrolled. Twenty-five patients were in stage 1 or 2 of CKD, 78 patients were in stage 3 or 4, 31 patients were in stage 5 without dialysis (referred to as CKD stage 5A), and 71 patients were in stage 5 with dialysis (referred to as CKD stage 5B). Body composition analysis showed that the patients had a phase angle (PA) of 5.02 ± 1.07°, a percentage of body fat (PBF) of 27.74 ± 8.8%, and a skeletal muscle mass index (SMI) of 7.4 ± 1.34 kg/m2. PBF peaked in the CKD stage 3/4 group and gradually decreased with the progression of CKD. The PA and SMI differed significantly between the CKD stage 1/2 and stage 5B groups. The proportion of low SMI did not differ significantly between the CKD stage 1/2 and stage 3/4 groups, but it was obviously higher in the CKD stage 5A and 5B groups. PTH was significantly correlated with BMI, hemoglobin, albumin, total cholesterol, triglycerides, and SMI. Binary logistic regression of low SMI showed that the odds ratio for PTH levels was greater than the upper limit of the normal range, which was 11.769 (p=0.043, 95% confidence interval: 1.078-128.536), and the model predictive power was 0.986 after correction for age, sex, height, weight, hemoglobin, serum calcium, serum phosphorus, serum total cholesterol, serum triglyceride, and basal metabolic rate. Conclusions: Bioelectrical impedance analysis might be useful in estimating the nutritional status of CKD patients in terms of fat and muscle parameters. High levels of PTH are an independent risk factor for developing low SMI in CKD patients.

Sirtuin 3 regulates mitochondrial protein acetylation and metabolism in tubular epithelial cells during renal fibrosis.

Proximal tubular epithelial cells (TECs) demand high energy and rely on mitochondrial oxidative phosphorylation as the main energy source. However, this is disturbed in renal fibrosis. Acetylation is an important post-translational modification for mitochondrial metabolism. The mitochondrial protein NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates mitochondrial metabolic function. Therefore, we aimed to identify the changes in the acetylome in tubules from fibrotic kidneys and determine their association with mitochondria. We found that decreased SIRT3 expression was accompanied by increased acetylation in mitochondria that have separated from TECs during the early phase of renal fibrosis. Sirt3 knockout mice were susceptible to hyper-acetylated mitochondrial proteins and to severe renal fibrosis. The activation of SIRT3 by honokiol ameliorated acetylation and prevented renal fibrosis. Analysis of the acetylome in separated tubules using LC-MS/MS showed that most kidney proteins were hyper-acetylated after unilateral ureteral obstruction. The increased acetylated proteins with 26.76% were mitochondrial proteins which were mapped to a broad range of mitochondrial pathways including fatty acid ß-oxidation, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation. Pyruvate dehydrogenase E1α (PDHE1α), which is the primary link between glycolysis and the TCA cycle, was hyper-acetylated at lysine 385 in TECs after TGF-ß1 stimulation and was regulated by SIRT3. Our findings showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in TECs. The deacetylation of PDHE1α by SIRT3 at lysine 385 plays a key role in metabolic reprogramming associated with renal fibrosis.

SS31 Ameliorates Podocyte Injury via Inhibiting OMA1-Mediated Hydrolysis of OPA1 in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is currently one of the leading causes of end-stage renal disease (ESRD). Mitochondrial dysfunction in podocyte is involve in DKD development. However, whether early mitochondrial stabilization delays or reverses DKD progression has not been elucidated. SS31 is a novel tetrapeptide compound that targets the inner mitochondrial membrane and protects mitochondria by reducing ROS and inhibiting cardiolipin oxidation. Our study discovered that SS31 might have a long-term podocyte protection in DKD. In this study, we examined the glomerular pathological damage and proteinuria at different stages of diabetes. Results revealed that podocyte mitochondrial injury appeared at the early stage of DKD. Early treatment with SS31 could protect podocyte and alleviate the development of DKD via inhibiting OMA1-mediated hydrolysis of OPA1. Those data indicate that SS31 might be a promising agent in delaying the development of DKD and OMA1-mediated hydrolysis of OPA1 in mitochondria, and SS31 is a novel therapeutic target for the treatment of DKD.

