Renal Medullary Overexpression of Sphingosine-1-Phosphate Receptor 1 Transgene Attenuates Deoxycorticosterone Acetate (DOCA)-Salt Hypertension.

BACKGROUND: Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. METHODS: Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. RESULTS: S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the ßENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and ßENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. CONCLUSIONS: These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel.

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice.

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Inactivation of fatty acid amide hydrolase protects against ischemic reperfusion injury-induced renal fibrogenesis.

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-ß1)-induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah-/- mice further validated the protection against TGF-ß1-induced damages. The CB 1 or CB2 receptor antagonist and exogenous FAAH metabolite arachidonic acid failed to reverse the protective effects of FAAH inactivation in HK-2 cells. However, a substrate-selective inhibitor of AEA-cyclooxygenase-2 (COX-2) pathway significantly suppressed the anti-profibrogenic actions of FAAH inhibition. Further, the AEA-COX-2 metabolite, prostamide E2 exerted anti-fibrogenesis effect. These findings suggest that FAAH activation and the consequent reduction of AEA contribute to the renal fibrogenesis, and that FAAH inhibition protects against fibrogenesis in renal cells independently of CB receptors via the AEA-COX-2 pathway by the recovery of reduced AEA.

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.

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.

Regulation of TRPML1 channel activity and inflammatory exosome release by endogenously produced reactive oxygen species in mouse podocytes.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase-produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of inflammatory cytokines produced by the NLRP3 inflammasome in podocytes after Hcy stimulation. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with Hcy compared with control podocytes, which was abolished by pre-treatment with the NADPH oxidase inhibitors, gp91 ds-tat peptide and diphenyleneiodonium (DPI). In addition, Hcy induced activation in podocytes of NLRP3 inflammasomes and the formation of multivesicular bodies (MVBs) containing inflammatory cytokines, which were prevented by treatment with gp91 ds-tat or the ROS scavenger, catalase. Given the importance of the transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited by endogenously produced ROS in podocytes after Hcy stimulation. By GCaMP3 Ca2+ imaging, we confirmed the inhibition of TRPML1 channel activity by Hcy which was remarkably ameliorated by catalase and gp91 ds-tat peptide. By structured illumination microscopy (SIM) and nanoparticle tracking analysis (NTA), we found that ML-SA1, a TRPML1 channel agonist, significantly enhanced lysosome-MVB interaction and reduced exosome release in podocytes, which were attenuated by Hcy. Pre-treatment of podocytes with catalase or gp91 ds-tat peptide restored ML-SA1-induced changes in lysosome-MVB interaction and exosome secretion. Moreover, we found that hydrogen peroxide (H2O2) mimicked the effect of Hcy on TRPML1 channel activity, lysosome-MVB interaction, and exosome secretion in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes through inhibition of TRPML1 channel activity, which may contribute to the initiation of glomerular inflammation during hHcy.

Contribution of podocyte inflammatory exosome release to glomerular inflammation and sclerosis during hyperhomocysteinemia.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis in response to hyperhomocysteinemia (hHcy). However, it remains unknown how the products of NLRP3 inflammasome in cytoplasm are secreted out of podocytes. In the present study, we tested whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in hHcy-induced glomerular injury. By various approaches, we found that hHcy induced NLRP3 inflammasome activation and neutrophil infiltration in glomeruli of WT/WT mice. Lysosome-MVB interaction in glomeruli remarkably decreased in WT/WT mice fed with FF diet, leading to elevation of urinary exosome excretion of these mice. Podocyte-derived exosomes containing pro-inflammatory cytokines increased in urine of WT/WT mice in response to hHcy. The release of inflammatory exosomes from podocytes was prevented by Smpd1 gene deletion but enhanced by podocyte-specific Smpd1 gene overexpression (Smpd1 encodes Asm in mice). Pathologically, hHcy-induced podocyte injury and glomerular sclerosis were blocked by Smpd1 gene knockout but amplified by podocyte-specific Smpd1 gene overexpression. Taken together, our results suggest that Asm-ceramide signaling pathway contributes to NLRP3 inflammasome activation and robust release of inflammatory exosomes in podocytes during hHcy, which together trigger local glomerular inflammation and sclerosis.

Collecting duct-specific knockout of sphingosine-1-phosphate receptor 1 aggravates DOCA-salt hypertension in mice.

