Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Mitigation of cisplatin-induced acute kidney injury through oral administration of FAAH Inhibitor PF-04457845.

Fatty acid amide hydrolase (FAAH) serves as the primary enzyme responsible for degrading the endocannabinoid anandamide (AEA). Inhibition of FAAH, either through pharmacological means or genetic manipulation, can effectively reduce inflammation in various organs, including the brain, colon, heart, and kidneys. Infusion of a FAAH inhibitor into the kidney medulla has been shown to induce diuretic and natriuretic effects. FAAH knockout mice have shown protection against both post-ischemia reperfusion injury and cisplatin-induced acute kidney injury (AKI), although through distinct mechanisms. The present study was based on the hypothesis that pharmacological inhibition of FAAH activity could mitigate cisplatin-induced AKI, exploring potential renoprotective mechanism. Male wild type C57BL/6 were administered an oral gavage of a FAAH inhibitor (PF-04457845, 5mg/kg) or vehicle (10% PEG200+5% Tween80+normal saline) at 72, 48, 24, and 2 hours before and 24 and 48 hours after a single intraperitoneal injection of cisplatin (Cis, 25 mg/kg). Mice were euthanatized 72 hours after cisplatin treatment. Compared to vehicle-treated mice, PF-04457845-treated mice showed a decrease of cisplatin-induced plasma creatinine, blood urea nitrogen levels, kidney injury biomarkers (NGAL and KIM-1) and renal tubular damage. The renal protection from oral gavage of PF-04457845 against cisplatin-induced nephrotoxicity was associated with an enhanced AEA tone and reduced levels of DNA damage response biomarkers p53 and p21. Our work demonstrates that PF-04457845 effectively alleviates cisplatin-induced nephrotoxicity in mice, underscoring the potential of orally targeting FAAH as a novel strategy to prevent cisplatin nephrotoxicity. Significance Statement Oral administration of FAAH inhibitor, can reduce cisplatin-induced DNA damage response, tubular damages, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to prevent cisplatin-induced nephrotoxicity.

Dual-loaded nano pesticide system based on industrial grade scaleable carrier materials with combinatory efficacy and improved safety.

Repeated and widespread use of single chemical pesticides raises concerns about efficiency and safety, developing multi-component synergistic pesticides provides a new route for efficient control of diseases. Most commercial compound formulations are open systems with non-adjustable released rates, resulting in a high frequency of applications. Meanwhile, although nano pesticide delivery systems constructed with different carrier materials have been extensively studied, realizing their actual scale-up production still has important practical significance due to the large-scale field application. In this study, a boscalid and pyraclostrobin dual-loaded nano pesticide system (BPDN) was constructed with industrial-grade carrier materials to facilitate the realization of large-scale production. The optimal industrial-scale preparation mechanism of BPDN was studied with surfactants as key factors. When agricultural emulsifier No.600 and polycarboxylate are used as the ratio of 1:2 in the preparation process, the BPDN has a spherical structure with an average size of 270 nm and exhibits superior physical stability. Compared with commercial formulation, BPDN maintains rate-stabilized release up to 5 times longer, exhibits better dispersion and spreading performance on foliar, has more than 20% higher deposition amounts, and reduces loss. A single application of BPDN could efficiently control tomato gray mold during the growing period of tomatoes due to extended duration and combinatory effectiveness, reducing two application times and labor costs. Toxicology tests on various objects systematically demonstrated that BPDN has improved safety for HepG2 cells, and nontarget organism earthworms. This research provides insight into creating safe, efficient, and environmentally friendly pesticide production to reduce manual operation times and labor costs. Accompanied by production strategies that can be easily scaled up industrially, this contributes to the efficient use of resources for sustainable agriculture.

Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates Cisplatin-Induced Nephropathy in Mice.