Tuberous sclerosis 1 (Tsc1) mediated mTORC1 activation promotes glycolysis in tubular epithelial cells in kidney fibrosis.

Energy reprogramming to glycolysis is closely associated with the development of chronic kidney disease. As an important negative regulatory factor of the mammalian target of rapamycin complex 1 (mTORC1) signal, tuberous sclerosis complex 1 (Tsc1) is also a key regulatory point of glycolysis. Here, we investigated whether Tsc1 could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells. We induced mTORC1 signal activation in tubular epithelial cells in kidneys with fibrosis via unilateral ureteral occlusion. This resulted in increased tubular epithelial cell proliferation and glycolytic enzyme upregulation. Prior incubation with rapamycin inhibited mTORC1 activation and abolished the enhanced glycolysis and tubular epithelial cell proliferation. Furthermore, knockdown of Tsc1 expression promoted glycolysis in the rat kidney epithelial cell line NRK-52E. Specific deletion of Tsc1 in the proximal tubules of mice resulted in enlarged kidneys characterized by a high proportion of proliferative tubular epithelial cells, dilated tubules with cyst formation, and a large area of interstitial fibrosis in conjunction with elevated glycolysis. Treatment of the mice with the glycolysis inhibitor 2-deoxyglucose notably ameliorated tubular epithelial cell proliferation, cystogenesis, and kidney fibrosis. Thus, our findings suggest that Tsc1-associated mTORC1 signaling mediates the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells.

Urinary mitochondrial DNA: A potential early biomarker of diabetic nephropathy.

BACKGROUND: Mitochondrial dysfunction and chronic sterile inflammation are common features of type 2 diabetes. Therefore, we aimed to investigate whether mitochondrial DNA (mtDNA) could be a biomarker implicated in the progression of type 2 diabetes and diabetic nephropathy and explore the underlying mechanism. MATERIAL AND METHODS: We developed a method for relative quantification of mtDNA content in clinical practice. qRT-PCR was used to measure the mtDNA content both in vivo in CD-1 mice with diabetes induction by streptozotocin and in vitro in murine endothelial cells and conditionally immortalized mouse podocytes. By pumping mtDNA into the mouse circulation, the effect of mtDNA on the kidney was assessed in mice. In patients with type 2 diabetes (n = 42; 24 males; mean age 57.9 ± 12.00 years), plasma mtDNA was evaluated. RESULTS: Plasma mtDNA content was significantly decreased in patients with type 2 diabetes, particularly those with significant proteinuria. In vitro, high glucose treatment suppressed intracellular mtDNA content and facilitated the extracellular release of mtDNA, so excessive circulatory mtDNA induced by high glucose might be filtered through the kidney and then into urine. Indeed, urinary mtDNA content was significantly increased in both diabetic patients and mice. Moreover, by pumping excess mtDNA into circulation in mice, filtered mtDNA could trigger inflammation and induce kidney injury. CONCLUSION: Excessive mtDNA filtered through the kidney under diabetic conditions may be involved in chronic renal inflammation. Reduced plasma mtDNA content and increased urinary mtDNA/creatinine ratio might play a potential role as an early biomarker of diabetic nephropathy.

UCP2-dependent improvement of mitochondrial dynamics protects against acute kidney injury.

Acute kidney injury (AKI) is a public health concern, with high morbidity and mortality rates in hospitalized patients and because survivors have an increased risk of progression to chronic kidney disease. Mitochondrial damage is the critical driver of AKI-associated dysfunction and loss of tubular epithelial cells; however, the pathways that mediate these events are poorly defined. Here, in murine ischemia/reperfusion (I/R)-induced AKI, we determined that mitochondrial damage is associated with the level of renal uncoupling protein 2 (UCP2). In hypoxia-damaged proximal tubular cells, a disruption of mitochondrial dynamics demonstrated by mitochondrial fragmentation and disturbance between fusion and fission was clearly indicated. Ucp2-deficient mice (knockout mice) with I/R injury experienced more severe AKI and mitochondrial fragmentation than wild-type mice. Moreover, genetic or pharmacological treatment increased UCP2 expression, improved renal function, reduced tubular injury and limited mitochondrial fission. In cultured proximal tubular epithelial cells, hypoxia-induced mitochondrial fission was exacerbated in cells with UCP2 deletion, whereas an increase in UCP2 ameliorated the hypoxia-induced disturbance of the balance between mitochondrial fusion and fission. Furthermore, results following modulation of UCP2 suggested it has a role in preserving mitochondrial integrity by preventing loss of membrane potential and reducing subsequent mitophagy. Taken together, our results indicate that UCP2 is protective against AKI and suggest that enhancing UCP2 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The feedback loop between miR-21, PDCD4 and AP-1 functions as a driving force for renal fibrogenesis.

