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The findings of our study are significant in several ways: 1) loss of an amino acid chaperone in the proximal tubule is sufficient to cause hypertension, 2) the results in global and proximal tubule-specific collectrin knockout mice support the notion that vascular dysfunction is required for salt sensitivity or that impaired renal tubule function causes hypertension but is not sufficient to cause salt sensitivity, and 3) our study is the first to implicate a role of collectrin in human hypertension.

Collectrin (

Acute kidney injury (AKI) is a frequent complication of sepsis and an important cause of morbidity and mortality worldwide. A cornerstone of sepsis-associated AKI is dysregulated inflammation, leading to increased tissue oxidative stress and free radical formation, which leads to multiple forms of cell death. DJ-1 is a peroxiredoxin protein with multiple functions, including its ability to control cellular oxidative stress. Although DJ-1 is expressed prominently by renal tubules, its role in AKI has not been investigated. In the present study, we examined the effect of DJ-1 deficiency in a murine model of endotoxin-induced AKI. Endotoxemia induced greater kidney injury in DJ-1-deficient mice. Furthermore, DJ-1 deficiency increased renal oxidative stress associated with increased renal tubular apoptosis and with expression of death domain-associated protein (DAXX). Similar to the in vivo model, in vitro experiments using a medullary collecting duct cell line (mIMCD3) and cytotoxic serum showed that serum obtained from wild-type mice resulted in increased expression of s100A8/s100A9, DAXX, and apoptosis in DJ-1-deficient mIMCD3 cells. Our findings demonstrate a novel renal protective role for renal tubular DJ-1 during endotoxemia through control of oxidative stress, renal inflammation, and DAXX-dependent apoptosis.

Lupus nephritis is the end organ manifestation of systemic lupus erythematosus. Iron metabolism and its master regulator, hepcidin, are known to regulate cell proliferation and inflammation, but their direct role in the pathophysiology of lupus nephritis remains under-investigated. Exogenous hepcidin reduced the severity of lupus nephritis in MRL/lpr mice, a preclinical model of spontaneous systemic lupus erythematosus without worsening anemia of inflammation. Hepcidin treatment reduced renal iron accumulation, systemic and intrarenal cytokines, and renal immune cell infiltration, independent of glomerular immune complex deposits and circulating autoantibodies. Hepcidin increased renal H-ferritin (a ferroxidase), reduced expression of free iron dependent DNA synthesis enzymes, Ribonucleotide Reductase 1 and 2, and intra-renal macrophage proliferation. These findings were recapitulated in vitro upon treatment of macrophages with hepcidin and murine colony stimulation factor-1. Furthermore, hepcidin-treated macrophages secreted less IL-1β and IL-6 upon stimulation with the TLR3 agonist polyinosine-polycytidylic acid. Of clinical relevance, hepcidin reduced progression and severity of nephritis in old mice with established systemic autoimmunity and overt proteinuria, highlighting its therapeutic potential. Thus, our findings provide a proof-of-concept that targeting cellular iron metabolism with hepcidin represents a promising therapeutic strategy in lupus nephritis.

Background GSTM1 encodes glutathione S-transferase μ-1 (GSTM1), which belongs to a superfamily of phase 2 antioxidant enzymes. The highly prevalent GSTM1 deletion variant is associated with kidney disease progression in human cohorts: the African American Study of Kidney Disease and Hypertension and the Atherosclerosis Risk in Communities (ARIC) Study. Methods We generated a Gstm1 knockout mouse line to study its role in a CKD model (involving subtotal nephrectomy) and a hypertension model (induced by angiotensin II). We examined the effect of intake of cruciferous vegetables and GSTM1 genotypes on kidney disease in mice as well as in human ARIC study participants. We also examined the importance of superoxide in the mediating pathways and of hematopoietic GSTM1 on renal inflammation. Results Gstm1 knockout mice displayed increased oxidative stress, kidney injury, and inflammation in both models. The central mechanism for kidney injury is likely mediated by oxidative stress, because treatment with Tempol, an superoxide dismutase mimetic, rescued kidney injury in knockout mice without lowering BP. Bone marrow crosstransplantation revealed that Gstm1 deletion in the parenchyma, and not in bone marrow-derived cells, drives renal inflammation. Furthermore, supplementation with cruciferous broccoli powder rich in the precursor to antioxidant-activating sulforaphane significantly ameliorated kidney injury in Gstm1 knockout, but not wild-type mice. Similarly, among humans (ARIC study participants), high consumption of cruciferous vegetables was associated with fewer kidney failure events compared with low consumption, but this association was observed primarily in participants homozygous for the GSTM1 deletion variant. Conclusions Our data support a role for the GSTM1 enzyme in the modulation of oxidative stress, inflammation, and protective metabolites in CKD.