Cisplatin is a potent first-line therapy for many solid malignancies, such as breast, ovarian, lung, testicular, and head and neck cancer. However, acute kidney injury (AKI) is a major dose-limiting toxicity in cisplatin therapy, which often hampers the continuation of cisplatin treatment. The endocannabinoid system, consisting of anandamide (AEA) and 2-arachidonoylglycerol and cannabinoid receptors, participates in different kidney diseases. Inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme for the degradation of AEA and AEA-related N-acylethanolamines, elicits anti-inflammatory effects; however, little is known about its role in cisplatin nephrotoxicity. The current study tested the hypothesis that genetic deletion of Faah mitigates cisplatin-induced AKI. Male wild-type C57BL6 (WT) and Faah-/- mice were administered a single dose of intraperitoneal injection of cisplatin (30 mg/kg) and euthanatized 72 hours later. Faah-/- mice showed a reduction of cisplatin-induced blood urea nitrogen, plasma creatinine levels, kidney injury markers, and tubular damage in comparison with WT mice. The renal protection from Faah deletion was associated with enhanced tone of AEA-related N-acylethanolamines (palmitoylethanolamide and oleoylethanolamide), attenuated nuclear factor-κB/p65 activity, DNA damage markers p53 and p21, and decreased expression of the inflammatory cytokine interleukin-1ß, as well as infiltration of macrophages and leukocytes in the kidneys. Notably, a selective FAAH inhibitor (PF-04457845) did not interfere with or perturb the antitumor effects of cisplatin in two head and neck squamous cell carcinoma cell lines, HN30 and HN12. Our work highlights that FAAH inactivation prevents cisplatin-induced nephrotoxicity in mice and that targeting FAAH could provide a novel strategy to mitigate cisplatin-induced nephrotoxicity. SIGNIFICANCE STATEMENT: Mice lacking the Faah gene are protected from cisplatin-induced inflammation, DNA damage response, tubular damage, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to mitigate cisplatin-induced nephrotoxicity.

Exosome Biogenesis and Lysosome Function Determine Podocyte Exosome Release and Glomerular Inflammatory Response during Hyperhomocysteinemia.

Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation in podocytes is reportedly associated with enhanced release of exosomes containing NLRP3 inflammasome products from these cells during hyperhomocysteinemia (hHcy). This study examined the possible role of increased exosome secretion during podocyte NLRP3 inflammasome activation in the glomerular inflammatory response. Whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during hHcy in mice and upon stimulation of homocysteine (Hcy) in podocytes was tested. By nanoparticle tracking analysis, treatments of mice with amitriptyline (acid sphingomyelinase inhibitor), GW4869 (exosome biogenesis inhibitor), and rapamycin (lysosome function enhancer) were found to inhibit elevated urinary exosomes during hHcy. By examining NLRP3 inflammasome activation in glomeruli during hHcy, amitriptyline (but not GW4869 and rapamycin) was shown to have an inhibitory effect. However, all treatments attenuated glomerular inflammation and injury during hHcy. In cell studies, Hcy treatment stimulated exosome release from podocytes, which was prevented by amitriptyline, GW4869, and rapamycin. Structured illumination microscopy revealed that Hcy inhibited lysosome-multivesicular body interactions in podocytes, which was prevented by amitriptyline or rapamycin but not GW4869. Thus, the data from this study shows that activation of exosome biogenesis and dysregulated lysosome function are critically implicated in the enhancement of exosome release from podocytes leading to glomerular inflammation and injury during hHcy.

Loss of sphingosine kinase 2 protects against cisplatin-induced kidney injury.