Renal fibrosis is a final common pathway of chronic kidney disease. Sustained activation of fibroblasts is considered to play a key role in perpetuating renal fibrosis but the driving force in the perpetuation stage is only partially understood. To date, some investigations have specifically identified overexpression of microRNA 21 (miR-21) in the progression of kidney fibrosis. Nevertheless, the precise role of miR-21 in fibroblast activation remains largely unknown. In this study, we found that miR-21 was significantly upregulated in activated fibroblasts and that it maintained itself at constant high levels by employing an auto-regulatory loop between miR-21, PDCD4 and AP-1. Persistently upregulated miR-21 suppressed protein expression of Smad7 and, eventually, enhanced the TGF-ß1/Smad pathway to promote fibroblast activation. More importantly, we found miR-21 sequestration with miR-21 antagomir or AP-1 inhibitors attenuated unilateral ureteral obstruction (UUO)-induced renal fibrosis. miR-21-knockout mice also suffered far less interstitial fibrosis in response to kidney injury. Altogether, these data suggest that miR-21 is a main driving force of fibroblast activation and keeps its high expression level by employing a double negative autoregulatory loop. Targeting this aberrantly activated feedback loop may provide new therapeutic strategy in treating fibrotic kidneys.

PDE/cAMP/Epac/C/EBP-ß Signaling Cascade Regulates Mitochondria Biogenesis of Tubular Epithelial Cells in Renal Fibrosis.

AIMS: Cyclic adenosine 3'5'-monophosphate (cAMP) is a universal second messenger that plays an important role in intracellular signal transduction. cAMP is synthesized by adenylate cyclases from adenosine triphosphate and terminated by the phosphodiesterases (PDEs). In the present study, we investigated the role of the cAMP pathway in tubular epithelial cell mitochondrial biogenesis in the pathogenesis of renal fibrosis. RESULTS: We found that the cAMP levels were decreased in fibrotic kidney tissues, and replenishing cAMP could ameliorate tubular atrophy and extracellular matrix deposition. The downregulation of cAMP was mainly attributed to the increased PDE4 expression in tubular epithelial cells. The inhibition of PDE4 by PDE4 siRNA or the specific inhibitor, rolipram, attenuated unilateral ureteral obstruction-induced renal interstitial fibrosis and transforming growth factor (TGF)-ß1-stimulated primary tubular epithelial cell (PTC) damage. The Epac1/Rap1 pathway contributed to the main effect of cAMP on renal fibrosis. Rolipram could restore C/EBP-ß and PGC-1α expression and protect the mitochondrial function and structure of PTCs under TGF-ß1 stimulation. The antifibrotic role of rolipram in renal fibrosis relies on C/EBP-ß and PGC-1α expression in tubular epithelial cells. Innovation and Conclusion: The results of the present study indicate that cAMP signaling regulates the mitochondrial biogenesis of tubular epithelial cells in renal fibrosis. Restoring cAMP by the PDE4 inhibitor rolipram may ameliorate renal fibrosis by targeting C/EBP-ß/PGC1-α and mitochondrial biogenesis. Antioxid. Redox Signal. 29, 637-652.

Erythropoietin protects the tubular basement membrane by promoting the bone marrow to release extracellular vesicles containing tPA-targeting miR-144.

Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing a mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of the tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in the obstructed kidney. Instead of acting directly on tPA in the kidney, EPO strongly increased the level of circulating microRNA (miR)-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target the tPA 3'-untranslated region and suppress tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1(+)CD44(+)CD11b(-)CD19(-) cells to secrete miR-144-containing EVs, which markedly suppressed tPA expression, as well as metalloproteinase 9 (MMP9) level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secrete miR-144, which, in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of the tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in CKD, is a potential therapeutic strategy for renal fibrosis.