Cross-presentation is a modular series of intracellular events dictating the internalization and subsequent MHC class I (MHC I) display of extracellular Ags. This process has been defined in dendritic cells and plays a fundamental role in the induction of CD8+ T cell immunity during viral, intracellular bacterial, and antitumor responses. Herein, acute viral infection of murine liver with adenovirus, a model for intrahepatic cross-presentation, confirms hepatocytes directly contribute to cross-presentation of Ags and priming the pool of naive CD8+ T cells within the liver microenvironment. Processing of soluble and cell-associated Ags into peptide displayed by MHC I is however defective in hepatocytes lacking collectrin, an intracellular chaperone protein that localizes within the endoplasmic reticulum–Golgi intermediate compartment. Loss of hepatic collectrin expression leads to the diminished cross-priming and expansion of cytolytic antiviral CD8+ T cells. This study demonstrates that collectrin positively regulates processing of engulfed Ags into MHC I:peptide complexes within hepatocytes. Collectrin-mediated cross-presentation supports intrahepatic adaptive antiviral immune responses and may lead to insights into the nature of how the liver acts as a primary site of CD8+ T cell activation.

Collectrin, encoded by the Tmem27 gene, is a transmembrane glycoprotein with approximately 50% homology with angiotensin converting enzyme 2, but without a catalytic domain. Collectrin is most abundantly expressed in the kidney proximal tubule and collecting duct epithelia, where it has an important role in amino acid transport. Collectrin is also expressed in endothelial cells throughout the vasculature, where it regulates L-arginine uptake. We previously reported that global deletion of collectrin leads to endothelial dysfunction, augmented salt sensitivity, and hypertension. Here, we performed kidney crosstransplants between wild-type (WT) and collectrin knockout (Tmem27Y/- ) mice to delineate the specific contribution of renal versus extrarenal collectrin on BP regulation and salt sensitivity. On a high-salt diet, WT mice with Tmem27Y/- kidneys had the highest systolic BP and were the only group to exhibit glomerular mesangial hypercellularity. Additional studies showed that, on a high-salt diet, Tmem27Y/- mice had lower renal blood flow, higher abundance of renal sodium-hydrogen antiporter 3, and lower lithium clearance than WT mice. In WT mice, administration of angiotensin II for 2 weeks downregulated collectrin expression in a type 1 angiotensin II receptor-dependent manner. This downregulation coincided with the onset of hypertension, such that WT and Tmem27Y/- mice had similar levels of hypertension after 2 weeks of angiotensin II administration. Altogether, these data suggest that salt sensitivity is determined by intrarenal collectrin, and increasing the abundance or activity of collectrin may have therapeutic benefits in the treatment of hypertension and salt sensitivity.

Hypertension affects over 1 billion people worldwide and increases the risk for heart failure, stroke, and chronic kidney disease. Despite high prevalence and devastating impact, its etiology still remains poorly understood for most hypertensive cases. Rcn2, which encodes reticulocalbin 2, is a candidate gene for atherosclerosis that we have previously reported in mice. Here, we identified Rcn2 as a novel regulator of blood pressure in mice. Rcn2 was abundantly expressed in the endothelium and adventitia of normal arteries and was dramatically upregulated in the medial layer of the artery undergoing structural remodeling. Deletion of Rcn2 lowered basal blood pressure and attenuated ANG II-induced hypertension in C57BL/6 mice. siRNA knockdown of Rcn2 dramatically increased production of the nitric oxide (NO) breakdown products nitrite and nitrate by endothelial cells but not by smooth muscle cells. Isolated carotid arteries from Rcn2−/−mice showed an increased sensitivity to the ACh-induced NO-mediated relaxant response compared with arteries of Rcn2+/+mice. Analysis of a recent meta-data set showed associations of genetic variants near RCN2 with blood pressure in humans. These data suggest that Rcn2 regulates blood pressure and contributes to hypertension through actions on endothelial NO synthase.

