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The quantum yield and fluorescence properties of carbon dots are key issues for environmental detection. In this study, nitrogen–sulfur-doped carbon dots (N,S-CDs) were prepared hydrothermally by adding thiourea to provide the N source. By adjusting the ratio of citric acid (CA) to thiourea (N,S) and adding anhydrous ethanol, blue fluorescent doped carbon dots with a quantum yield of up to 53.80% were obtained. The particle morphology and crystalline organization of the N,S-CDs were analyzed using transmission electron microscopy (TEM) and X-ray diffraction (XRD). Fourier transform infrared (FTIR) spectroscopy was used to illuminate distinct functional units through the recording of typical vibration bands. The luminescence properties of the N,S-CDs were investigated using ultraviolet–visible (UV-vis) absorption spectroscopy and steady-state fluorescence spectroscopy (PL). In addition, the fluorescence stability of the N,S-CDs was studied in detail. The results showed that the functional groups of the N,S-CDs chelate Fe3+ ions to quench the fluorescence of carbon dots. This shows that the N,S-CDs exhibit high selectivity for Fe3+ ions. With the addition of Fe3+ in the concentration of 0–100 µM, the fluorescence intensity of the N,S-CDs exhibited distinct and linear dependence upon the Fe3+ concentration (R2 = 0.9965), and the detection limit (D = 3ơ/m) was measured as 0.2 µM. The excellent optical properties and Fe3+ selectivity of the N,S-CDs provide a huge boost for application in the field of environmental monitoring.

Vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation induced by high-phosphate is a crucial step in the development of arterial medial calcification (AMC) in patients with chronic kidney disease (CKD), and previous studies implicate Wnt/β-catenin signaling in osteogenic transdifferentiation of VSMCs and AMC. Given that resveratrol's ability to modulate Wnt/β-catenin signaling in other types of cell, we tested the effect of resveratrol on high-phosphate-induced osteogenic transdifferentiation of VSMCs and AMC in CKD. Resveratrol ameliorated AMC in rats with chronic renal failure and calcium deposition in aortic rings and VSMCs cultured in a high-phosphate environment. Resveratrol also diminished high-phosphate-induced osteogenic transdifferentiation of VSMCs in cultured aortic rings and VSMCs. In vitro, resveratrol attenuated the activation of β-catenin induced by high-phosphate and inhibited the expression of Runx2, a downstream effector of Wnt/β-catenin signaling during osteogenic transdifferentiation of VSMCs. Intriguingly, resveratrol inhibited high-phosphate-induced phosphorylation of LRP6 (Ser1490), but didn't inhibit Wnt3a-induced phosphorylation of LRP6 (Ser1490) and Runx2 expression. The expression of several Wnts was induced by high-phosphate, but the expression of Wnt7a, not Wnt2b and Wnt10a could be suppressed by resveratrol. In addition, the expression of both porcupine and wntless, two obligatory proteins for Wnt secretion, was induced by high-phosphate in cultured aortic rings and VSMCs, which could be suppressed by resveratrol. In summary, these findings suggest that resveratrol possesses a vascular protective effect on retarding high-phosphate-induced osteogenic transdifferentiation of VSMCs and AMC in CKD by targeting Wnt/β-catenin signaling, which may, to a large extent, via impeding Wnt secretion.

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 β-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-β1 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.

Impaired orthostatic blood pressure (BP) stabilization is prevalent in patients with chronic kidney disease (CKD) and is associated with adverse outcomes. We aimed to test the hypothesis that reduced hemoglobin is an important contributor to orthostatic intolerance in CKD in the present study. This study included 262 patients with non‐dialysis‐dependent CKD. Seated and standing BP was measured, and orthostatic BP reduction was calculated for both systolic BP (∆ SBP) and diastolic BP (∆ DBP). The association between orthostatic BP reduction and hemoglobin was determined by multiple linear regression models. We also performed mediation analysis to test to what extent the effect of renal dysfunction on impaired orthostatic BP stabilization can be explained by reduced hemoglobin. The mean age of the patients was 57.7 (±14.5) years, and 61.5% were male. Both ∆ SBP and ∆ DBP correlated negatively with estimated glomerular filtration rate (eGFR). With adjustment for age and sex, hemoglobin level was negatively associated with ∆ SBP (β = −1.4, SE = 0.4, P

