Pre-emptive pharmacological inhibition of fatty acid–binding protein 4 attenuates kidney fibrosis by reprogramming tubular lipid metabolism

Kidney fibrosis is a hallmark of chronic kidney disease (CKD) progression that is caused by tubular injury and dysregulated lipid metabolism. Genetic abolition fatty acid-binding protein 4 (FABP4), a key lipid transporter, has been reported to suppress kidney interstitial fibrosis. However, the role and underlying mechanism of chemical inhibition of FABP4 in fibrotic kidney have not been well-documented. Here, we examined preemptive the effect of a FABP4 inhibitor, BMS309403, on lipid metabolism of tubular epithelial cells (TECs) and progression of kidney fibrosis. The expression of FABP4 was significantly elevated, concomitated with the accumulation of lipid droplets in TECs during kidney fibrosis. Treatment with BMS309403 alleviated lipid deposition of TECs, as well as interstitial fibrotic responses both in unilateral ureteral obstruction (UUO)-engaged mice and TGF-β-induced TECs. Moreover, BMS309403 administration enhanced fatty acid oxidation (FAO) in TECs by regulating peroxisome proliferator-activated receptor γ (PPARγ) and restoring FAO-related enzyme activities; In addition, BMS309403 markedly reduced cell lipotoxicity, such as endoplasmic reticulum (ER) stress and apoptosis in fibrotic kidney. Taken together, our results suggest that preemptive pharmacological inhibition of FABP4 by BMS309403 rebalances abnormal lipid metabolism in TECs and attenuates the progression of kidney fibrosis, thus may hold therapeutic potential for the treatment of fibrotic kidney diseases.Subject terms: Metabolism, Chronic kidney disease     

Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis.     

Inflammatory stress exacerbates lipid-mediated renal injury in ApoE/CD36/SRA triple knockout mice

Both lipids and inflammation play important roles in the progression of kidney disease. This study was designed to investigate whether inflammation exacerbates lipid accumulation via LDL receptors (LDLr), thereby causing renal injury in C57BL/6J mice, apolipoprotein E (ApoE) knockout (KO) mice, and ApoE/CD36/scavenger receptor A triple KO mice. The mice were given a subcutaneous casein injection to induce inflammatory stress. After 14 wk, terminal blood samples were taken for renal function, lipid profiles, amyloid A (SAA), and IL-6 assays. Lipid accumulation in kidneys was visualized by oil red O staining. Fibrogenic molecule expression in kidneys was examined. There was a significant increase in serum SAA and IL-6 in the all casein-injected mice compared with respective controls. Casein injection reduced serum total cholesterol, LDL cholesterol, and HDL cholesterol and caused lipid accumulation in kidneys from three types of mice. The expression of LDLr and its regulatory proteins sterol-responsive element-binding protein (SREBP) 2 and SREBP cleavage-activating protein (SCAP) were upregulated in inflamed mice compared with controls. Casein injection induced renal fibrosis accompanied by increased expression of fibrogenic molecules in the triple KO mice. These data imply that inflammation exacerbates lipid accumulation in the kidney by diverting lipid from the plasma to the kidney via the SCAP-SREBP2-LDLr pathway and causing renal injury. Low blood cholesterol levels, resulting from inflammation, may be associated with high risk for chronic renal fibrosis.     

C-X-C motif chemokine receptor 4 aggravates renal fibrosis through activating JAK/STAT/GSK3β/β-catenin pathway

Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC-8) to examine the expression of C-X-C motif chemokine receptor 4 (CXCR4) and β-catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up-regulated in tubules, with a concomitant activation of β-catenin. CXCR4 expression level was positively correlated with the expression of β-catenin target MMP-7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and β-catenin signalling, protected against tubular injury and renal fibrosis. CXCR4-induced renal fibrosis was inhibited by treatment with ICG-001, an inhibitor of β-catenin signalling. In HKC-8 cells, overexpression of CXCR4 induced activation of β-catenin and deteriorated cell injury. These effects were inhibited by ICG-001. Stromal cell–derived factor (SDF)-1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC-8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3β mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF-1α-induced activation of β-catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and β-catenin activation in renal fibrosis in association with JAK/STAT/GSK3β pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.     