Cisplatin is an established chemotherapeutic drug for treatment of solid-organ cancers and is the primary drug used in the treatment of head and neck cancer; however, cisplatin-induced nephrotoxicity largely limits its clinical use. Inhibition of sphingosine kinase 2 (SphK2) has been demonstrated to alleviate various kidney diseases. Therefore, we hypothesized that inhibition of SphK2 could also protect against cisplatin-induced nephrotoxicity. Results from the present study showed that the SphK2 inhibitor ABC294640 or knockdown of SphK2 by siRNA blocked the cisplatin-induced increase of cellular injury markers (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cleaved caspase-3) by Western blot analysis in HK-2 cells, a human renal tubular cell line. In addition, SphK2 inhibition blocked cisplatin-induced activation of NF-κB by Western blot analysis and immunostaining analysis. Furthermore, SphK2 inhibition suppressed cisplatin-induced increases of proinflammatory markers (NLR family pyrin domain containing 3, interleukin-1ß, and interleukin-6). Genetic deletion of the SphK2 gene in mice further confirmed that inhibition of SphK2 protected against cisplatin-induced kidney damage in vivo. Compared with wild-type mice, SphK2 knockout mice exhibited less renal dysfunction and reduced promotion of kidney injury markers, inflammatory factors, tubular morphology damage, and fibrotic staining. At the same time, the SphK2 inhibitor ABC294640 failed to interfere with the activity of cisplatin or radiation in two cell culture models of head and neck cancer. It is concluded that inhibition of Sphk2 protects against cisplatin-induced kidney injury. SphK2 may be used as a potential therapeutic target for the prevention or treatment of cisplatin-induced kidney injury.NEW & NOTEWORTHY The present study provides new findings that sphingosine kinase 2 (SphK2) is highly expressed in renal tubules, cisplatin treatment increases the expression of SphK2 in proximal tubular cells and kidneys, and inhibition of SphK2 alleviates cisplatin-induced kidney injury by suppressing the activation of NF-κB, production of inflammatory factors, and apoptosis. SphK2 may serve as a potential therapeutic target for the prevention or treatment of cisplatin-induced nephrotoxicity.

Nanomaterials and nanotechnology for the delivery of agrochemicals: strategies towards sustainable agriculture.

Nanomaterials (NMs) have received considerable attention in the field of agrochemicals due to their special properties, such as small particle size, surface structure, solubility and chemical composition. The application of NMs and nanotechnology in agrochemicals dramatically overcomes the defects of conventional agrochemicals, including low bioavailability, easy photolysis, and organic solvent pollution, etc. In this review, we describe advances in the application of NMs in chemical pesticides and fertilizers, which are the two earliest and most researched areas of NMs in agrochemicals. Besides, this article concerns with the new applications of NMs in other agrochemicals, such as bio-pesticides, nucleic acid pesticides, plant growth regulators (PGRs), and pheromone. We also discuss challenges and the industrialization trend of NMs in the field of agrochemicals. Constructing nano-agrochemical delivery system via NMs and nanotechnology facilitates the improvement of the stability and dispersion of active ingredients, promotes the precise delivery of agrochemicals, reduces residual pollution and decreases labor cost in different application scenarios, which is potential to maintain the sustainability of agricultural systems and improve food security by increasing the efficacy of agricultural inputs.

Kidney-Targeted Delivery of Prolyl Hydroxylase Domain Protein 2 Small Interfering RNA with Nanoparticles Alleviated Renal Ischemia/Reperfusion Injury.