Background The reactive hyperemia index (RHI), measured by peripheral arterial tonometry (PAT), is a novel measurement of endothelial function and has been proven to be valuable in cardiovascular risk stratification in several populations. The current study aims to explore its relation to renal function and its association with traditional cardiovascular risk factors in patients with chronic kidney disease (CKD). Methods Subjects with non-dialysis dependent CKD were recruited and 252 of them had a successful PAT test. In addition to general demographic and medical information, carotid-femoral pulse wave velocity (cfPWV), carotid-radial pulse wave velocity (crPWV) and augmentation index (AIx) were recorded. Results The mean age of the study population was 57.7 (±14.7) years and 155 (61.5%) were males. The average RHI was 1.92 (±14.7) with no difference noted between males and females. There was no statistically significant correlation between RHI and eGFR (r = − 0.107, p = 0.089) or urine protein-to-creatinine ratio (r = 0.036, p = 0.570). With adjustment for age and sex, RHI was associated with systolic blood pressure (BP) (β = 0.006, p = 0.001), diastolic BP (β = 0.008, p = 0.010), heart rate (β = − 0.007, p = 0.015) crPWV (β = 0.037, p = 0.022) and AIx (β = 0.006, p = 0.001), but not with cfPWV or any other conventional risk factors analyzed. Systolic BP remained the only predictor for RHI in the stepwise regression analysis. Conclusions RHI did not decline with reduced renal function in CKD patients and had a modest association with traditional cardiovascular risk factors. Further studies are warranted to determine if RHI could predict cardiovascular outcome in CKD patients.

Kidney fibroblasts play a crucial role in dictating tubular cell fate and the outcome of acute kidney injury (AKI). The underlying mechanisms remain to be determined. Here, we found that mTOR signaling was activated in fibroblasts from mouse kidneys with ischemia/reperfusion injury (IRI). Ablation of fibroblast Rheb or Rictor promoted, while ablation of fibroblast Tsc1 protected against tubular cell death and IRI in mice. In tubular cells cultured with conditioned media (CM) from Rheb(−/−) or Rictor(−/−) fibroblasts, less hepatocyte growth factor (HGF) receptor c-met signaling activation or staurosporine-induced cell apoptosis was observed. While CM from Tsc1(−/−) fibroblasts promoted tubular cell c-met signaling activation and inhibited staurosporine-induced cell apoptosis. In kidney fibroblasts, blocking mTOR signaling downregulated the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and HGF. Downregulating fibroblast HGF expression or blocking tubular cell c-met signaling facilitated tubular cell apoptosis. Notably, renal PPARγ and HGF expression was less in mice with fibroblast Rheb or Rictor ablation, but more in mice with fibroblast Tsc1 ablation than their littermate controls, respectively. Together, these data suggest that mTOR signaling activation in kidney fibroblasts protects against tubular cell death and dictates the outcome of AKI through stimulating PPARγ and HGF expression.

Our previously published study demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling mediates TGFβ1-induced fibroblast activation. However, the underlying mechanisms for mTORC2 in stimulating fibroblast activation remain poorly understood. Here, we found that TGFβ1 could stimulate mTORC2 and Yap/Taz activation in NRK-49F cells. Blocking either mTORC2 or Yap/Taz signaling diminished TGFβ1-induced fibroblast activation. In addition, blockade of mTORC2 could down-regulate the expression of Yap/Taz, connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). Overexpression of constitutively active Taz (Taz-S89A) could restore fibroblast activation suppressed by PP242, an mTOR kinase inhibitor in NRK-49F cells. In mouse kidneys with unilateral ureter obstructive (UUO) nephropathy, both mTORC2 and Yap/Taz were activated in the interstitial myofibroblasts. Ablation of Rictor in fibroblasts/pericytes or blockade of mTOR signaling with PP242 attenuated Yap/Taz activation and UUO nephropathy in mice. Together, this study uncovers that targeting mTORC2 retards fibroblast activation and kidney fibrosis through suppressing Yap/Taz activation.