Sirt3 modulates fatty acid oxidation and attenuates cisplatin‐induced AKI in mice

Fatty acid oxidation (FAO) dysfunction is one of the important mechanisms of renal fibrosis. Sirtuin 3 (Sirt3) has been confirmed to alleviate acute kidney injury (AKI) by improving mitochondrial function and participate in the regulation of FAO in other disease models. However, it is not clear whether Sirt3 is involved in regulating FAO to improve the prognosis of AKI induced by cisplatin. Here, using a murine model of cisplatin‐induced AKI, we revealed that there were significantly FAO dysfunction and extensive lipid deposition in the mice with AKI. Metabolomics analysis suggested reprogrammed energy metabolism and decreased ATP production. In addition, fatty acid deposition can increase reactive oxygen species (ROS) production and induce apoptosis. Our data suggested that Sirt3 deletion aggravated FAO dysfunction, resulting in increased apoptosis of kidney tissues and aggravated renal injury. The activation of Sirt3 by honokiol could improve FAO and renal function and reduced fatty acid deposition in wide‐type mice, but not Sirt3‐defective mice. We concluded that Sirt3 may regulate FAO by deacetylating liver kinase B1 and activating AMP‐activated protein kinase. Also, the activation of Sirt3 by honokiol increased ATP production as well as reduced ROS and lipid peroxidation through improving mitochondrial function. Collectively, these results provide new evidence that Sirt3 is protective against AKI. Enhancing Sirt3 to improve FAO may be a potential strategy to prevent kidney injury in the future.     

Proximal tubular RAGE mediated the renal fibrosis in UUO model mice via upregulation of autophagy

Previous studies reported that RAGE participated in the process of kidney fibrosis, but the function and regulation pathway of RAGE in proximal tubular cells in this process remains unclear. Here, we found that expression of RAGE was increased by TGF-β1 treatment and unilateral ureteral obstruction (UUO). Knock down of RAGE ameliorated renal fibrosis by TGF-β1 treatment, the expression of vimentin, Collagen I&III, and fibronectin are decreased. Mechanistically, RAGE mediated TGF-β1-induced phosphorylation of Stat3 and directly upregulated the Atg7 to increase the level of autophagy, and ultimately resulting in renal fibrosis. Furthermore, PT-RAGE-KO mice reduced kidney fibrosis in UUO model via inhibiting Stat3/Atg7 axis by knocking down RAGE. Furthermore, the above findings were confirmed in kidney of patients with obstructive nephropathy. Collectively, RAGE in proximal tubular cells promotes the autophagy to increase renal fibrosis via upregulation of Stat3/Atg7 axis.Subject terms: Autophagy, Urogenital diseases     

microRNA-206 overexpression inhibits epithelial-mesenchymal transition and glomerulosclerosis in rats with chronic kidney disease by inhibiting JAK/STAT signaling pathway

Chronic kidney disease (CKD) is a traumatic disease with significant psychic consequences to the patient's overall physical condition. microRNA-206 (miR-206) has been reported to play an essential role in the development of various diseases. The purpose of the present study is to investigate the effect of miR-206 through the JAK/STAT signaling pathway on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and glomerulosclerosis in rats with CKD. The targeting relationship between miR-206 and ANXA1 was verified. To explore the role of miR-206 in CKD, the model of CKD rats was established to detect glomerular sclerosis index (GSI), contents of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1), and expression of type IV collagen. Moreover, to further determine the roles of both miR-206 and the JAK/STAT signaling pathway in CKD, the gain- and loss-of function approaches were performed with the expression of ANXA1, α-SMA, E-cadherin, vimentin, N-cadherin, and the JAK/STAT signaling pathway-related genes detected. miR-206 negatively targeted ANXA1. Overexpressed miR-206 inhibited the degeneration and interstitial fibrosis of renal tubular epithelial cells, decreased GSI of rats, and the expression of type IV collagen, TGF-β1 and IL-6. Overexpressed miR-206 inhibited the degeneration of renal tubular epithelial cells, the expression of ANXA1, α-SMA, TGF-β1, p-STAT3, STAT3, p-STAT1, STAT1, p-JAK2, and JAK2, while promoted the expression of E-cadherin. Taken together the results, miR-206 inhibits EMT of renal tubular epithelial cells and glomerulosclerosis by inactivating the JAK/STAT signaling pathway via ANXA1 in CKD.     