Inhibition of hypoxia-inducible factor-prolyl hydroxylase (PHD) has been shown to protect against various kidney diseases. However, there are controversial reports on the effect of PHD inhibition in renoprotection. The present study determined whether delivery of PHD2 small interfering RNA (siRNA) using an siRNA carrier, folic acid (FA)-decorated polyamidoamine dendrimer generation 5 (G5-FA), would mainly target kidneys and protect against renal ischemia/reperfusion injury (I/R). The renal I/R was generated by clipping the renal pedicle for 30 minutes in uninephrectomized mice. Mice were sacrificed 48 hours after I/R. Normal saline or G5-FA complexed with control or PHD2 siRNA was injected via tail vein 24 hours before ischemia. After the injection of near-infrared fluorescent dye-labeled G5-FA, the fluorescence was mainly detected in kidneys but not in other organs. The reduction of PHD2 mRNA and protein was only observed in kidneys but not in other organs after injection of PHD2-siRNA-G5-FA complex. The injection of PHD2-siRNA-G5-FA significantly alleviated renal I/R injury, as shown by the inhibition of increases in serum creatinine and blood urea nitrogen, the blockade of increases in kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, and the improvement of histologic damage compared with mice treated with control siRNA. PHD2 siRNA can be delivered specifically into kidneys using G5-FA, and that local knockdown of PHD2 gene expression within the kidney alleviates renal I/R injury. Therefore, G5-FA is an efficient siRNA carrier to deliver siRNA into the kidney, and that local inhibition of PHD2 within the kidney may be a potential strategy for the management of acute I/R injury. SIGNIFICANCE STATEMENT: Folic acid (FA)-decorated polyamidoamine dendrimer generation 5 (G5-FA) was demonstrated to be an effective carrier to deliver small interfering RNA (siRNA) into kidneys. Delivery of prolyl hydroxylase domain protein 2 siRNA with G5-FA effectively protected the kidneys against the acute renal ischemia/reperfusion injury.

Mechanism of Diuresis and Natriuresis by Cannabinoids: Evidence for Inhibition of Na+-K+-ATPase in Mouse Kidney Thick Ascending Limb Tubules.

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na+ transport in isolated mouse TAL tubules was studied using the Na+-sensitive dye, SBFI-AM. Switching from 0 Na+ solution to control Ringer's solution (CR) rapidly increased TAL cell [Na+]i Addition of CP to CR produced a further elevation, similar in magnitude to that of ouabain, a Na+-K+-ATPase inhibitor. This [Na+]i-elevating effect of CP was time-dependent, required the presence of Na+ in the bathing solution, and was insensitive to Na+-K+-2Cl- cotransporter inhibition. Addition of PF to CR elevated [Na+]i in FAAH wild-type but not FAAH knockout (KO) TALs, whereas the additions of CP and AEA to PF-treated FAAH KO TALs increased [Na+]i An interaction between cannabinoidimetics and ouabain (Ou) was observed. Ou produced less increase in [Na+]i after cannabinoidimetic treatment, whereas cannabinoidimetics had less effect after Ou treatment. It is concluded that cannabinoidimetics, including CP and AEA, inhibit Na+ transport in TALs by inhibiting Na+ exit via Na+-K+-ATPase. SIGNIFICANCE STATEMENT: Cannabinoids including endocannabinoids induce renal urine and salt excretion and are proposed to play a physiological role in the regulation of blood pressure. Our data suggest that the mechanism of the cannabinoids involves inhibition of the sodium pump, Na+-K+-ATPase, in thick ascending limb cells and, likely, other proximal and distal tubular segments of the kidney nephron.

Endothelial SIRT6 Is Vital to Prevent Hypertension and Associated Cardiorenal Injury Through Targeting Nkx3.2-GATA5 Signaling.

RATIONALE: Endothelial dysfunction is an important determinant risk factor for the development of hypertension and its complications. Thus, identification of potential therapeutic targets for preventing endothelial dysfunction has major clinical importance. Emerging evidence indicates that epigenetic modifications are closely associated with the regulation of endothelial function. Among them, HDAC (histone deacetylase)-mediated epigenetic processes in vascular homeostasis and cardiovascular disease have attracted much attention. SIRT6 (sirtuin 6) is one member of SIRTs (class III HDAC) that are highly conserved NAD+-dependent deacetylases. OBJECTIVE: This study was designed to elucidate the role of SIRT6 in the pathogenesis of hypertension, discover the new targets of SIRT6, and explore related mechanisms on the regulation of endothelial function. METHODS AND RESULTS: The levels of endothelial SIRT6 were significantly reduced in 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Utilizing genetically engineered endothelial-specific SIRT6 knockout (Cre+/SIRT6fl/fl) mice, we found that endothelial-specific deletion of SIRT6 significantly enhanced blood pressure, exacerbated endothelial dysfunction and cardiorenal injury in experimental hypertension. Functionally, SIRT6 has pleiotropic protective actions in endothelial cells, which include promoting endothelium-dependent vasodilatation and vascular NO bioavailability, reducing cellular permeability, ameliorating endothelial senescence and apoptosis, and facilitating autophagy. Mechanistically, SIRT6 induced the expression of GATA5 (GATA-binding protein 5), a novel regulator of blood pressure, through inhibiting Nkx3.2 (NK3 homeobox 2) transcription by deacetylating histone H3K9 (histone H3 lysine 9), thereby regulating GATA5-mediated signaling pathways to prevent endothelial injury. Finally, we provide direct evidence for the therapeutic potential of SIRT6 in desoxycorticosterone acetate/salt-induced hypertensive mice by overexpression of SIRT6 in vivo. CONCLUSIONS: This study for the first time demonstrates that SIRT6 prevents hypertension and its complications by maintaining endothelial function. Pharmacological targeting of SIRT6 may be an innovative therapeutic strategy for treating patients with hypertension.

Detrimental role of sphingosine kinase 1 in kidney damage in DOCA-salt hypertensive model: evidence from knockout mice.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingolipids and produced by sphingosine kinases (SphK1 and SphK2). SphK1/S1P pathway is implicated in the progression of chronic kidney disease. However, the role of SphK1/S1P pathway in renal injury in hypertension has not been reported. This study tested the hypothesis that SphK1/S1P pathway mediates the kidney damage in DOCA-salt hypertensive mice. METHODS: Male wild type (WT) C57BL6 and SphK1 knockout (KO) mice were subjected to unilateral nephrectomy, subcutaneous implant containing 50 mg of deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water for 7 weeks. At the end of experiments, blood pressure data, 24 h urine and kidney samples were collected. Renal mRNA levels of SphK1 were measured by real-time RT-PCR. Markers for fibrogenesis and immune cell infiltration in kidneys were detected using Western blot and immunohistochemistray analysis, respectively. The glomerular morphological changes were examined in kidney tissue slides stained with Periodic-Acid Schiff. Four groups were studied: wild type control (WT-C), WT-DOCA, KO-C and KO-DOCA. RESULTS: The renal SphK1 mRNA expression was significantly upregulated in WT-DOCA mice, whereas this upregulation of renal SphK1 mRNA was blocked in KO-DOCA mice. There was no difference in DOCA-salt-induced hypertension between WT and KO mice. The urinary albumin was increased in both DOCA-salt groups. However, the albuminuria was significantly lower in KO-DOCA than in WT-DOCA group. There were increases in glomerulosclerosis indices in both DOCA-salt groups, whereas the increases were also significantly lower in KO-DOCA than in WT-DOCA mice. Renal protein levels of α-smooth muscle actin were upregulated in both DOCA-salt groups, but the increase was significant lower in KO-DOCA than in WT-DOCA group. The increased staining areas of collagen detected by Sirius Red-staining in kidney tissue sections were also attenuated in KO-DOCA compared with WT-DOCA mice. In contrast, the increased infiltration of CD43+ (a T cell marker) or CD68+ (a macrophage marker) cells in DOCA-salt kidneys showed no significant difference between WT-DOCA and KO-DOCA mice. CONCLUSIONS: SphK1/S1P signaling pathway mediates kidney damage in DOCA-salt hypertensive mice independent of blood pressure and immune modulation.

Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells.