OBJECTIVE: We have previously reported that renal medullary sphingosine-1-phosphate (S1P) regulates sodium excretion via the S1P type-1 receptor (S1PR1). As S1PR1 is predominantly expressed in collecting ducts (CD), the present study tested the hypothesis that the CD-S1PR1 pathway plays a critical role in sodium excretion and contributes to salt-sensitive hypertension. METHODS: CD-specific S1PR1 knockout mice were generated by crossing aquaporin-2-Cre mice with S1PR1-floxed mice. Renal sodium excretion and arterial pressure were compared between wild type and KO mice in response to high-salt challenges and treatment of deoxycorticosterone acetate (DOCA) salt. RESULTS: Protein levels of renal medullary S1PR1 were increased by 100% after high-salt intake, whereas DOCA treatment with high-salt intake blocked the increase of S1PR1 levels. Urinary sodium excretions in knockout mice were decreased by 60% compared with wild type mice after acute intravenous sodium loading (0.84 ±â€Š0.16 vs. 2.22 ±â€Š0.62 µmole/min per g kwt). The pressure natriuresis was impaired in knockout mice compared with wild type mice (4.32 ±â€Š1.04 vs. 8.73 ±â€Š0.19 µmole/min per g kwt). The chronic high-salt intake-induced positive sodium balance was enhanced in knockout mice compared with wild type mice (5.27 ±â€Š0.39 vs. 2.38 ±â€Š1.04 mmol/100 g BW per 24 h). After 10-day DOCA-salt treatment, knockout mice developed more severe hypertension than wild type mice (SBP 142 ±â€Š8 vs. 115 ±â€Š4 mmHg). CONCLUSION: The deletion of CD-S1PR1 reduced sodium excretion, promoted sodium retention, and accelerated DOCA-salt-induced salt-sensitive hypertension, suggesting that the CD-S1PR1 signaling is an important antihypertensive pathway by promoting sodium excretion and that impairment of renal medullary S1PR1 may represent a novel mechanism for salt-sensitive hypertension.

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.

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.

Podocytopathy and Nephrotic Syndrome in Mice with Podocyte-Specific Deletion of the Asah1 Gene: Role of Ceramide Accumulation in Glomeruli.

Lysosomal acid ceramidase (Ac) has been shown to be critical for ceramide hydrolysis and regulation of lysosome function and cellular homeostasis. In the present study, we generated a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of the α subunit (main catalytic subunit) of Ac. Although no significant morphologic changes in glomeruli were observed in these mice under light microscope, severe proteinuria and albuminuria were found in these podocyte-specific knockout mice compared with control genotype littermates. Transmission electron microscopic analysis showed that podocytes of the knockout mice had distinctive foot process effacement and microvillus formation. These functional and morphologic changes indicate the development of nephrotic syndrome in mice bearing the Asah1 podocyte-specific gene deletion. Ceramide accumulation determined by liquid chromatography-tandem mass spectrometry was demonstrated in isolated glomeruli of Asah1fl/fl/PodoCre mice compared with their littermates. By crossbreeding Asah1fl/fl/PodoCre mice with Smpd1-/- mice, we also produced a double knockout strain, Smpd1-/-/Asah1fl/fl/PodoCre, that also lacks Smpd1, the acid sphingomyelinase that hydrolyzes sphingomyelin to ceramide. These mice exhibited significantly lower levels of glomerular ceramide with decreased podocyte injury compared with Asah1fl/fl/PodoCre mice. These results strongly suggest that lysosomal Ac in podocytes is essential for the maintenance of the structural and functional integrity of podocytes.

The Certification of Standard Reference Material 1979: Powder Diffraction Line Profile Standard for Crystallite Size Analysis.

This rather long-standing project has resulted in a National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) for the analysis of crystallite size from a consideration of powder diffraction line profile broadening. It consists of two zinc oxide powders, one with a crystallite size distribution centered at approximately 15 nm, and a second centered at about 60 nm. These materials display the effects of stacking faults that broaden specific hkl reflections and a slight amount of microstrain broadening. Certification data were collected on the high-resolution powder diffractometer located at beamline 11-BM of the Advanced Photon Source, and on a NIST-built laboratory diffractometer equipped with a Johansson incident beam monochromator and position sensitive detector. Fourier transforms were extracted from the raw data using a modified, two-step profile fitting procedure that addressed the issue of accurate background determination. The mean column lengths, 〈L〉area and 〈L〉vol, were then computed from the Fourier transforms of the specimen contribution for each reflection. Data were also analyzed with fundamental parameters approach refinements using broadening models to yield 〈L〉area and 〈L〉vol values. These values were consistent with the model-independent Fourier transform results; however, small discrepancies were noted for the 〈L〉area values from both machines and both crystallite size ranges. The fundamental parameters approach fits to the laboratory data yielded the certified lattice parameters.

D-Ribose Induces Podocyte NLRP3 Inflammasome Activation and Glomerular Injury via AGEs/RAGE Pathway.

D-ribose levels are demonstrated to be increased in type II diabetes mellitus and increased blood D-ribose is involved in the development of diabetic complications such as diabetic encephalopathy and nephropathy. However, the mechanism mediating the pathogenic role of D-ribose in nephropathy remains poorly understood. Given that D-ribose was reported to induce advanced glycation end products (AGEs) formation, the present study tested whether D-ribose induces NLRP3 activation and associated glomerular injury via AGEs/receptor of AGEs (RAGE) signaling pathway. In vivo, C57BL/6J and Asc-/- mice were treated with D-ribose with or without AGEs inhibitor. Administration of D-ribose daily for 30 days was found to induce NLRP3 inflammasome formation in glomerular podocyte, as shown by increased co-localization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) or caspase-1. This D-ribose-induced NLRP3 inflammasome formation was accompanied by its activation as evidenced by increased IL-1ß production, a major product of NLRP3 inflammasome. Corresponding to NLRP3 inflammasome activation, D-ribose led to significant glomerular injury in mice. All these D-ribose-induced glomerular inflammasome and associated pathological changes were markedly attenuated by deletion of Asc gene. Furthermore, the accumulation of AGEs and RAGE was found increased in glomeruli of mice receiving D-ribose. In cell studies, we also confirmed that D-ribose induced NLRP3 inflammasome formation and activation in podocytes, which was significantly blocked by caspase-1 inhibitor, YvAD. Mechanically, AGEs formation inhibition and cleavage or silencing of RAGE gene were shown to suppress D-ribose-induced NLRP3 inflammasome formation and activation, as shown by significant reduction of NLRP3 inflammasome molecular aggregation, caspase-1 activity and IL-1ß production. These results strongly suggest that relatively long term administration of D-ribose induces NLRP3 inflammasome formation and activation in podocytes via AGEs/RAGE signaling pathway, which may be one of important triggering mechanisms leading to diabetic nephropathy.

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.

Formation of HETE-EAs and dihydroxy derivatives in mouse kidney tissue and analysis by high-performance liquid chromatography tandem mass spectrometry.

The kidneys play an important role in the long-term regulation of blood pressure by control of salt and water balance in the body through various systems including the endocannabinoid system. The endocannabinoid system consists of the two major cannabinoid receptor agonists, anandamide (AEA) and 2-arachidonylglycerol (2-AG), their hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid receptors, CB1 and CB2. AEA can be converted into 12- and 15(S)-hydroperoxyeicosatetraenoic acid ethanolamides by 12-LOX and 15-LOX, respectively and can form epoxyeicosatrienoic acid- (EET-EAs) (5,6-, 8,9-, 11,12-, 14,15-) and hydroxyeicosatetraenoic acid- (HETE) ethanolamides. Furthermore, the EET-EAs produce a secondary metabolism by microsomal epoxide hydrolase to form the corresponding dihydroxyeicosatetraenoic acid-EAs (DiHETE-EA). Reference material was not available for DiHETE-EA. These metabolites were synthesized by incubation of the corresponding EET-EAs with mouse liver cytosol containing epoxide hydrolases. Presented is a solid phase extraction and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the extraction and quantitation of AEA, 2-AG, their metabolites, oleoylethanolamide (OEA), and palmitoylethanolamide (PEA), and the in vivo formation of the DiHETE-EAs in kidney after a single intravenous bolus administration of 20 mg/kg of anandamide in C57BL/6 J and FAAH KO mice.

Lysosomal regulation of extracellular vesicle excretion during d-ribose-induced NLRP3 inflammasome activation in podocytes.

The NLRP3 inflammasome is activated in the cytoplasm of cells and its products such as IL-1ß are exported through a non-classical ER-Golgi pathway. Several mechanistically distinct models including exocytosis of secretory lysosomes, microvesicles (MVs) and extracellular vehicles (EVs) have been proposed for their release. In this study, we hypothesized that the NLRP3 inflammasome product, IL-1ß in response to exogenously administrated and endogenously produced d-ribose stimulation is released via extracellular vesicles including EVs via a sphingolipid-mediated molecular mechanisms controlling lysosome and multivesicular body (MVB) interaction. First, we demonstrated that both endogenous and exogenous d-ribose induced NLRP3 inflammasome activation to produce IL-1ß, which was released via EVs in podocytes. Then, we found that colocalization of marker MVB marker VPS16 with IL-1ß within podocytes increased upon d-ribose stimulation, which was accompanied by decreased colocalization of lysosome marker Lamp-1 and VPS16, suggesting decrease in MVB inclusion of IL-1ß due to reduced lysosome and MVB interaction. All these changes were mimicked and accelerated by lysosome v-ATPase inhibitor, bafilomycin. Moreover, ceramide in podocytes was found elevated upon d-ribose stimulation, and prior treatments of podocyte with acid sphingomyelinase (Asm) inhibitor, amitriptyline, acid ceramidase (AC) inducer, genistein, or AC CRISPR/cas9 activation plasmids were found to decrease d-ribose-induced ceramide accumulation, EVs release and IL-1ß secretion due to reduced interactions of lysosome with MVBs. These results suggest that inflammasome-derived products such as IL-1ß during d-ribose stimulation are released via EVs, in which lysosomal sphingolipid-mediated regulation of lysosome function plays an important role.

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.

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.

Inhibition of pannexin-1 channel activity by adiponectin in podocytes: Role of acid ceramidase activation.

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs+in/Na+out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P.