PP2Acα promotes macrophage accumulation and activation to exacerbate tubular cell death and kidney fibrosis through activating Rap1 and TNFα production

Macrophage accumulation and activation play an essential role in kidney fibrosis; however, the underlying mechanisms remain to be explored. By analyzing the kidney tissues from patients and animal models with kidney fibrosis, we found a large induction of PP2Acα in macrophages. We then generated a mouse model with inducible macrophage ablation of PP2Acα. The knockouts developed less renal fibrosis, macrophage accumulation, or tubular cell death after unilateral ureter obstruction or ischemic reperfusion injury compared to control littermates. In cultured macrophages, PP2Acα deficiency resulted in decreased cell motility by inhibiting Rap1 activity. Moreover, co-culture of PP2Acα^−/− macrophages with tubular cells resulted in less tubular cell death attributed to downregulated Stat6-mediated tumor necrosis factor α (TNFα) production in macrophages. Together, this study demonstrates that PP2Acα promotes macrophage accumulation and activation, hence accelerates tubular cell death and kidney fibrosis through regulating Rap1 activation and TNFα production.Subject terms: Cell biology, Inflammation     

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.Subject terms: Biochemistry, Cell biology     

Attenuation of Hyperlipidemia- and Diabetes-Induced Early-Stage Apoptosis and Late-Stage Renal Dysfunction via Administration of Fibroblast Growth Factor-21 Is Associated with Suppression of Renal Inflammation

Background

Lipotoxicity is a key feature of the pathogenesis of diabetic kidney disease, and is attributed to excessive lipid accumulation (hyperlipidemia). Increasing evidence suggests that fibroblast growth factor (FGF)21 has a crucial role in lipid metabolism under diabetic conditions.

Objective

The present study investigated whether FGF21 can prevent hyperlipidemia- or diabetes-induced renal damage, and if so, the possible mechanism.

Methods

Mice were injected with free fatty acids (FFAs, 10 mg/10 g body weight) or streptozotocin (150 mg/kg) to establish a lipotoxic model or type 1 diabetic model, respectively. Simultaneously the mice were treated with FGF21 (100 µg/kg) for 10 or 80 days. The kidney weight-to-tibia length ratio and renal function were assessed. Systematic and renal lipid levels were detected by ELISA and Oil Red O staining. Renal apoptosis was examined by TUNEL assay. Inflammation, oxidative stress, and fibrosis were assessed by Western blot.

Results

Acute FFA administration and chronic diabetes were associated with lower kidney-to-tibia length ratio, higher lipid levels, severe renal apoptosis and renal dysfunction. Obvious inflammation, oxidative stress and fibrosis also observed in the kidney of both mice models. Deletion of the fgf21 gene further enhanced the above pathological changes, which were significantly prevented by administration of exogenous FGF21.

Conclusion

These results suggest that FFA administration and diabetes induced renal damage, which was further enhanced in FGF21 knock-out mice. Administration of FGF21 significantly prevented both FFA- and diabetes-induced renal damage partially by decreasing renal lipid accumulation and suppressing inflammation, oxidative stress, and fibrosis.     