Overt dysregulation of the renin-angiotensin-aldosterone system (primary aldosteronism: APA and IHA) has long been known for its deleterious cardiovascular consequences. Recently, however, subclinical mild hyperaldosteronism was recognized as a significant risk factor for hypertension, and may represent an early stage in the disease spectrum. Previously, we developed mouse models of hyperaldosteronism by globally deleting subunits of the TWIK-related acid sensitive potassium channel (TASK). Global TASK-1 (Kcnk3) and TASK-3 (Kcnk9) KO mice markedly overproduce Ang II-independent (autonomous) aldosterone, that is not suppressed by high dietary sodium and is accompanied by low renin and elevated blood pressure. Here, we report a novel mouse model of mild hyperaldosteronism produced by the zona glomerulosa (zG)-specific deletion of TASK channels. To generate trigenic Cyp11b2Cre/+ :: Kcnk3f/f :: Kcnk9f/f mice (zG-TASK-KO), the Cyp11b2Cre/+ strain that expresses Cre-recombinase under the aldosterone synthase promoter was crossed with the Kcnk3f/f :: Kcnk9f/f strain. We confirmed restriction of Cre-expression to the zG layer by qRT-PCR and immunohistochemistry using a Cre-dependent reporter mouse (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J ). Despite normal plasma renin, zG-TASK-KO mice displayed a mild increase in 24-hr. urinary aldosterone excretion (11.8±0.8 KO vs 9.3±0.6 controls; ng/mg-aldosterone/creatinine, p

The renin-angiotensin system tightly controls aldosterone synthesis. Dysregulation is evident in hypertension (primary aldosteronism), low renin, and resistant hypertension) but also can exist in normotension. Whether chronic, mild aldosterone autonomy can elicit hypertension remains untested. Previously, we reported that global genetic deletion of 2 pore-domain TWIK-relative acid-sensitive potassium channels, TASK-1 and TASK-3, from mice produces striking aldosterone excess, low renin, and hypertension. Here, we deleted TASK-1 and TASK-3 channels selectively from zona glomerulosa cells and generated a model of mild aldosterone autonomy with attendant hypertension that is aldosterone-driven and Ang II (angiotensin II)–independent. This study shows that a zona glomerulosa–specific channel defect can produce mild autonomous hyperaldosteronism sufficient to cause chronic blood pressure elevation.

Background Lupus nephritis (LN) is an end-organ complication of Systemic lupus erythematosus and is more common in premenopausal women. Hepcidin, the master regulator of iron homeostasis, modulates inflammation and is negatively regulated by estrogen. Therefore, we hypothesized that exogenous hepcidin may reduce the severity and delay the onset of LN. Methods Pre-nephritic 8-week-old or nephritic 16-week-old MRL/lpr female mice were injected Hepcidin (50ug, i.p) or vehicle twice a week and markers of renal injury and inflammation were examined at 18 and 20 weeks of age. The direct effect of hepcidin on macrophages was studied in-vitro. Results Hepcidin reduced intrarenal iron accumulation, and increased H-ferritin. This was associated with reduced renal inflammation and immune cell infiltration, which collectively mitigated microalbuminuria and tubular injury, independent of immune complex deposits and autoantibodies. The increase in H-ferritin, was associated with a reduced number of renal Ki-67+-F4/80+ macrophages. In-vitro, hepcidin induced H-ferritin in macrophages and reduced labile (Fe2+) iron. H-ferritinhi macrophages proliferated less upon Mcsf-1 stimulation and secreted less IL-1b, and IL-6 upon TLR-3 activation. Hepcidin reduced microalbuminuria when administered to nephritic, 16-week-old mice without worsening lupus-associated anemia. Conclusions We have identified that hepcidin targets iron homeostasis and reduces cardinal pathogenic features of LN. Importantly, Hepcidin treatment ameliorates kidney disease in mice with established proteinuria. Thus, our data highlight that targeting cellular iron metabolism with hepcidin represents a promising and a new therapeutic strategy in LN.