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.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)-β1-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-β and PGC-1α expression and protect the mitochondrial function and structure of PTCs under TGF-β1 stimulation. The antifibrotic role of rolipram in renal fibrosis relies on C/EBP-β 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-β/PGC1-α and mitochondrial biogenesis. Antioxid. Redox Signal. 29, 637-652.

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome–lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

β‐Catenin signalling plays an important role in regulating tubular epithelial‐to‐mesenchymal transition (EMT), an indispensable programme for driving renal fibrosis. As an adapter protein, four and a half LIM domain protein 2 (FHL2) acts as a coregulator of β‐catenin in several other cell types. To determine whether FHL2 affects β‐catenin signalling and thus is involved in tubular EMT, we examined its expression and function in the process of TGF‐β1‐induced EMT. FHL2 mRNA and protein were induced by TGF‐β1 in rat tubular epithelial cells (NRK‐52E), an effect that intracellular Smad signalling was required. Ectopic expression of FHL2 inhibited E‐cadherin and enhanced α‐smooth muscle actin (α‐SMA) and fibronectin expression, whereas knockdown of FHL2 partially restored E‐cadherin and reduced α‐SMA and fibronectin induction stimulated by TGF‐β1. Overexpression of FHL2 increased β‐catenin dephosphorylation (Ser37/Thr41), nuclear translocation and β‐catenin‐mediated transcription and up‐regulated expression of β‐catenin target, EMT‐related genes, such as Snail, Twist, vimentin, plasminogen activator inhibitor‐1 and matrix metalloproteinase‐7. Conversely, knockdown of FHL2 increased β‐catenin phosphorylation (Ser33/37/Thr41), decreased its nuclear translocation and inhibited β‐catenin‐mediated transcription and target genes expression. TGF‐β1 induced a FHL2/β‐catenin interaction in NRK‐52E cells, especially in the nuclei. In a mouse model of obstructive nephropathy, FHL2 mRNA and protein were induced in a time‐dependent fashion, and the extent and pattern of renal β‐catenin activation were positively correlated with FHL2 induction. Collectively, this study suggests that FHL2, via modulating β‐catenin signalling, may implicate in regulation of TGF‐β1‐mediated tubular EMT and could be a potential therapeutic target for fibrotic kidney disease.

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4- or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor–/– macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor+/+ macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronic kidney disease (CKD) is a potent risk factor for acute kidney injury (AKI) and a modifier for the relationship between AKI and adverse outcome, while AKI increases the risk of de novo CKD and is an accelerator for the progression of underlying CKD. When patients with CKD develop dialysis-requiring AKI, available data suggest that the recovery of kidney function is less likely than in patients without CKD. Patients with AKI were more likely to develop CKD compared with matched control patients without AKI. For CKD patients at high risk for developing AKI, preventive measures for AKI include adequate fluid repletion in those with hypovolemia, avoidance of hypotension by providing inotropic support as needed, and readjustment of nephrotoxic medications based on close monitoring of renal function and drug levels, if available. For CKD patients developing AKI, the basic principles of general management include specific treatment of the underlying cause, fluid management, electrolyte management, adjustment of drug dosing, nutritional support, renal replacement therapy, and specific pharmacologic therapies.

Although the pregnancy rate of women with chronic kidney disease (CKD) and the overall survival rate of their fetuses have improved, pregnancy in women with CKD still have a high risk of adverse maternal and fetal outcomes. To achieve better outcomes for this particular population, managing pregnancy in women with CKD has become a considerable challenge shared by both nephrologists and obstetricians. Strengthened management, including prepregnancy preparation, pregnancy management, peripartum management, and postpartum care, could prevent or mitigate maternal renal damage and adverse maternal and fetal outcomes. Women with CKD require risk assessment of pregnancy before conceiving, close follow-up by both nephrologists and obstetricians to monitor disease activity and detect obstetric complications during pregnancy, evaluation of indications for termination of pregnancy and selection of delivery mode, and assessment of disease activity and emotional support to prevent depression after delivery.