[Continuous renal replacement therapy and negative fluid balance improves renal function and prognosis of patients with acute kidney injury in sepsis].

OBJECTIVE: To investigate the influence of fluid balance and model of renal replacement therapy (RRT) on renal function and prognosis of patients suffering from septic acute kidney injury (AKI). METHODS: A retrospective cohort analysis of 117 septic AKI patients who had undergone RRT between January 2009 and December 2014 was performed in the Second Affiliated Hospital of Nanjing Medical University. The patients were divided into positive fluid balance group (n = 52) and negative fluid balance group (n = 65) according to the total amount of fluid calculated from the difference between fluid administered and fluid lost during the first 1 week of RRT. The incidence of renal recovery and death of the patients by 60 days as the endpoint events were taken to judge the prognosis of two groups. RRT strategies included continuous renal replacement therapy (CRRT) and intermittent renal replacement therapy (IRRT). Multiple factors including estimated glomerular filtration rate (eGFR), sequential organ failure assessment (SOFA) score, RRT model, the accumulation of fluid before initiation of RRT, and negative fluid balance during RRT were analyzed for outcome predictors by Cox proportional hazards model. RESULTS: There were no differences between two groups regarding clinical characteristics. The percentage of receiving CRRT in the negative fluid balance group was slightly higher than that of the positive fluid balance group (52.31% vs. 36.54%, χ² = 2.899, P = 0.089). With Kaplan-Meier survival curves, it was shown that the patients of negative fluid balance group had a higher rate of recovery of renal function (χ² = 4.803, P = 0.028) and significantly lower mortality rate (χ² = 9.505, P = 0.002). The rate of recovery of renal function by 60 days was higher in the negative fluid balance group than that in the positive fluid balance group (47.69% vs. 28.85%, χ² = 3.991, P = 0.046), while the mortality rate was significantly lowered in the negative fluid balance group compared with that of the positive fluid balance group (40.00% vs. 67.31%, χ² = 4.378, P = 0.036). Cox multivariate regression was used for excluding confounding factors. After adjusting for the clinically relevant variables, RRT negative fluid balance was significantly associated with recovery of renal function [ hazard ratios (HR) = 2.440, 95% confidence intervals (95%CI) = 1.089-5.464, P = 0.030] and mortality (HR = 0.443, 95%CI = 0.238-0.822, P = 0.010]. Higher eGFR before RRT and CRRT were independent favorable factors for recovery of renal function (HR = 1.014, 95%CI = 1.003-1.026, P = 0.012; HR = 3.138, 95%CI = 1.765-7.461, P = 0.002), and higher SOFA score was associated with a significantly higher risk of death (HR = 1.115, 95%CI = 1.057-1.177, P < 0.001). CONCLUSIONS: Once the patients with septic AKI showed the signs of fluid overload, timely RRT and effective removal of excessive liquid may reverse the adverse prognosis. RRT with negative fluid balance is beneficial for the recovery of renal function, and reduce the mortality in patients with septic AKI, and CRRT model is a good choice.

Circulatory mitochondrial DNA is a pro-inflammatory agent in maintenance hemodialysis patients.

Chronic inflammation is highly prevalent in maintenance hemodialysis (MHD) patients, and it has been shown to be a strong predictor of morbidity and mortality. Mitochondrial DNA (mtDNA) released into circulation after cell damage can promote inflammation in patients and animal models. However, the role and mechanisms of circulatory mtDNA in chronic inflammation in MHD patients remain unknown. Sixty MHD patients and 20 health controls were enrolled in this study. The circulatory mtDNA was detected by quantitative real-time PCR assay. Plasma interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were quantitated by ELISA assay. Dialysis systems in MHD patients and in vitro were used to evaluate the effect of different dialysis patterns on circulatory mtDNA. Circulatory mtDNA was elevated in MHD patients comparing to that of health control. Regression analysis demonstrated that plasma mtDNA was positively associated with TNF-α and the product of serum calcium and phosphorus, while negatively associated with hemoglobin and serum albumin in MHD patients. MtDNA induced the secretion of IL-6 and TNF-α in the THP-1 cells. Single high-flux hemodialysis (HF-HD) and on line hemodiafiltration (OL-HDF) but not low-flux hemodialysis (LF-HD) could partially reduce plasma mtDNA in MHD patients. In vitro, both HD and hemofiltration (HF) could fractional remove mtDNA. Collectively, circulatory mtDNA is elevated and its level is closely correlated with chronic inflammation in MHD patients. HF-HD and HDF can partially reduce circulatory mtDNA in MHD patients.

Circulating MiR-133a as a biomarker predicts cardiac hypertrophy in chronic hemodialysis patients.

BACKGROUND: MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. MicroRNAs are present in the blood in a remarkably stable form and have emerged as potential diagnostic markers in patients with cardiovascular disease. Our study aimed to assess circulating miR-133a levels in MHD patients and the relation of miR-133a to cardiac hypertrophy. METHODS: We profiled miRNAs using RNA isolated from the plasma of participants. The results were validated in 64 MHD patients and 18 healthy controls. RESULTS: Levels of plasma miR-133a decreased in MHD patients with LVH compared with those in healthy controls. Plasma miR-133a concentrations were negatively correlated with LVMI and IVS. After single hemodialytic treatment, plasma miR-133a levels remained unchanged. Cardiac Troponin I and T were not associated with LVMI and IVS. CONCLUSIONS: Our observations supplied the possibility that circulating miR-133a could be a surrogate biomarker of cardiac hypertrophy in MHD patients.

Ets-1 targeted by microrna-221 regulates angiotensin II-induced renal fibroblast activation and fibrosis.

BACKGROUND: Fibroblast activation is one of the most important mechanisms for Angiotensin II (Ang II) in promoting renal fibrosis. Transcription factor Ets-1 is recognized to play a key role in kidney diseases. However, the role and mechanisms of Ets-1 in Ang-II induced fibroblast activation and kidney fibrosis are not fully understood. METHODS: Mice were treated with Ang II via osmotic mini-pumps or Ang II expression plasmid (pAng II). Cultured normal rat kidney interstitial fibroblast (NRK-49F) cells were incubated with Ang II. Role of Ets-1 in renal fibrosis and fibroblast activation were assessed by Western blot, Immunohistochemical staining'MTT, Boyden chamber and Immunofluorescence staining. Effects of miR-221 on Ets-1 and fibroblast activation were investigated by MTT, Boyden chamber, Western blot and Q-PCR. RESULTS: We found that Ets-1 was up-regulated in fibrotic kidneys. Similarly, Ang II could activate NRK-49F cells as demonstrated by up-regulated α-SMA and fibronectin(FN) expression and enhanced cell proliferation and migration. Ang II also induced Ets-1 expression in NRK-49F cells in a dose and time dependent manner. Knock-down of Ets-1 by RNA interference attenuated Ang II-induced activation of NRK-49F cells. Ets-1 was previously reported as a target of microRNA-221 (miR-221). In Ang II-induced fibrotic kidney, miR-221 was down-regulated. Similar results were observed in Ang II treated NRK-49F cells. Ectopic expression of miR-221 mimic attenuated the up-regulation of Ets-1 by Ang II in NRK-49F cells, which further prevented the activation of NRK-49F cells. However, the inhibitor of miR-221 aggravated Ang II induced Ets-1 expression and NRK-49F cells activation. CONCLUSIONS: Our study suggests that miR-221/Ets-1 axis takes an important role in mediating AngII induced interstitial fibroblast activation and renal fibrosis.

miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment.

OBJECTIVES: Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by ß-glycerophosphoric acid. APPROACH AND RESULTS: Primary cultured rat aortic VSMCs were treated with ß-glycerophosphoric acid for different periods of time. In VSMCs, after ß-glycerophosphoric acid treatment, the expressions of cbf ß1, osteocalcin and osteopontin were significantly increased and SM-22ß expression was decreased. ALP activity was induced by ß-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with ß-glycerophosphoric acid. Overexpression of miR-125b could inhibit ß-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited ß-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification. CONCLUSION: Our study suggests that down-regulation of miR-125b after ß-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1.

A study of the immunoloregulation of double filtration plasmapheresis in maintenance hemodialysis patients with chronic hepatitis C.