Growth differentiation factor (GDF)11 has been reported to reverse age-related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The present study attempted to test a hypothesis that GDF11 counteracts the pathologic dedifferentiation of mouse carotid arterial smooth muscle cells (CASMCs) due to deficient autophagy. By real-time RT-PCR and Western blot analysis, exogenously administrated GDF11 was found to promote CASMC differentiation with increased expression of various differentiation markers (α-smooth muscle actin, myogenin, myogenic differentiation, and myosin heavy chain) as well as decreased expression of dedifferentiation markers (vimentin and proliferating cell nuclear antigen). Upregulation of the GDF11 gene by trichostatin A (TSA) or CRISPR-cas9 activating plasmids also stimulated the differentiation of CASMCs. Either GDF11 or TSA treatment blocked 7-ketocholesterol-induced CASMC dedifferentiation and autophagosome accumulation as well as lysosome inhibitor bafilomycin-induced dedifferentiation and autophagosome accumulation. Moreover, in CASMCs from mice lacking the CD38 gene, an autophagy deficiency model in CASMCs, GDF11 also inhibited its phenotypic transition to dedifferentiation status. Correspondingly, TSA treatment was shown to decrease GDF11 expression and reverse CASMC dedifferentiation in the partial ligated carotid artery of mice. The inhibitory effects of TSA on dedifferentiation of CASMCs were accompanied by reduced autophagosome accumulation in the arterial wall, which was accompanied by attenuated neointima formation in partial ligated carotid arteries. We concluded that GDF11 promotes CASMC differentiation and prevents the phenotypic transition of these cells induced by autophagosome accumulation during different pathological stimulations, such as Western diet, lysosome function deficiency, and inflammation. NEW & NOTEWORTHY The present study demonstrates that growth differentiation factor (GDF)11 promotes autophagy and subsequent differentiation in carotid arterial smooth muscle cells. Upregulation of GDF11 counteracts dedifferentiation under different pathological conditions. These findings provide novel insights into the regulatory role of GDF11 in the counteracting of sclerotic arterial diseases and also suggest that activation or induction of GDF11 may be a new therapeutic strategy for the treatment or prevention of these diseases.

Diuretic, Natriuretic, and Vasodepressor Activity of a Lipid Fraction Enhanced in Medium of Cultured Mouse Medullary Interstitial Cells by a Selective Fatty Acid Amide Hydrolase Inhibitor.

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not yet understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured, and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase, PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide). Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. High-performance liquid chromatography (HPLC) analyses of lipid extracts of MMIC culture medium revealed a 205-nm absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 area units under the peak/10 min) stimulated diuresis and natriuresis, whereas these parameters returned toward baseline at higher doses but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, the glomerular filtration rate, and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urine parameters. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid that acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Knockout of Sphingosine Kinase 1 Attenuates Renal Fibrosis in Unilateral Ureteral Obstruction Model.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in various diseases. S1P also plays significant roles in the differentiation of fibroblasts into myofibroblasts, being implicated in fibrotic diseases. S1P is produced by the phosphorylation of sphingosine catalyzed by sphingosine kinases (SphK1 and SphK2). It remains unclear if the activation of endogenous SphK1 contributes to fibrogenesis in kidneys. The present study determined the effect of SphK1 gene knockout (KO) on fibrotic markers in kidneys. METHODS: The renal fibrosis was produced using the unilateral ureteral obstruction (UUO) model in wild-type (WT) and SphK1 gene KO mice. Renal mRNA levels of SphK1 and S1P receptors (S1PR) were measured by real-time RT-PCR. Fibrotic and immune cell markers in kidneys were measured by Western blot analysis and immunostaining, respectively. Renal morphological damage was examined by Periodic-Acid Schiff staining. RESULTS: The mRNA levels of SphK1 and S1PRs were dramatically increased in renal tissues of WT-UUO mice, whereas the increase in renal SphK1 mRNA was blocked in KO-UUO mice. Interestingly, the increased levels of fibrotic markers, collagen and α-smooth muscle actin, in kidneys were significantly attenuated in KO-UUO versus WT-UUO mice. Meanwhile, kidney damage indices were remarkably attenuated in KO-UUO mice compared with WT-UUO mice. However, increased numbers of CD43+ and CD48+ cells, markers for T cell and macrophage, respectively, showed no significant difference between -WT-UUO and KO-UUO kidneys. CONCLUSION: The activation of the SphK1-S1P pathway may contribute to tubulointerstitial fibrosis in UUO kidneys by affecting fibrotic signaling within renal cells independent of immune modulation.

Gpr97 Exacerbates AKI by Mediating Sema3A Signaling.

Background G protein-coupled receptors (GPCRs) participate in a variety of physiologic functions, and several GPCRs have critical physiologic and pathophysiologic roles in the regulation of renal function. We investigated the role of Gpr97, a newly identified member of the adhesion GPCR family, in AKI.Methods AKI was induced by ischemia-reperfusion or cisplatin treatment in Gpr97-deficient mice. We assessed renal injury in these models and in patients with acute tubular necrosis by histologic examination, and we conducted microarray analysis and in vitro assays to determine the molecular mechanisms of Gpr97 function.Results Gpr97 was upregulated in the kidneys from mice with AKI and patients with biopsy-proven acute tubular necrosis compared with healthy controls. In AKI models, Gpr97-deficient mice had significantly less renal injury and inflammation than wild-type mice. Gpr97 deficiency also attenuated the AKI-induced expression of semaphorin 3A (Sema3A), a potential early diagnostic biomarker of renal injury. In NRK-52E cells subjected to oxygen-glucose deprivation, siRNA-mediated knockdown of Gpr97 further increased the expression of survivin and phosphorylated STAT3 and reduced toll-like receptor 4 expression. Cotreatment with recombinant murine Sema3A protein counteracted these effects. Finally, additional in vivo and in vitro studies, including electrophoretic mobility shift assays and luciferase reporter assays, showed that Gpr97 deficiency attenuates ischemia-reperfusion-induced expression of the RNA-binding protein human antigen R, which post-transcriptionally regulates Sema3A expression.Conclusions Gpr97 is an important mediator of AKI, and pharmacologic targeting of Gpr97-mediated Sema3A signaling at multiple levels may provide a novel approach for the treatment of AKI.

Sphingosine-1-phosphate pathway in renal fibrosis.

Renal fibrosis is defined as the excessive deposition and modification of extracellular matrix (ECM) in the renal parenchyma in response to injury and inflammation, resulting in renal function loss. This condition is common to many chronic kidney diseases occurring under diverse pathological conditions, such as diabetic and hypertensive nephropathy. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in the regulation of cardiovascular functions and the pathogenesis of various cardiovascular diseases. S1P has also been considered an important regulator of fibrotic diseases, playing significant roles in the differentiation of fibroblasts to myofibroblasts and in the induction of inflammatory responses during the early stages of fibrotic diseases. This minireview summarizes recent research findings regarding the importance of the sphingosine kinase-1-S1P-S1P receptor axis and its interactions with other classic fibrotic signaling pathways and the immune inflammatory response to reveal novel therapeutic targets for the treatment or prevention of renal fibrosis.

Downregulation of microRNA-429 contributes to angiotensin II-induced profibrotic effect in rat kidney.

MicroRNA (miR) 429 has been shown to inhibit epithelial-to-mesenchymal transition (EMT) in cancer cells. However, the role of miR429 in EMT in non-cancer cells has not been defined, especially in the kidneys. The present study determined whether miR429 participated in angiotensin (ANG) II-induced EMT and fibrogenesis in renal cells. In NRK-52E cells, a rat proximal tubular cell line, incubation of ANG II (10-9 M) for 24 h significantly reduced the level of miR429 by 60% and increased the protein levels of mesenchymal markers α-smooth muscle actin and fibroblast-specific protein-1 by threefold and decreased epithelial marker E-cadherin by 60%, which was blocked by losartan, an AT1 receptor antagonist. Treatment of cells with miR429 inhibitor produced similar changes in the above EMT markers to that induced by ANG II. In cells overexpressed with miR429 transgene, ANG II-induced increases in collagen were abolished. Male Sprague-Dawley rats were infused with ANG II (200 ng·kg-1·min-1) for 12 days, and the levels of miR429 in the kidneys were reduced by 75%. Intrarenal transfection of lentivirus expressing miR429 also reversed the ANG II-induced changes in the EMT markers and collagen in the kidneys. The ANG II-induced increase in urinary albumin was significantly inhibited by miR429 transgene. There was no difference in the increases of blood pressure between ANG II- and ANG II+miR429 transgene-treated rats. These data suggest that ANG II-induced inhibition of miR429 contributes to ANG II-induced transdifferentiation and fibrogenesis in renal cells and that miR429 protects against ANG II-induced kidney damages.