Anemia is an important and frequent complication in patients with chronic kidney disease (CKD), and its prevalence increases as renal function declines. The etiology of anemia in CKD is multifactorial, including absolute or relative lack of erythropoietin, iron deficiency, blood loss, shortened red blood cell survival, and other factors. Anemia in CKD is associated with impaired quality of life and elevated morbidity and mortality of cardiovascular diseases. Patients with CKD should monitor hemoglobin levels regularly. If the degree of anemia is not consistent with that of renal dysfunction in patients with CKD, it is necessary to rule out the existence of other diseases that can cause anemia. Erythropoiesis-stimulating agent (ESA) and iron supplementation are the primary treatment for anemia in CKD. Hemoglobin levels and iron status should be monitored regularly during treatment. It is generally recommended that hemoglobin concentration not exceed 115 g/L in patients undergoing ESA maintenance; the monthly hemoglobin growth rate should be limited in ≥10 g/L, but

The high sensitivity to changes in oxygen tension makes kidney vulnerable to hypoxia. Both acute kidney injury and chronic kidney disease are almost always accompanied by hypoxia. Tubular epithelial cells (TECs), the dominant intrinsic cells in kidney tissue, are believed to be not only a victim in the pathological process of various kidney diseases, but also a major contributor to kidney damage. Hypoxia inducible factor-1 (HIF-1) is the main regulator of adaptive response of cells to hypoxia. Under various clinical and experimental kidney disease conditions, HIF-1 plays a pivotal role in modulating multiple cellular processes in TECs, including apoptosis, autophagy, inflammation, metabolic pattern alteration, and cell cycle arrest. A comprehensive understanding of the mechanisms by which HIF-1 regulates these cellular processes in TECs may help identify potential therapeutic targets to improve the outcome of acute kidney injury and delay the progression of chronic kidney disease.

Four-and-a-half LIM domains protein 2 (FHL2) has been proposed involving in β-catenin activity. We previously reported that FHL2 mediates TGF-β1-induced tubular epithelial-to-mesenchymal transition through activating Wnt/β-catenin signaling. However, the potential role and mechanism for FHL2 in TGF-β1-induced fibroblast activation and kidney fibrosis remains unknown. Here, we initially observed higher levels of FHL2 expression in fibrotic kidneys from both patients and mice, especially in α-smooth muscle actin (α-SMA)-positive cells in the interstitium. In cultured interstitial fibroblasts, FHL2 expression was induced by TGF-β1. Knockdown of FHL2 remarkably suppressed TGF-β1-induced α-SMA, type I collagen, and fibronectin expression, while overexpression of FHL2 was sufficient to activate fibroblasts. In mice, fibroblast-specific deletion of FHL2 diminished renal induction of α-SMA, type I collagen, and fibronectin and interstitial extracellular matrix deposition at 2 weeks after ureteral obstruction. We next investigated Wnt/β-catenin activity and found that β-catenin was activated in most FHL2-positive cells in renal interstitium from mice with obstructive nephropathy. In vitro, TGF-β1 induced a physical interaction between FHL2 and β-catenin, especially in the nucleus. Downregulation of FHL2 inhibited TGF-β1-induced active β-catenin upregulation, β-catenin nuclear translocation, and β-catenin-mediated transcription, whereas overexpression of FHL2 was able to activate Wnt/β-catenin signaling. FHL2 overexpression-induced β-catenin-mediated gene transcription could be hindered by ICG-001, but FHL2 overexpression-induced upregulation of active β-catenin could not be. Collectively, this study reveals that the signal regulatory effect of FHL2 on β-catenin plays an important role in TGF-β1-induced fibroblast activation and 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.