Astaxanthin protects against renal fibrosis through inhibiting myofibroblast activation and promoting CD8+ T cell recruitment

BackgroundRenal fibrosis is a common pathological hallmark of chronic kidney disease, and no effective treatment is clinically available to manage its progression. Astaxanthin was recently found to be anti-fibrotic, but its effect on renal fibrosis remains unclear.MethodsC57BL/6J mice were subjected to unilateral ureteral obstruction and intragastrically administered astaxanthin. Histopathology and immunohistochemistry were performed to evaluate renal fibrosis. Flow cytometry was used to examine lymphocyte accumulation in the fibrotic kidneys. Western blotting, real-time qPCR, and immunofluorescence were performed to cover the underlying mechanism concerning astaxanthin treatment during renal fibrosis.ResultsOral administration of astaxanthin effectively alleviates renal fibrosis in mice. In vitro, astaxanthin inhibited fibroblast activation by modulating Smad2, Akt and STAT3 pathways and suppressed epithelial-to-mesenchymal transition in renal tubular epithelial cells through Smad2, snail, and β-catenin. Moreover, astaxanthin significantly induced the rapid accumulation of CD8⁺ T cells in fibrotic kidneys, which was accompanied by elevated expression of IFN-γ. Accordingly, the depletion of CD8⁺ T cells strongly diminished the protective effect of astaxanthin. Further investigation showed that astaxanthin increased the population of CD8⁺ T cells by upregulating the expression of CCL5 in macrophages.ConclusionsThese findings highlight the beneficial effect of astaxanthin on fibroblast activation, epithelial-to-mesenchymal transition, and CD8⁺ T cell recruitment during renal fibrosis.General significanceThese data indicate that astaxanthin could serve as a therapeutic strategy to treat renal fibrotic conditions.     

PI3K/Akt pathway mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells through regulation of SREBP-1 and TGF-β1

Previous studies have shown that high glucose stimulates renal SREBP-1 gene expression and increases renal tubular cells lipid metabolism, however, the mechanisms remain elusive. In the present study we demonstrated that PI3K/Akt pathway was activated in human renal proximal tubular cell line (HKC) exposed to high glucose accompanied with up-regulation of SREBP-1, TGF-β1, lipid droplets deposits and extracellular matrix production. Inhibition of PI3K/Akt pathway by chemical LY294002 or specific short hairpin RNA (shRNA) vector prevented SREBP-1 and TGF-β1 up-regulation, as well as ameliorated HKC cells lipogenesis and extracellular matrix accumulation. These findings indicate that PI3K/Akt pathway potentially mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells.     

Niacin improves renal lipid metabolism and slows progression in chronic kidney disease

Background

Mounting evidence points to lipid accumulation in the diseased kidney and its contribution to progression of nephropathy. We recently found heavy lipid accumulation and marked dysregulation of lipid metabolism in the remnant kidneys of rats with chronic renal failure (CRF). Present study sought to determine efficacy of niacin supplementation on renal tissue lipid metabolism in CRF.

Methods

Kidney function, lipid content, and expression of molecules involved in cholesterol and fatty acid metabolism were determined in untreated CRF (5/6 nephrectomized), niacin-treated CRF (50 mg/kg/day in drinking water for 12 weeks) and control rats.

Results

CRF resulted in hypertension, proteinuria, renal tissue lipid accumulation, up-regulation of scavenger receptor A1 (SR-A1), acyl-CoA cholesterol acyltransferase-1 (ACAT1), carbohydrate-responsive element binding protein (ChREBP), fatty acid synthase (FAS), acyl-CoA carboxylase (ACC), liver X receptor (LXR), ATP binding cassette (ABC) A-1, ABCG-1, and SR-B1 and down-regulation of sterol responsive element binding protein-1 (SREBP-1), SREBP-2, HMG-CoA reductase, PPAR-α, fatty acid binding protein (L-FABP), and CPT1A. Niacin therapy attenuated hypertension, proteinuria, and tubulo-interstitial injury, reduced renal tissue lipids, CD36, ChREBP, LXR, ABCA-1, ABCG-1, and SR-B1 abundance and raised PPAR-α and L-FABP.

Conclusions and general significance

Niacin administration improves renal tissue lipid metabolism and renal function and structure in experimental CRF.     

