External Validation of the International Prognosis Prediction Model of IgA Nephropathy.

BACKGROUND: The International IgA Nephropathy (IgAN) Network developed and validated two prognostic prediction models for IgAN, one incorporating a race parameter. These models could anticipate the risk of a 50% reduction in estimated glomerular filtration rate (eGFR) or progression to end-stage renal disease (ESRD) subsequent to an IgAN diagnosis via renal biopsy. This investigation aimed to validate the International IgA Nephropathy Prediction Tool (IIgANPT) within a contemporary Chinese cohort. METHODS: Within this study,185 patients diagnosed with IgAN via renal biopsy at the Center for Kidney Disease, Second Affiliated Hospital of Nanjing Medical University, between January 2012 and December 2021, were encompassed. Each patient's risk of progression was assessed utilizing the IIgANPT formula. The primary outcome, a 50% decline in eGFR or progression to ESRD, was examined. Two predictive models, one inclusive and the other exclusive of a race parameter, underwent evaluation via receiver-operating characteristic (ROC) curves, subgroup survival analyses, calibration plots, and decision curve analyses. RESULTS: The median follow-up duration within our cohort spanned 5.1 years, during which 18 patients encountered the primary outcome. The subgroup survival curves exhibited distinct separations, and the comparison of clinical and histological characteristics among the risk subgroups revealed significant differences. Both models demonstrated outstanding discrimination, evidenced by the areas under the ROC curve at five years: 0.882 and 0.878. Whether incorporating the race parameter or not, both prediction models exhibited acceptable calibration. Decision curve analysis affirmed the favorable clinical utility of both models. CONCLUSIONS: Both prognostic risk evaluation models for IgAN exhibited remarkable discrimination, sound calibration, and acceptable clinical utility.

Protocol for a single-blind randomized clinical trial to test the efficacy of bilateral transcranial magnetic stimulation on upper extremity motor function in patients recovering from stroke.

BACKGROUND: No consensus currently exists regarding the optimal protocol for repetitive transcranial magnetic stimulation (rTMS) treatment of upper-extremity motor dysfunction after stroke. Studies have shown that combined low- and high-frequency stimulation (LF-HF-rTMS) of the bilateral cerebral hemispheres is more effective than sham stimulation or stimulation of one cerebral hemisphere alone in treating motor dysfunction in the subacute stage of stroke. The efficacy of this protocol in the convalescence phase of stroke has rarely been reported, and its mechanism of action has not been clarified. In this study, we designed a prospective, single-blind, randomized controlled trial to investigate the efficacy and safety of different stimulation regimens for the treatment of upper extremity motor disorders in patients with convalescent stage stroke and aimed to explore the underlying mechanisms based on biomarkers such as brain-derived neurotrophic factor (BDNF). METHODS: Seventy-six subjects will be randomly divided into combined, low-frequency, high-frequency, and control groups based on the proportion of 1:1:1:1, with 19 cases in each group. All groups will have conventional rehabilitation, on top of which the combined group will receive 1 Hz rTMS in the unaffected hemisphere and 10 Hz rTMS in the affected hemisphere. The low-frequency group will be administered 1 Hz rTMS in the unaffected hemisphere and sham stimulation in the contralateral hemisphere. The high-frequency group will be administered 10 Hz rTMS in the affected hemisphere and contralateral sham stimulation. The control group will receive bilateral sham stimulation. Assessments will be performed at baseline, after 2 weeks of treatment, and at post-treatment follow-up at week 6. The primary outcomes are FMA-UE (Fugl-Meyer assessment-upper extremity), latency, and serum BDNF levels. The secondary outcomes are the National Institute of Health Stroke Scale (NIHSS), Brunnstrom staging (BS), modified Ashworth scale (MAS), Modified Barthel Index (MBI), central motor conduction time (CMCT), precursor proteins of mature BDNF (proBDNF), and matrix metalloproteinase-9 (MMP-9) levels. Adverse events, such as headaches and seizures, will be recorded throughout the study. DISCUSSION: The findings of this study will help develop optimal stimulation protocols for motor recovery in stroke patients and identify biomarkers that respond to post-stroke motor rehabilitation, for better guidance of clinical treatment. TRIAL REGISTRATION: The study protocol was passed by the Medical Research Ethics Committee of the General Hospital of Ningxia Medical University on January 1, 2022 (no. KYLL-2021-1082). It was registered into the Chinese Clinical Trials Registry on May 22, 2022 (no. ChiCTR2200060201). This study is currently in progress.

Increased thoracic fluid content is associated with higher risk for pneumonia in patients undergoing maintenance hemodialysis.

BACKGROUND: Pneumonia is the most common infectious disease in patients undergoing maintenance hemodialysis (MHD). The aim of this study is to determine the possible predictive value of thoracic fluid content (TFC) for pneumonia in this population. METHOD: Clinical data were recorded for 1412 MHD patients who were hospitalized for certain comorbidities or complications. Each patient underwent an impedance cardiography (ICG) examination before next dialysis session after admission. Patients were divided into Having-, Will-have-, and Non-pneumonia groups based on whether they had pneumonia at the time of ICG examination after the admission and within five months after the examination. Hemodynamic parameters and other clinical data were compared and analyzed. RESULTS: Patients who were going to develop pneumonia were older, and had a higher proportion of diabetes, poorer nutritional status, a higher level of inflammatory, poorer cardiac function, and more fluid volume load than those who did not develop pneumonia. Multivariate binary logistic analysis revealed that for each 1/KΩ increase in TFC and 1 increase in neutrophil-to-lymphocyte ratio (NLR), the risk of the development of pneumonia increased by 3.1% (p ˂ 0.01) and 7.2% (p = 0.035), respectively, whereas for each 1 g/L increase in hemoglobin and 1 g/L increase in serum albumin, the risk of the development of pneumonia decreased by 1.3% (p = 0.034) and 5% (p = 0.048), respectively. CONCLUSIONS: TFC, NLR, hemoglobin, and serum albumin were independent risk factors for the development of pneumonia in MHD patients. Given the advantages of ICG, TFC can be used clinically as a helpful predictor of pneumonia in MHD patients.

Effects of 1-MCP Treatment on Physiology and Storage Quality of Root Mustard at Ambient Temperature.

Root mustard is plentiful in vitamins and minerals but shrivels and molds easily. In this study, freshly harvested root mustard was fumigated with various concentrations of 1-Methycyclopropene(1-MCP) (1 µL L-1, 1.5 µL L-1, and 2.0 µL L-1) for 24 h and stored at ambient temperature (17 ± 1 °C) for 35 d. Our data showed that 1-MCP fumigation had a striking preservation effect on maintaining weight loss, fruit firmness, lignin, Vc content, and moisture content, inhibiting respiratory intensity and ethylene release rate, as well as decreasing cell permeability and malondialdehyde (MDA) accumulation and maintaining cell membrane integrity of root mustard. In addition, lipoxygenase (LOX), pyruvate dehydrogenase (PDH), and polyphenol oxidase (PPO) activities were significantly reduced throughout the storage period. In contrast, the activities of succinate dehydrogenase (SDH), superoxide dismutase (SOD), ascorbate peroxidase (APX), phenylalanine deaminase (PAL), and peroxidase (POD) remained at high levels. Results showed that 1-MCP treatments were effective in maintaining the quality of root mustard, and the preservation effect of 1.0 µL·L-1 1-MCP was better than other concentrations of 1-MCP. This study could serve as a theoretical reference for root mustard preservation.

Resveratrol ameliorates high-phosphate-induced VSMCs to osteoblast-like cells transdifferentiation and arterial medial calcification in CKD through regulating Wnt/ß-catenin signaling.

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/ß-catenin signaling in osteogenic transdifferentiation of VSMCs and AMC. Given that resveratrol's ability to modulate Wnt/ß-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 ß-catenin induced by high-phosphate and inhibited the expression of Runx2, a downstream effector of Wnt/ß-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/ß-catenin signaling, which may, to a large extent, via impeding Wnt secretion.

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.

Improving the Dysregulation of FoxO1 Activity Is a Potential Therapy for Alleviating Diabetic Kidney Disease.

A substantial proportion of patients with diabetes will develop kidney disease. Diabetic kidney disease (DKD) is one of the most serious complications in diabetic patients and the leading cause of end-stage kidney disease worldwide. Although some mechanisms have been revealed to contribute to the understanding of the pathogenesis of DKD and some drugs currently in use have been shown to be beneficial, prevention and management of DKD remain tricky and challenging. FoxO1 transcriptional factor is a crucial regulator of cellular homeostasis and posttranslational modification is a major mechanism to alter FoxO1 activity. There is increasing evidence that FoxO1 is involved in the regulation of various cellular processes such as stress resistance, autophagy, cell cycle arrest, and apoptosis, thereby playing an important role in the pathogenesis of DKD. Improving the dysregulation of FoxO1 activity by natural compounds, synthetic drugs, or manipulation of gene expression may attenuate renal cell injury and kidney lesion in the cells cultured under a high-glucose environment and in diabetic animal models. The available data imply that FoxO1 may be a potential clinical target for the prevention and treatment of DKD.

Emerging Therapeutic Strategies for Attenuating Tubular EMT and Kidney Fibrosis by Targeting Wnt/ß-Catenin Signaling.

Epithelial-mesenchymal transition (EMT) is defined as a process in which differentiated epithelial cells undergo phenotypic transformation into myofibroblasts capable of producing extracellular matrix, and is generally regarded as an integral part of fibrogenesis after tissue injury. Although there is evidence that the complete EMT of tubular epithelial cells (TECs) is not a major contributor to interstitial myofibroblasts in kidney fibrosis, the partial EMT, a status that damaged TECs remain inside tubules, and co-express both epithelial and mesenchymal markers, has been demonstrated to be a crucial stage for intensifying fibrogenesis in the interstitium. The process of tubular EMT is governed by multiple intracellular pathways, among which Wnt/ß-catenin signaling is considered to be essential mainly because it controls the transcriptome associated with EMT, making it a potential therapeutic target against kidney fibrosis. A growing body of data suggest that reducing the hyperactivity of Wnt/ß-catenin by natural compounds, specific inhibitors, or manipulation of genes expression attenuates tubular EMT, and interstitial fibrogenesis in the TECs cultured under profibrotic environments and in animal models of kidney fibrosis. These emerging therapeutic strategies in basic researches may provide beneficial ideas for clinical prevention and treatment of chronic kidney disease.

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.

Deletion of FHL2 in fibroblasts attenuates fibroblasts activation and kidney fibrosis via restraining TGF-ß1-induced Wnt/ß-catenin signaling.

Four-and-a-half LIM domains protein 2 (FHL2) has been proposed involving in ß-catenin activity. We previously reported that FHL2 mediates TGF-ß1-induced tubular epithelial-to-mesenchymal transition through activating Wnt/ß-catenin signaling. However, the potential role and mechanism for FHL2 in TGF-ß1-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-ß1. Knockdown of FHL2 remarkably suppressed TGF-ß1-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/ß-catenin activity and found that ß-catenin was activated in most FHL2-positive cells in renal interstitium from mice with obstructive nephropathy. In vitro, TGF-ß1 induced a physical interaction between FHL2 and ß-catenin, especially in the nucleus. Downregulation of FHL2 inhibited TGF-ß1-induced active ß-catenin upregulation, ß-catenin nuclear translocation, and ß-catenin-mediated transcription, whereas overexpression of FHL2 was able to activate Wnt/ß-catenin signaling. FHL2 overexpression-induced ß-catenin-mediated gene transcription could be hindered by ICG-001, but FHL2 overexpression-induced upregulation of active ß-catenin could not be. Collectively, this study reveals that the signal regulatory effect of FHL2 on ß-catenin plays an important role in TGF-ß1-induced fibroblast activation and kidney fibrosis.

The regulatory role of HIF-1 in tubular epithelial cells in response to kidney injury.

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.

Impaired orthostatic blood pressure stabilization and reduced hemoglobin in chronic kidney disease.

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 (ß = -1.4, SE = 0.4, P < .001) and ∆ DBP (ß = -0.6, SE = 0.2, P = .009). The associations remained significant with further adjustment for additional covariates. When eGFR was introduced as a covariate, it did not eliminate the significance (both P < .05). The associations remained essentially unchanged in a sensitivity analysis excluding those with concurrent erythropoietin use. Mediation analysis demonstrated that reduced hemoglobin accounted for 35.4% (P = .004) of the effect of eGFR on ∆ SBP and 47.7% (P = .032) on ∆ DBP. Our study suggests that reduced hemoglobin is a potentially important contributor to the development of orthostatic hypotension in CKD.

Digital microvascular reactivity does not decline with impaired renal function in chronic kidney disease.

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) (ß = 0.006, p = 0.001), diastolic BP (ß = 0.008, p = 0.010), heart rate (ß = - 0.007, p = 0.015) crPWV (ß = 0.037, p = 0.022) and AIx (ß = 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.

Fibroblast mTOR/PPARγ/HGF axis protects against tubular cell death and acute kidney injury.

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.

The signaling protein Wnt5a promotes TGFß1-mediated macrophage polarization and kidney fibrosis by inducing the transcriptional regulators Yap/Taz.

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 ß1 (TGFß1)-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 TGFß1-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.

Yap/Taz mediates mTORC2-stimulated fibroblast activation and kidney fibrosis.

Our previously published study demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling mediates TGFß1-induced fibroblast activation. However, the underlying mechanisms for mTORC2 in stimulating fibroblast activation remain poorly understood. Here, we found that TGFß1 could stimulate mTORC2 and Yap/Taz activation in NRK-49F cells. Blocking either mTORC2 or Yap/Taz signaling diminished TGFß1-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.

Protein kinase Cα drives fibroblast activation and kidney fibrosis by stimulating autophagic flux.

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 ß1 (TGFß1) 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 TGFß1-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.

Identification of macrophage-related candidate genes in lupus nephritis using bioinformatics analysis.

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.

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.

FHL2 promotes tubular epithelial-to-mesenchymal transition through modulating ß-catenin signalling.

ß-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 ß-catenin in several other cell types. To determine whether FHL2 affects ß-catenin signalling and thus is involved in tubular EMT, we examined its expression and function in the process of TGF-ß1-induced EMT. FHL2 mRNA and protein were induced by TGF-ß1 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-ß1. Overexpression of FHL2 increased ß-catenin dephosphorylation (Ser37/Thr41), nuclear translocation and ß-catenin-mediated transcription and up-regulated expression of ß-catenin target, EMT-related genes, such as Snail, Twist, vimentin, plasminogen activator inhibitor-1 and matrix metalloproteinase-7. Conversely, knockdown of FHL2 increased ß-catenin phosphorylation (Ser33/37/Thr41), decreased its nuclear translocation and inhibited ß-catenin-mediated transcription and target genes expression. TGF-ß1 induced a FHL2/ß-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 ß-catenin activation were positively correlated with FHL2 induction. Collectively, this study suggests that FHL2, via modulating ß-catenin signalling, may implicate in regulation of TGF-ß1-mediated tubular EMT and could be a potential therapeutic target for fibrotic kidney disease.