Our previous study determined that prominent cerebral vasospasm (CVS) may occur in an in vivo model of subarachnoid hemorrhage (SAH) in rats. Matrix metalloproteinase 9 (MMP‑9) expression levels in basilar arteries were upregulated in a similar manner to the development of CVS following SAH. To identify the changes that occur in the contractility of cerebrovascular smooth muscle cells and the expression levels of MMP‑9 in an in vitro model of SAH, rat cerebrovascular smooth muscle cells were isolated, cultured, and then stimulated with hemolysate. Additionally, 2-[(4-phenoxyphenylsulfonyl)methyl]thiirane (SB-3CT), a selective MMP-9 inhibitor, was used to determine the effect of MMP‑9 on the contractility of cerebrovascular smooth muscle cells. Cerebrovascular smooth muscle cells were successfully isolated and cultured in vitro, and hemolysate stimulation enhanced their contractility and increased MMP‑9 expression levels. The present study also revealed that pretreatment with SB‑3CT decreased MMP‑9 expression levels in cerebrovascular smooth muscle cells, and reduced their contractility upon hemolysate treatment. Therefore, the current study confirmed that MMP‑9 is important for the enhancement of the contractility of cerebrovascular smooth muscle cells in an in vitro rat model of SAH.

M2 macrophage polarization is known to underlie kidney fibrosis. We previously reported that most of the members of the Wnt family of signaling proteins are induced in fibrotic kidneys. Dysregulation of the signaling protein Wnt5a is associated with fibrosis, but little is known about the role of Wnt5a in regulating M2 macrophage activation that results in kidney fibrosis. Here, using murine Raw 264.7 cells and bone marrow–derived macrophages, we found that Wnt5a enhanced transforming growth factor β1 (TGFβ1)-induced macrophage M2 polarization as well as expression of the transcriptional regulators Yes-associated protein (Yap)/transcriptional coactivator with PDZ-binding motif (Taz). Verteporfin blockade of Yap/Taz inhibited both Wnt5a- and TGFβ1-induced macrophage M2 polarization. In mouse models of kidney fibrosis, shRNA-mediated knockdown of Wnt5a expression diminished kidney fibrosis, macrophage Yap/Taz expression, and M2 polarization. Moreover, genetic ablation of Taz in macrophages attenuated kidney fibrosis and macrophage M2 polarization in mice. Collectively, these results indicate that Wnt5a promotes kidney fibrosis by stimulating Yap/Taz-mediated macrophage M2 polarization.

Lupus nephritis (LN) is a chronic autoimmune disorder. Here we try to identify the candidate genes in macrophages related to LN. We performed a systematic search in the Gene Expression Omnibus (GEO) database for microarray in human mononuclear cells and mouse macrophages of LN. The results of clustering and venn analysis of different GEO datasets showed that 8 genes were up-regulated and 2 genes down-regulated in samples from both human and mouse LN. The data from gene network and GO analysis revealed that CD38 and CCL2 were localized in the core of the network. Immunofluorescence staining showed that CD38 expression was markedly increased in macrophages from kidneys with LN. Our study identifies the gene expression profile for macrophages and demonstrated the induction of CCL2 and CD38 in macrophages from patients with LN. However, regarding the limited patient number included in this study, the results are preliminary and more studies are still needed to further decipher the macrophage-related candidate genes for the pathogenesis of LN.

Fibrosis is defined as an excessive accumulation of extracellular matrix components that lead to the destruction of organ architecture and impairment of organ function. Moreover, fibrosis is an intricate process attributable to a variety of interlaced fibrogenic signals and intrinsic mechanisms of activation of myofibroblasts. Being the dominant matrix-producing cells in organ fibrosis, myofibroblasts may be differentiated from various types of precursor cells. Identification of the signal pathways that play a key role in the pathogenesis of fibrotic diseases may suggest potential therapeutic targets. Here, we emphasize several intracellular signaling pathways that control the activation of myofibroblasts and matrix production.