Although a large number of drugs have been used to treat chronic hepatitis C (CHC), there still remains a great challenge to treat maintenance hemodialysis (MHD) patients with chronic hepatitis C. To clarify the immunnoloregulation of double filtration plasmapheresis (DFPP) in MHD patients with CHC, DFPP was performed in 20 MHD patients with CHC (HCV-antibody positive, serum HCV RNA >500 IU/ml more than 6 months and HCV genotype 1b). The clinical data was collected and peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at the time of hour 0, hour 3, day 3, day 7 and day 28 after the DFPP, respectively. Serum HCV particles could be removed partially by the DFPP. The titer of serum HCV RNA could remain in a lower level even 28 days after the treatment. Compared to MHD patients without HCV infection, the frequencies of innate immune cells were similar in MHD patients with CHC, while Th1/Th2 was elevated and the frequencies of regulatory T (Treg) cells were higher in those MHD patients with CHC. The frequencies of monocytes and natural killer (NK) cells remained after the DFPP in MHD patients with CHC. There were no significant changes of Th1, Th2 and Th1/Th2 in PBMC after DFPP. DFPP could reduce the frequencies of Th17 cells and Treg cells in PBMC from 7 days after DFPP in MHD patients with CHC. DFPP could partially remove the serum HCV particles mechanically. The titer of HCV RNA could remain in a lower level at least for 28 days probably due to the redistribution of the immunocytes in circulation.

Involvement of endoplasmic reticulum stress in albuminuria induced inflammasome activation in renal proximal tubular cells.

Albuminuria contributes to the progression of tubulointerstitial fibrosis. Although it has been demonstrated that ongoing albuminuria leads to tubular injury manifested by the overexpression of numerous proinflammatory cytokines, the mechanism remains largely unknown. In this study, we found that the inflammasome activation which has been recognized as one of the cornerstones of intracellular surveillance system was associated with the severity of albuminuria in the renal biopsies specimens. In vitro, bovine serum albumin (BSA) could also induce the activation of NLRP3 inflammasome in the cultured kidney epithelial cells (NRK-52E). Since there was a significant overlap of NLRP3 with the ER marker calreticulin, the ER stress provoked by BSA seemed to play a crucial role in the activation of inflammasome. Here, we demonstrated that the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA) which was proved to be an enhancer for the adaptive capacity of ER could attenuate the inflammasome activation induced by albuminuria not only in vitro but also in diabetic nephropathy. Taken together, these data suggested that ER stress seemed to play an important role in albuminuria-induced inflammasome activation, elimination of ER stress via TUDCA might hold promise as a novel avenue for preventing inflammasome activation ameliorating kidney epithelial cells injury induced by albuminuria.

Sp1 mediates microRNA-29c-regulated type I collagen production in renal tubular epithelial cells.

Specificity protein 1 (Sp1), a ubiquitously expressed transcription factor, plays a potential pathogenic role for fibrotic disease in many organs by regulating the expression of several fibrosis-related genes, however, its role in kidney fibrosis and the mechanisms regulating its expression remain incompletely clarified. Here, we found that Sp1 was markedly induced and closely correlated with interstitial type I collagen accumulation in kidney tubular epithelia from obstructive nephropathy. In vitro, both Sp1 and type I collagen expression were up-regulated in TGF-ß1-treated kidney tubular epithelial cells (NRK-52E), whereas knockdown of Sp1 largely abolished TGF-ß1-induced type I collagen production, suggesting that Sp1 induction is partially responsible for type I collagen expression. In addition, we found that miR-29c expression was remarkably reduced in either the tubular epithelial cells from kidney with UUO nephropathy or TGF-ß1-treated NRK-52E cells. Knockdown of miR-29c could sufficiently induce Sp1 and type I collagen expression, whereas ectopic expression of miR-29c largely abolished their expression stimulated by TGF-ß1 in NRK-52E cells. Furthermore, knockdown of Sp1 effectively hindered type I collagen induction stimulated by miR-29c down-regulation. Collectively, this study demonstrates that Sp1 acts as an essential mediator for miR-29c in regulating type I collagen production in tubular epithelial cells, which may provide a novel mechanistic insight about miR-29c in renal fibrosis.