Modulation of mean arterial pressure and diuresis by renomedullary infusion of a selective inhibitor of fatty acid amide hydrolase.

The kidneys contribute to the control of body fluid and electrolytes and the long-term regulation of blood pressure through various systems, including the endocannabinoid system. Previously, we showed that inhibition of the two major endocannabinoid-hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, in the renal medulla increased the rate of urine excretion (UV) and salt excretion without affecting mean arterial pressure (MAP). The present study evaluated the effects of a selective FAAH inhibitor, N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidine carboxamide (PF-3845) on MAP and renal functions. Infusion of PF-3845 into the renal medulla of C57BL/6J mice reduced MAP during the posttreatment phases and increased UV at 15 and 30 nmol/min per gram kidney weight (g kwt), relative to the pretreatment control phase. Intravenous PF-3845 administration reduced MAP at the 7.5, 15, and 30 doses and increased UV at the 15 and 30 nmol⋅min-1⋅g-1 kwt doses. PF-3845 treatment elevated sodium and potassium urinary excretion and medullary blood flow. Homozygous FAAH knockout mice were refractory to intramedullary PF-3845-induced changes in MAP, but UV was increased. Both MAP and UV responses to intramedullary PF-3845 in C57BL/6J mice were diminished by pretreatment with the cannabinoid type 1 receptor-selective antagonist, rimonabant (3 mg/kg, ip) but not the cyclooxygenase 2-selective inhibitor, celecoxib (15 mg/kg, iv). Liquid chromatography-tandem mass spectrometry analyses showed increased anandamide in kidney tissue and 2-arachidonoyl glycerol in plasma after intramedullary PF-3845. These data suggest that inhibition of FAAH in the renal medulla leads to both a diuretic and blood pressure-lowering response mediated by elevated anandamide in kidney tissue or 2-arachidonoyl glycerol in plasma.

Role of Nitric Oxide in the Cardiovascular and Renal Systems.

The gasotransmitters are a family of gaseous signaling molecules which are produced endogenously and act at specific receptors to play imperative roles in physiologic and pathophysiologic processes. As a well-known gasotransmitter along with hydrogen sulfide and carbon monoxide, nitric oxide (NO) has earned repute as a potent vasodilator also known as endothelium-derived vasorelaxant factor (EDRF). NO has been studied in greater detail, from its synthesis and mechanism of action to its physiologic, pathologic, and pharmacologic roles in different disease states. Different animal models have been applied to investigate the beneficial effects of NO as an antihypertensive, renoprotective, and antihypertrophic agent. NO and its interaction with different systems like the renin⁻angiotensin system, sympathetic nervous system, and other gaseous transmitters like hydrogen sulfide are also well studied. However, links that appear to exist between the endocannabinoid (EC) and NO systems remain to be fully explored. Experimental approaches using modulators of its synthesis including substrate, donors, and inhibitors of the synthesis of NO will be useful for establishing the relationship between the NO and EC systems in the cardiovascular and renal systems. Being a potent vasodilator, NO may be unique among therapeutic options for management of hypertension and resulting renal disease and left ventricular hypertrophy. Inclusion of NO modulators in clinical practice may be useful not only as curatives for particular diseases but also for arresting disease prognoses through its interactions with other systems.

Simvastatin promotes NPC1-mediated free cholesterol efflux from lysosomes through CYP7A1/LXRα signalling pathway in oxLDL-loaded macrophages.

Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.