The CD38, possessing ADP-ribosyl cyclase (ADPR-cyclase) and cyclic ADP-ribose hydrolase (cADPR-hydrolase), is able to regulate a variety of cellular activities. However, the role and mechanisms for CD38 in macrophage activation and sepsis-induced acute kidney injury (AKI) remain to be determined. Here we report that in cultured macrophages, Lipopolysaccharide (LPS) could upregulate CD38 expression in time and dose dependent manner. Knocking down or blockade of CD38 in macrophages could inhibit LPS-induced macrophage M1 polarization accompanied by diminished NF-κB signaling activation. In mouse model with LPS-induced acute kidney injury, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection. Together, these results demonstrate that CD38 mediates LPS-induced macrophage activation and AKI, which may be treated as a therapeutic target for sepsis-induced AKI in patients.

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4– or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro. Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4– or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor

Podocytes play a key role in the formation of cellular crescents in experimental and human diseases. However, the underlying mechanisms for podocytes in promoting crescent formation need further investigation. Here, we demonstrated that mammalian target of rapamycin complex 1 (mTORC1) signaling was remarkably activated and hypoxia-inducible factor (HIF) 1α expression was largely induced in cellular crescents from patients with crescentic glomerular diseases. Specific deletion of Tsc1 in podocytes led to mTORC1 activation in podocytes and kidney dysfunction in mice. Interestingly, 33 of 36 knockouts developed cellular or mixed cellular and fibrous crescents at 7 wk of age (14.19 ± 3.86% of total glomeruli in knockouts vs. 0% in control littermates, n = 12–36, P = 0.04). All of the seven knockouts developed crescents at 12 wk of age (30.92 ± 11.961% of total glomeruli in knockouts vs. 0% in control littermates, n = 4–7, P = 0.002). Most notably, bridging cells between the glomerular tuft and the parietal basement membrane as well as the cellular crescents were immunostaining positive for WT1, p-S6, HIF1α, and Cxcr4. Furthermore, continuously administrating rapamycin starting at 7 wk of age for 5 wk abolished crescents as well as the induction of p-S6, HIF1α, and Cxcr4 in the glomeruli from the knockouts. Together, it is concluded that mTORC1 activation in podocytes promotes cellular crescent formation, and targeting this signaling may shed new light on the treatment of patients with crescentic glomerular diseases.

The mammalian target of rapamycin (mTOR) plays a critical role for cell growth and survival in many cell types. While substantial progress has been made in understanding the abnormal activation of mTORC1 in the pathogenesis of kidney disease, little is known about mTORC2 in kidney disease such as acute kidney injury (AKI). To study this, we generated a mouse model with tubule-specific deletion of Rictor (Tubule-Rictor-/-). The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 2 months of birth. However, ablation of Rictor in the tubular cells exacerbated cisplatin-induced AKI compared to that in the control littermates. As expected, tubular cell apoptosis, Akt phosphorylation (Ser473), and autophagy were induced in the kidneys from the control littermates by cisplatin treatment. Less cell autophagy or Akt phosphorylation and more cell apoptosis in the kidneys of the knockout mice were identified compared with those in the control littermates. In NRK-52E cells in vitro , Rictor siRNA transfection sensitized cell apoptosis to cisplatin but with reduced cisplatin-induced autophagy. Metformin, an inducer of autophagy, abolished cell death induced by Rictor siRNA and cisplatin. Thus, endogenous Rictor/mTORC2 protects against cisplatin-induced AKI, probably mediated by promoting cell survival through Akt signaling activation and induction of autophagy.

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 β-glycerophosphoric acid. Approach and results Primary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbf β1, osteocalcin and osteopontin were significantly increased and SM-22β expression was decreased. ALP activity was induced by β-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 β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-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 β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification. Conclusion Our study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1.

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-β1-treated kidney tubular epithelial cells (NRK-52E), whereas knockdown of Sp1 largely abolished TGF-β1-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-β1-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-β1 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.

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFβ1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFβ1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease.