Paraoxonase 1 Ameliorates Renal Lipotoxicity by Activating Lipophagy and Inhibiting Pyroptosis.

Several studies in recent years have shown that lipid overload causes lipotoxic damage to the kidney, and oxidative stress, inflammation, and autophagic arrest are all important mechanisms of renal lipotoxicity. However, effective measures with therapeutic effects on renal lipotoxicity are limited. The present study indicated the protective effect of the paraoxonase 1 (PON1) against renal lipotoxicity in high-fat diet-fed scavenger receptor class B type I-deficient (SR-BI-/-) mice. The results showed that SR-BI-/- mice exhibited significant renal pathologic characteristics, such as oxidative stress, inflammation, and fibrosis, under a normal chow diet, and were accompanied by dyslipidemia and reduced plasma PON1 activity and renal PON1 levels. PON1 overexpression significantly attenuated the above pathologic changes in the kidneys of SR-BI-/- mice fed with a high-fat diet. Mechanistically, PON1 may ameliorate renal oxidative stress by reducing reactive oxygen species production, reduce renal lipid accumulation by inhibiting AKT/mechanistic target of rapamycin kinase pathway to activate lipophagy, and reduce the occurrence of inflammation and cell death by inhibiting Nod-like receptor family protein 3 inflammasome-mediated pyroptosis. The present study is the first to show that PON1 overexpression can effectively alleviate renal lipotoxicity injury, and PON1 may be a promising therapeutic strategy for the treatment of renal lipotoxicity-related diseases.

The automatic detection of diabetic kidney disease from retinal vascular parameters combined with clinical variables using artificial intelligence in type-2 diabetes patients.

BACKGROUND: Diabetic kidney disease (DKD) has become the largest cause of end-stage kidney disease. Early and accurate detection of DKD is beneficial for patients. The present detection depends on the measurement of albuminuria or the estimated glomerular filtration rate, which is invasive and not optimal; therefore, new detection tools are urgently needed. Meanwhile, a close relationship between diabetic retinopathy and DKD has been reported; thus, we aimed to develop a novel detection algorithm for DKD using artificial intelligence technology based on retinal vascular parameters combined with several easily available clinical parameters in patients with type-2 diabetes. METHODS: A total of 515 consecutive patients with type-2 diabetes mellitus from Xiangyang Central Hospital were included. Patients were stratified by DKD diagnosis and split randomly into either the training set (70%, N = 360) or the testing set (30%, N = 155) (random seed = 1). Data from the training set were used to develop the machine learning algorithm (MLA), while those from the testing set were used to validate the MLA. Model performances were evaluated. RESULTS: The MLA using the random forest classifier presented optimal performance compared with other classifiers. When validated, the accuracy, sensitivity, specificity, F1 score, and AUC for the optimal model were 84.5%(95% CI 83.3-85.7), 84.5%(82.3-86.7), 84.5%(82.7-86.3), 0.845(0.831-0.859), and 0.914(0.903-0.925), respectively. CONCLUSIONS: A new machine learning algorithm for DKD diagnosis based on fundus images and 8 easily available clinical parameters was developed, which indicated that retinal vascular changes can assist in DKD screening and detection.

Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness.

Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-ß1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-ß1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.

Src-mediated ligand release-independent EGFR transactivation involves TGF-ß-induced Smad3 activation in mesangial cells.

A great deal of evidence highlighted the pathophysiologic importance of TGF-ß1/Smad3 pathway in masangial extracellular matrix (ECM) accumulation, but some alternative signaling pathways are also involved. TGF-ß was shown recently to induce rapid and transient epidermal-like growth factor receptor (EGFR) transactivation and subsequent fibronectin expression via heparin-binding epidermal-like growth factors (HB-EGF) release and binding in mesangial cells, which is independent of Smad2 activation. However, whether TGF-ß could induce persistent EGFR transactivation remains to be identified. The present study demonstrates that in addition to transient EGFR transactivation, TGF-ß1 can also induce continuous EGFR transactivation by a non-ligand-dependent pathway in rat mesangial cells. This sustained EGFR transactivation is mainly due to Src kinase-mediated persistent EGFR tyrosine phosphorylation at Y845 rather than Y1173. TGF-ß1-induced early Smad3 phosphorylation is independent of transient EGFR transactivation and ERK1/2 activation initiated by HB-EGF release, whereas Src-mediated chronic EGFR transactivation and ERK1/2 activation participate in Smad3 activation in a relatively modest and delayed manner. Therefore, the present study further clarifies the mechanisms of EGFR transactivation in the TGF-ß-initiated ECM upregulation and raises the possibility that targeting EGFR may provide a viable alternative strategy for inhibiting TGF-ß in chronic kidney disease.

Parathyroid hormone inhibits TGF-ß/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells.

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-ß1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-ß1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-ß1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-ß1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-ß type II receptor (TßR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TßR II might reduce the amount of membrane TßR II, attenuate the sensitivity of mesangial cells to TGF-ß1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-ß1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-ß1 signaling more comprehensively.

Blocking VEGF/Caveolin-1 signaling contributes to renal protection of fasudil in streptozotocin-induced diabetic rats.

AIM: RhoA/ROCK signaling plays an important role in diabetic nephropathy, and ROCK inhibitor fasudil exerts nephroprotection in experimental diabetic nephropathy. In this study we investigated the molecular mechanisms underlying the protective actions of fasudil in a rat model of diabetic nephropathy. METHODS: Streptozotocin (STZ)-induced diabetic rats, to which fasudil or a positive control drug enalapril were orally administered for 8 months. Metabolic parameters and blood pressure were assessed during the treatments. After the rats were euthanized, kidney samples were collected for histological and molecular biological studies. VEGF, VEGFR1, VEGFR2 and fibronectin expression, and Src and caveolin-1 phosphorylation in the kidneys were assessed using RT-PCR, Western blot and immunohistochemistry assays. The association between VEGFR2 and caveolin-1 was analyzed with immunoprecipitation. RESULTS: Chronic administration of fasudil (30 and 100 mg·kg(-1)·d(-1)) or enalapril (10 mg/kg, bid) significantly attenuated the glomerular sclerosis and albuminuria in the diabetic rats. Furthermore, fasudil treatment prevented the upregulation of VEGF, VEGFR1, VEGFR2 and fibronectin, and the increased association between VEGFR2 and caveolin-1 in the renal cortices, and partially blocked Src activation and caveolin-1 phosphorylation on tyrosine 14 in the kidneys, whereas enalapril treatment had no effects on the VEGFR2/Src/caveolin-1 signaling pathway. CONCLUSION: Fasudil exerts protective actions in STZ-induced diabetic nephropathy by blocking the VEGFR2/Src/caveolin-1 signaling pathway and fibronectin upregulation. Thus, VEGFR2 may be a potential therapeutic target for the treatment of diabetic nephropathy.

Akt and RhoA activation in response to high glucose require caveolin-1 phosphorylation in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic renal disease. Serine/threonine kinase PKC-ß1 mediates glucose-induced Akt S473 phosphorylation, RhoA activation, and transforming growth factor (TGF)-ß1 upregulation and finally leads to matrix upregulation in mesangial cells (MCs). It has been reported that glucose-induced PKC-ß1 activation is dependent on caveolin-1 and the presence of intact caveolae in MCs; however, whether activated PKC-ß1 regulates caveolin-1 expression and phosphorylation are unknown. Here, we showed that, although the caveolin-1 protein level had no significant change, the PKC-ß-specific inhibitor LY-333531 blocked caveolin-1 Y14 phosphorylation in high glucose (HG)-treated MCs and in the renal cortex of diabetic rats. The Src-specific inhibitor SU-6656 prevented the HG-induced association between PKC-ß1 and caveolin-1 and PKC-ß1 membrane translocation, whereas PKC-ß1 small interfering RNA failed to block Src activation, indicating that Src kinase is upstream of PKC-ß1 activation. Although LY-333531 blocked PKC-ß1 membrane translocation, it had no effect on the PKC-ß1/caveolin-1 association, suggesting that PKC-ß1 activation requires the interaction of caveolin-1 and PKC-ß1. PKC-ß1-mediated Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation in response to HG were prevented by SU-6656 and nonphosphorylatable mutant caveolin-1 Y14A. In conclusion, Src activation by HG mediates the PKC-ß1/caveolin-1 association and PKC-ß1 activation, which assists in caveolin-1 Y14 phosphorylation by Src kinase. The downstream effects, including Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation, require caveolin-1 Y14 phosphorylation. Caveolin-1 is thus an important mediator of the profibrogenic process in diabetic renal disease.

Maternal glucocorticoid elevation and associated blood metabonome changes might be involved in metabolic programming of intrauterine growth retardation in rats exposed to caffeine prenatally.

Our previous studies demonstrated that prenatal caffeine exposure causes intrauterine growth retardation (IUGR), fetuses are over-exposed to high levels of maternal glucocorticoids (GC), and intrauterine metabolic programming and associated metabonome alteration that may be GC-mediated. However, whether maternal metabonomes would be altered and relevant metabolite variations might mediate the development of IUGR remained unknown. In the present studies, we examined the dose- and time-effects of caffeine on maternal metabonome, and tried to clarify the potential roles of maternal GCs and metabonome changes in the metabolic programming of caffeine-induced IUGR. Pregnant rats were treated with caffeine (0, 20, 60 or 180 mg/kg·d) from gestational days (GD) 11 to 20, or 180 mg/kg·d caffeine from GD9. Metabonomes of maternal plasma on GD20 in the dose-effect study and on GD11, 14 and 17 in the time-course study were analyzed by ¹H nuclear magnetic resonance spectroscopy, respectively. Caffeine administration reduced maternal weight gains and elevated both maternal and fetal corticosterone (CORT) levels. A negative correlation between maternal/fetal CORT levels and fetal bodyweight was observed. The maternal metabonome alterations included attenuated metabolism of carbohydrates, enhanced lipolysis and protein breakdown, and amino acid accumulation, suggesting GC-associated metabolic effects. GC-associated metabolite variations (α/ß-glucoses, high density lipoprotein-cholesterol, ß-hydroxybutyrate) were observed early following caffeine administration. In conclusion, prenatal caffeine exposure induced maternal GC elevation and metabonome alteration, and maternal GC and relevant discriminatory metabolites might be involved in the metabolic programming of caffeine-induced IUGR.

Inhibition of mitochondrial over-division by (+)-14,15-Dehydrovincamine attenuates cisplatin-induced acute kidney injury via the JNK/Mff pathway.

Cisplatin-induced acute kidney injury (AKI) is characterized by mitochondrial damage and apoptosis, and safe and effective therapeutic agents are urgently needed. Renal tubular epithelial cells, the main site of AKI, are enriched with a large number of mitochondria, which are crucial for the progression of AKI with an impaired energy supply. Vincamine has anti-inflammatory and antioxidant effects in mouse AKI models. As a natural compound derived from Tabernaemontana pandacaqui, (+)-14, 15-Dehydrovincamine and Vincamine differ in structure by only one double bond, and the role and exact mechanism of (+)-14, 15-Dehydrovincamine remains to be elucidated in AKI. The present study demonstrated that (+)-14,15-Dehydrovincamine significantly ameliorated mitochondrial dysfunction and maintained mitochondrial homeostasis in a cisplatin-induced AKI model. Furthermore, (+)-14,15-Dehydrovincamine ameliorates cytochrome C-dependent apoptosis in renal tubular epithelial cells. c-Jun NH2-terminal kinase (JNK) was identified as a potential target protein of (+)-14,15-Dehydrovincamine attenuating AKI by network pharmacological analysis. (+)-14,15-Dehydrovincamine inhibited cisplatin-induced JNK activation, mitochondrial fission factor (Mff) phosphorylation, and dynamin-related protein 1 (Drp1) translocation to the mitochondria in renal tubular epithelial cells. Meanwhile, the JNK activator anisomycin restored Mff phosphorylation and Drp1 translocation, counteracting the protective effect of (+)-14,15-Dehydrovincamine on mitochondrial dysfunction in cisplatin-induced TECs injury. In conclusion, (+)-14,15-Dehydrovincamine reduced mitochondrial fission, maintained mitochondrial homeostasis, and attenuated apoptosis by inhibiting the JNK/Mff/Drp1 pathway, which in turn ameliorated cisplatin-induced AKI.

Scavenger Receptor Class B Type I Deficiency Induces Iron Overload and Ferroptosis in Renal Tubular Epithelial Cells via Hypoxia-Inducible Factor-1α/Transferrin Receptor 1 Signaling Pathway.

Aims: Scavenger receptor class B type I (SRBI) promotes cell cholesterol efflux and the clearance of plasma cholesterol. Thus, SRBI deficiency causes abnormal cholesterol metabolism and hyperlipidemia. Studies have suggested that ferroptosis is involved in lipotoxicity; however, whether SRBI deficiency could induce ferroptosis remains to be investigated. Results: We knocked down or knocked out SRBI in renal HK-2 cells and C57BL/6 mice to determine the expression levels of ferroptosis-related regulators. Our results demonstrated that SRBI deficiency upregulates transferrin receptor 1 (TFR1) expression and downregulates ferroportin expression, which induces iron overload and subsequent ferroptosis in renal tubular epithelial cells. TFR1 is known to be regulated by hypoxia-inducible factor-1α (HIF-1α). Next, we investigated whether SRBI deletion affected HIF-1α. SRBI deletion upregulated the mRNA and protein expression of HIF-1α, and promoted its translocation to the nucleus. To determine whether HIF-1α plays a key role in SRBI-deficiency-induced ferroptosis, we used HIF-1α inhibitor and siHIF-1α in HK-2 cells, and found that downregulation of HIF-1α prevented SRBI-silencing-induced TFR1 upregulation and iron overload, and eventually reduced ferroptosis. The underlying mechanism of HIF-1α activation was explored next, and the results showed that SRBI knockout or knockdown may upregulate the expression of HIF-1α, and promote HIF-1α translocation from the cytoplasm into the nucleus via the PKC-ß/NF-κB signaling pathway. Innovation and Conclusion: Our study showed, for the first time, that SRBI deficiency induces iron overload and subsequent ferroptosis via the HIF-1α/TFR1 pathway.

A potential role for caveolin-1 in VEGF-induced fibronectin upregulation in mesangial cells: involvement of VEGFR2 and Src.

VEGF is known to be an endothelial cell mitogen that stimulates angiogenesis by promoting endothelial cell survival, proliferation, migration, and differentiation. Recent studies have suggested that VEGF may play a pivotal role in glomerular sclerosis through extracellular matrix protein (ECM) accumulation, although the signaling mechanism is still unclear. The GTPase RhoA has been implicated in VEGF-induced type IV collagen accumulation in some settings. Here we study the role of different VEGF receptors and membrane microdomain caveolae in VEGF-induced RhoA activation and fibronectin upregulation in mesangial cells (MCs). In primary rat MC, VEGF time and dose dependently increased fibronectin production. Rho pathway inhibition blocked VEGF-induced fibronectin upregulation. VEGF-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. VEGF stimulation led to a markedly increased VEGFR2/caveolin-1 but failed to increase VEGFR1/caveolin-1 association. VEGF also increased caveolin-1/Src association and activated Src, and Src inhibitor blocked RhoA activation and fibronectin upregulation. Src-mediated phosphorylation of caveolin-1 on Y14 has also been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented VEGF-induced RhoA activation and fibronectin upregulation. In vivo, although VEGFR1 and VEGFR2 protein levels were both increased in the kidney cortices of diabetic rats, VEGFR2/caveolin-1 association but not VEGFR1/caveolin-1 association was significantly increased. In conclusion, VEGF-induced RhoA activation and fibronectin upregulation require caveolae and caveolin-1 interaction with VEGFR2 and Src. Interference with caveolin/-ae signaling may provide new avenues for the treatment of fibrotic renal disease.

Association of Obesity Indices with Diabetic Kidney Disease and Diabetic Retinopathy in Type 2 Diabetes: A Real-World Study.

Background: Diabetic microvascular complications mainly include diabetic kidney disease (DKD) and diabetic retinopathy (DR). Obesity was recognized as a risk factor for DKD, while the reported relationship between obesity and DR was inconsistent. Moreover, whether the associations can be attributed to C-peptide levels is unknown. Methods: Data from 1142 sequential inpatients with T2DM at Xiangyang Central Hospital between June 2019 and March 2022 were extracted retrospectively from the electronic medical record system. The associations between four obesity indices (body mass index (BMI), waist-hip circumference ratio (WHR), visceral fat tissue area (VFA), and subcutaneous fat tissue area (SFA)) and DKD and DR were evaluated. Whether the associations can be attributed to C-peptide levels was also explored. Results: Obesity was a risk factor for DKD after adjusting for sex, HbA1c, TG, TC, HDL, LDL, smoking history, education, duration of diabetes, and insulin use (obesity indices: BMI (OR 1.050: 95% CI: 1.008-1.094; P = 0.020); WHR (OR 10.97; 95% CI: 1.250-92.267; P = 0.031); VFA (OR 1.005; 95% CI: 1.001-1.008; P = 0.008)), but it became insignificant after further adjusting for fasting C-peptide. The associations between BMI, WHR, VFA, and DKD might be U-shaped. Obesity and FCP tended to protect against DR; however, they became insignificant after adjusting for multiple potential confounders. C2/C0 (the ratio of the postprandial serum C-peptide to fasting C-peptide) was a protective factor for both DKD (OR 0.894, 95% CI: 0.833-0.959, P < 0.05) and DR (OR 0.851, 95% CI: 0.787-0.919; P < 0.05). Conclusions: Obesity was a risk factor for DKD, and the effect may be attributable to C-peptide, which represents insulin resistance. The protective effect of obesity or C-peptide on DR was not independent and could be confounded by multiple factors. Higher C2/C0 was associated with both decreased DKD and DR.

Empagliflozin attenuates the renal tubular ferroptosis in diabetic kidney disease through AMPK/NRF2 pathway.

Renal tubular damage plays a key role in the pathogenesis of diabetic kidney disease (DKD), and one of the main pathological process associated with DKD in diabetic mice is the ferroptosis, a novel form of cell death caused by iron-dependent lipid peroxidation. Several researches suggested that empagliflozin may treat renal injury, but its effects on diabetic-related ferroptosis and underlying mechanisms were not fully elucidated. In this study, the influence of empagliflozin on renal injury was evaluated in vivo and in vitro in a mouse model and in high-glucose (HG) or Erastin-stimulated renal HK-2 cell line, respectively. Ferroptosis-related markers were assessed, including GSH, labile iron levels, and ferroptosis regulators by Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence. The level of malondialdehyde (MDA) and the fluorescence intensity of BODIPY probe indicated the level of lipid peroxidation. It was demonstrated that solute carrier family 7, member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were less expressed in renal biopsy samples from patients affected by DKD than in those from non-diabetic renal disease patients (NDRD), proving the ferroptosis of tubular epithelial cells in case of DKD. Furthermore, empagliflozin markedly decreased the ferroptosis impairment in DKD mice, as well as in HG model of HK-2 cells. Our investigations showed the ability of empagliflozin to suppress ferroptosis was partially countered by AMP-activated protein kinase (AMPK) inhibitor, which led to a reduction of the nuclear translocation of the antioxidant transcription factor NFE2-related factor 2 (NRF2) and downregulation of target genes such as GPX4, ferritin heavy chain 1 (FTH1), and SLC7A11, while AMPK agonists were responsible for the enhancement of the protective effects of empagliflozin. Taken together, our findings showed that empagliflozin may prevent the development of ferroptosis by promoting the AMPK-mediated NRF2 activation pathway, providing important insights for possible novel treatment approaches for DKD.

Fetal rat metabonome alteration by prenatal caffeine ingestion probably due to the increased circulatory glucocorticoid level and altered peripheral glucose and lipid metabolic pathways.

The aims of this study were to clarify the metabonome alteration in fetal rats after prenatal caffeine ingestion and to explore the underlying mechanism pertaining to the increased fetal circulatory glucocorticoid (GC). Pregnant Wistar rats were daily intragastrically administered with different doses of caffeine (0, 20, 60 and 180 mg/kg) from gestational days (GD) 11 to 20. Metabonome of fetal plasma and amniotic fluid on GD20 were analyzed by ¹H nuclear magnetic resonance-based metabonomics. Gene and protein expressions involved in the GC metabolism, glucose and lipid metabolic pathways in fetal liver and gastrocnemius were measured by real-time RT-PCR and immunohistochemistry. Fetal plasma metabonome were significantly altered by caffeine, which presents as the elevated α- and ß-glucose, reduced multiple lipid contents, varied apolipoprotein contents and increased levels of a number of amino acids. The metabonome of amniotic fluids showed a similar change as that in fetal plasma. Furthermore, the expressions of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD-2) were decreased, while the level of blood GC and the expressions of 11ß-HSD-1 and glucocorticoid receptor (GR) were increased in fetal liver and gastrocnemius. Meanwhile, the expressions of insulin-like growth factor 1 (IGF-1), IGF-1 receptor and insulin receptor were decreased, while the expressions of adiponectin receptor 2, leptin receptors and AMP-activated protein kinase α2 were increased after caffeine treatment. Prenatal caffeine ingestion characteristically change the fetal metabonome, which is probably attributed to the alterations of glucose and lipid metabolic pathways induced by increased circulatory GC, activated GC metabolism and enhanced GR expression in peripheral metabolic tissues.

Regulator of calcineurin 1 deletion attenuates mitochondrial dysfunction and apoptosis in acute kidney injury through JNK/Mff signaling pathway.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.

PKC-beta1 mediates glucose-induced Akt activation and TGF-beta1 upregulation in mesangial cells.

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. The serine/threonine kinase Akt mediates glucose-induced upregulation of collagen I in mesangial cells through transactivation of the EGF receptor (EGFR). In addition, in renal tubular cells, glucose-induced secretion of TGF-beta requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-beta expression, but the mechanisms of Akt activation and its involvement in TGF-beta regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C beta (PKC-beta)-dependent manner. Glucose led to PKC-beta1 membrane translocation and association with Akt, and PKC-beta1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-beta1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-beta and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-beta1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-beta1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-beta1. In vivo, the PKC-beta inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-beta1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-beta1 transcriptional upregulation requires EGFR/PKC-beta1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation.

RCAN1.4 mediates high glucose-induced matrix production by stimulating mitochondrial fission in mesangial cells.

High glucose (HG)-induced mitochondrial dynamic changes and oxidative damage are closely related to the development and progression of diabetic kidney disease (DKD). Recent studies suggest that regulators of calcineurin 1 (RCAN1) is involved in the regulation of mitochondrial function in different cell types, so we investigate the role of RCAN1 in mitochondrial dynamics under HG ambience in rat glomerular mesangial cells (MCs). MCs subjected to HG exhibited an isoform-specific up-regulation of RCAN1.4 at both mRNA and protein levels. RCAN1.4 overexpression induced translocation of Dynamin related protein 1 (Drp1) to mitochondria, mitochondrial fragmentation and depolarization, accompanied by increased matrix production under normal glucose and HG ambience. In contrast, decreasing the expression of RCAN1.4 by siRNA inhibited HG-induced mitochondrial fragmentation and matrix protein up-regulation. Moreover, both mitochondrial fission inhibitor Mdivi-1 and Drp1 shRNA prevented RCAN1.4-induced fibronectin up-regulation, suggesting that RCAN1.4-induced matrix production is dependent on its modulation of mitochondrial fission. Although HG-induced RCAN1.4 up-regulation was achieved by activating calcineurin, RCAN1.4-mediated mitochondrial fragmentation and matrix production is independent of calcineurin activity. These results provide the first evidence for the HG-induced RCAN1.4 up-regulation involving increased mitochondrial fragmentation, leading to matrix protein up-regulation.

Parathyroid hormone-related protein induces fibronectin up-regulation in rat mesangial cells through reactive oxygen species/Src/EGFR signaling.