There is emerging evidence that extracellular vesicles (EVs) may be novel bio-markers and bio-activators in the pathogenesis of HTN. We hypothesize that the phenotypes of EVs from the circulation and kidney are altered in Angiotensin II (AII) induced HTN and normalize with anti-hypertensive treatment (Rx). AII was delivered via osmotic mini pumps (500 ng/kg/min) alone, or with candesartan (C) @ 10 mg/kg/day, or with hydralazine, hydrochlorothiazide, reserpine (HHR) @ 30/10/0.2 mg/kg/day in drinking water for 2 weeks to 129S6 mice (n = 5 each). Systolic blood pressure (SBP) was measured daily for 2 weeks by tail-cuff manometry. After 2 weeks, mice were euthanized and citrated blood and kidneys collected. Kidney EVs (KEVs) were isolated after incubation with Collagenase type A and subsequent DC. Both circulating EVs and KEVs were isolated through differential centrifugation and characterized by imaging flow cytometry (AmnisImage-StreamX Mark II) using PECAM-1 (CD31), leukocyte (CD45), platelet (CD41) and endothelial (CD105) markers. AII treated mice had significantly higher SBP (mmHg) (162.05 ± 7.20) than normotensive controls ((N) 120.25 ± 7.12), p=0.02. SBP was similarly reduced with C and HHR (134.12 ± 6.15 and 122.45 ± 5.20, respectively, p=NS), and significantly lower than AII, p=0.0026 and =0.03, respectively). Circulating leukocyte derived EVs (LEVs, CD31+/CD45+) increased after 2 weeks in AII treated mice compared to N (AII 2.01E+0.3, N 2.04E+0.4 particles/mL; p=0.0130). This effect was not observed with endothelial and platelet derived EVs. Numbers of circulating LEVs correlated significantly with SBP [(r 2 =0.675); p=0.0029]. Circulating LEVs were reduced after equal normalization of SBP, though no difference in EVs was observed between Rx with C and HHR. CD45+ KEVs in AII treated mice were significantly increased compared to N (AII 7.52E+05 vs N 4.79E+04; p= 0.0257). KEVs also correlated significantly with SBP [(r 2 =0.682); p=0.0032]. In conclusion, circulating and kidney derived EVs may play a role in the immune response in HTN and may be influenced by blood pressure threshold, independent of AT 1 receptor activation. Further studies are needed to confirm and to characterize specific subtype(s) of LEVs and dissect their functional role.

Glutathione- S -transferase μ-1 ( GSTM1 ) enzyme belongs to the superfamily of phase II antioxidant glutathione- S -transferases (GSTs) that are downstream targets of the Nrf2 antioxidant transcription factor. In humans, a common GSTM1 gene deletion variant, the null allele GSTM1(0) , results in decreased or complete absence of GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with accelerated kidney disease progression in the African Americans Study of Kidney Disease (AASK) participants. To understand the direct impact of GSTM1 deficiency on renal inflammation and oxidative stress, we generated Gstm1 deficient mice (KO) to determine their response to angiotensin II, delivered @ 1000 ng/kg/min for 4 weeks via mini-osmotic pump. Blood pressure (BP) was measured by radiotelemetery. Kidney histopathology was assessed by a renal pathologist blinded to genotype and experimental conditions. Renal leukocyte populations were analyzed quantitatively by flow cytometry. Gstm1 KO mice had significantly higher levels of baseline systolic BP (SBP) and ~ 17 mmHg higher SBP after 4 weeks of Ang II-HTN, compared to WT mice. Gstm1 KO mice have increased renal superoxide levels by nearly 3 folds - independent of activation of Nox2 and Nox4 NADPH oxidases or alteration in superoxide dismutase - and worse kidney injury. These changes were associated with significantly increased renal expression of genes involved in inflammation - chemokine ligand 1 (CXCL-1), monocyte chemotactic protein 1 (MCP-1), Interleukin-1β (IL-1β) and IL-6 ( P Gstm1 KO mice displayed ~ two fold increases in all leukocyte populations in the kidney, with the exception of the numbers of B cells that were not significantly different between groups. This increased renal inflammation was attenuated by dietary administration of broccoli powder that is rich in sulforaphane, a phytochemical that increases Nrf2 expression, independent of BP. In Ang II-hypertension, loss of GSTM1 enzyme may be deleterious by augmenting oxidative stress and inflammation in the kidney. Stimulation of the Nrf2 pathway in hypertension may be a novel therapeutic approach to prevent kidney disease progression in GSTM1 deficiency.