Farnesoid X Receptor Protects against Kidney Injury in Uninephrectomized Obese Mice

Activation of the farnesoid X receptor (FXR) has indicated a therapeutic potential for this nuclear bile acid receptor in the prevention of diabetic nephropathy and obesity-induced renal damage. Here, we investigated the protective role of FXR against kidney damage induced by obesity in mice that had undergone uninephrectomy, a model resembling the clinical situation of kidney donation by obese individuals. Mice fed a high-fat diet developed the core features of metabolic syndrome, with subsequent renal lipid accumulation and renal injury, including glomerulosclerosis, interstitial fibrosis, and albuminuria. The effects were accentuated by uninephrectomy. In human renal biopsies, staining of 4-hydroxynonenal (4-HNE), glucose-regulated protein 78 (Grp78), and C/EBP-homologous protein, markers of endoplasmic reticulum stress, was more prominent in the proximal tubules of 15 obese patients compared with 16 non-obese patients. In mice treated with the FXR agonist obeticholic acid, renal injury, renal lipid accumulation, apoptosis, and changes in lipid peroxidation were attenuated. Moreover, disturbed mitochondrial function was ameliorated and the mitochondrial respiratory chain recovered following obeticholic acid treatment. Culturing renal proximal tubular cells with free fatty acid and FXR agonists showed that FXR activation protected cells from free fatty acid-induced oxidative stress and endoplasmic reticulum stress, as denoted by a reduction in the level of reactive oxygen species staining and Grp78 immunostaining, respectively. Several genes involved in glutathione metabolism were induced by FXR activation in the remnant kidney, which was consistent with a decreased glutathione disulfide/glutathione ratio. In summary, FXR activation maintains endogenous glutathione homeostasis and protects the kidney in uninephrectomized mice from obesity-induced injury.     

A mouse model of airway disease: oncostatin M-induced pulmonary eosinophilia, goblet cell hyperplasia, and airway hyperresponsiveness are STAT6 dependent, and interstitial pulmonary fibrosis is STAT6 independent

Oncostatin M (OSM), a pleiotropic cytokine of the gp130 cytokine family, has been implicated in chronic allergic inflammatory and fibrotic disease states associated with tissue eosinophilia. Mouse (m)OSM induces airway eosinophilic inflammation and interstitial pulmonary fibrosis in vivo and regulates STAT6 activation in vitro. To determine the requirement of STAT6 in OSM-induced effects in vivo, we examined wild-type (WT) and STAT6-knockout (STAT6(-/-)) C57BL/6 mouse lung responses to transient ectopic overexpression of mOSM using an adenoviral vector (AdmOSM). Intratracheal AdmOSM elicited persistent eosinophilic lung inflammation that was abolished in STAT6(-/-) mice. AdmOSM also induced pronounced pulmonary remodeling characterized by goblet cell hyperplasia and parenchymal interstitial fibrosis. Goblet cell hyperplasia was STAT6 dependent; however, parenchymal interstitial fibrosis was not. OSM also induced airway hyperresponsiveness in WT mice that was abolished in STAT6(-/-) mice. OSM stimulated an inflammatory signature in the lungs of WT mice that demonstrated STAT6-dependent regulation of Th2 cytokines (IL-4, IL-13), chemokines (eotaxin-1/2, MCP-1, keratinocyte chemoattractant), and extracellular matrix modulators (tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-13), but STAT6-independent regulation of IL-4Rα, total lung collagen, collagen-1A1, -1A2 mRNA, and parenchymal collagen and α smooth muscle actin accumulation. Thus, overexpression of mOSM induces STAT6-dependent pulmonary eosinophilia, mucous/goblet cell hyperplasia, and airway hyperresponsiveness but STAT6-independent mechanisms of lung tissue extracellular matrix accumulation. These results also suggest that eosinophil or neutrophil accumulation in mouse lungs is not required for OSM-induced lung parenchymal collagen deposition and that OSM may have unique roles in the pathogenesis of allergic and fibrotic lung disease.