Parathyroid hormone-related protein (PTHrP) is known to be up-regulated in both glomeruli and tubules in patients with diabetic kidney disease (DKD), but its role remains unclear. Previous studies show that PTHrP-induced hypertrophic response in mesangial cells (MCs) and epithelial-mesenchymal transition (EMT) in tubuloepithelial cells can be mediated by TGF-ß1. In the present study, although long-term PHTrP (1-34) treatment increased the mRNA and protein level of TGF-ß1 in primary rat MCs, fibronectin up-regulation occurred earlier, suggesting that fibronectin induction is independent of TGF-ß1/Smad signaling. We thus evaluated the involvement of epidermal growth factor receptor (EGFR) signaling and found that nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species mediates PTHrP (1-34)-induced Src kinase activation. Src phosphorylates EGFR at tyrosine 845 and then transactive EGFR. Subsequent PI3K activation mediates Akt and ERK1/2 activation. Akt and ERK1/2 discretely lead to excessive protein synthesis of fibronectin. Our study thus demonstrates the new role of PTHrP in fibronectin up-regulation for the first time in glomerular MCs. These data also provided new insights to guide development of therapy for glomerular sclerosis.

Caveolin-1 phosphorylation is required for stretch-induced EGFR and Akt activation in mesangial cells.

Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled in vitro by exposure of mesangial cells (MC) to cyclic mechanical strain. We have recently shown that Akt mediates the stretch-induced production of type I collagen, an important contributor to sclerosis, in MC. Here we studied the upstream mediators of Akt activation. Primary rat MC were exposed to 1 Hz cyclic strain for 10 min, previously shown to induce maximal Akt activation. Neither the integrin inhibitor GRDGSP nor cytoskeletal disruptors had any effect on stretch-induced Akt activation. Akt activation was, however, mediated by transactivation of the epidermal growth factor receptor (EGFR), and this required receptor kinase activity since Akt activation did not occur in cells expressing kinase-dead EGFR (K721A). Src was further shown to be upstream of the EGFR, with its inhibitor SU6656 preventing both EGFR and Akt activation. The membrane microdomains caveolae were found to be required for this signaling to occur. Chemical disruption of caveolae with cyclodextrin or filipin prevented Akt activation, and both EGFR and Akt activation were lost in caveolin-1 (cav-1) knockout MC. The latter was rescued with reexpression of cav-1. Further, Src-mediated phosphorylation of cav-1 on Y14 was required for stretch-induced EGFR and Akt activation, since these were abrogated in MC expressing the nonphosphorylatable cav-1 Y14A mutant. Thus, mechanical strain-induced activation of Akt in MC is independent of integrin activation and the actin cytoskeleton, but depends upon EGFR transactivation. EGFR transactivation requires intact caveolae and the Src-mediated phosphorylation of cav-1 on Y14. These studies define a novel function for cav-1 and caveolae in EGFR transactivation leading to Akt activation by mechanical stress.

Dual roles of parathyroid hormone related protein in TGF-ß1 signaling and fibronectin up-regulation in mesangial cells.

Little is known about the cross-talk between parathyroid hormone (PTH) related protein (PTHrP) and TGF-ß1 in mesangial cells (MCs). Our results showed that PTHrP treatment (≤3 h) induced internalization of PTH1R (PTH/PTHrP receptor)-TßRII (TGF-ß type 2 receptor) complex and suppressed TGF-ß1-mediated Smad2/3 activation and fibronectin (FN) up-regulation. However, prolonged PTHrP treatment (12-48 h) failed to induce PTH1R-TßRII association and internalization. Total protein levels of PTH1R and TßRII were unaffected by PTHrP treatment. These results suggest that internalization of PTH1R and TßRII after short PTHrP treatment might not lead to their proteolytic destruction, allowing the receptors to be recycled back to the plasma membrane during prolonged PTHrP exposure. Receptor re-expression at the cell surface allows PTHrP to switch from its initial inhibitory effect to promote induction of FN. Our study thus demonstrates the dual roles of PTHrP on TGF-ß1 signaling and FN up-regulation for the first time in glomerular MCs. These data also provided new insights to guide development of therapy for diabetic kidney disease (DKD).