In humans, a common deletion variant of the GSTM1 gene, the GSTM1(0) null allele, results in decreased GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with increased risks of chronic kidney disease (CKD) progression and death in the African Americans Study of Kidney Disease. To establish a causal relationship between GSTM1 deficiency and CKD progression, we deleted Gstm1 in the mouse, and determined its effect in the sub-total nephrectomy (Nx) model of CKD. After the 8 th week after Nx, 4 out of 8 Gstm1 knockout (KO) mice began to die, whereas wild-type (WT) mice had 100% survival by the study endpoint at 13 th week. Remaining KO mice had significantly higher plasma creatinine (p < 0.05), suggestive of lower GFR. Due to early mortality in KO mice after the 8th week after Nx, we phenotyped mice at the 8 th week (n=6 each). KO mice had significantly higher 1) mean systolic blood pressure (by radiotelemetry): KO 157.2 ± 5, WT 141.1 ± 2.4 mmHg, 2) urine albumin/creatinine ratio (μg/mg): KO 748.4 ± 187.9, WT 235.1 ± 24.6, and 3) renal superoxide (O2·-) levels (counts/min/mg dry tissue): KO 249.7 ± 63; WT 74.5 ± 14.7; P ≤ 0.02. Hydrogen peroxide levels were not different. Renal mRNA levels of the superoxide dismutase isoforms SOD1, SOD2, and SOD3 were significantly lower in KO mice (KO 7.78 ± 0.047, 0.446 ± 0.062, 0.18 ± 0.03; WT 10.64 ± 0.43, 2.432 ± 0.386, 1.188 ± 0.124, respectively, P ≤ 0.003. Total kidney pathology score (KO 18.7 ± 1.5; WT 8 ± 0.6; P

Intronic variants of the MYH9 gene that encodes the nonmuscle myosin heavy chain IIA are associated with diabetic nephropathy in European Americans and with sickle cell disease-associated nephropathy. However, the causal functional variants of MYH9 have remained elusive. Rare missense mutations in MYH9 cause macrothrombocytopenia and are occasionally associated with development of nephropathy. The E1841K mutation is among the common MYH9 missense mutations and has been associated with nephropathy in some carriers. To determine the contribution of the E1841K mutation in kidney disease, we studied the effects of the E1841K mutation in mice subjected to high salt or angiotensin II (Ang II) as models of hypertension and in mice subjected to renal mass reduction as a model of CKD. Despite similar levels of BP among wild-type (MYH9+/+ ) mice and mice heterozygous (MYH9+/E1841K ) and homozygous (MYH9E1841K/E1841K ) for the mutation in each model, MYH9E1841K/E1841K mice exhibited mildly increased albuminuria in response to high salt; severe albuminuria, nephrinuria, FSGS, and podocyte foot effacement in Ang II-induced hypertension; and early mortality in the renal mass reduction model. Treatment with candesartan during Ang II-induced hypertension attenuated kidney disease development in MYH9E1841K/E1841K mice. In vitro, isolated primary podocytes from MYH9E1841K/E1841K mice exhibited increased lamellipodia formation and reorganization of F-actin stress fibers. Wound healing assays revealed that MYH9+/+ podocytes had the lowest migration rate, followed by MYH9+/E1841K then MYH9E1841K/E1841K podocytes. In conclusion, the MYH9 E1841K variant alters podocyte cytoskeletal structure and renders podocytes more susceptible to injury after a damaging stimulus.

Background: Collectrin ( Tmem27 ) is a key regulator of blood pressure (BP) and modulator of the bioavailability of nitric oxide (NO) and superoxide. It is highly expressed in the kidney in the proximal tubule (PT), collecting duct, and throughout the vascular endothelium. We reported that collectrin plays a critical role as a chaperone for the reabsorption of all amino acids (AAs) in the PT, and for the uptake of the cationic AA L-arginine (L-Arg) in endothelial cells. Global collectrin knockout ( Tmem27 Y/- ) mice display baseline hypertension (HTN), augmented salt-sensitive hypertension (SSH), and decreased renal blood flow. Objective and Methods: To determine the PT-specific effect of collectrin on BP homeostasis and salt sensitivity, we used the Cre -loxP approach and PEPCK-Cre to generate a mouse line lacking collectrin specifically in the PT-- PEPCK-Cre + Tmem27 Y/Flox mice. PEPCK-Cre - Tmem27 Y/Flox mice were used as control. Radiotelemetry was used to measure BP for 2 weeks at baseline and 2 weeks on high salt diet (HSD). Renal blood flow at baseline and on HSD was measured using contrast enhanced ultrasound in the same mice. Results: Successful deletion of collectrin in the PT was confirmed by assessing mRNA levels using real-time RT-PCR, immunohistochemistry staining of renal tissues using anti-collectrin antibody, and quantitation of protein from kidney cortex by Western analysis. Compared to control PEPCK-Cre - Tmem27 Y/Flox mice (n=6), PEPCK-Cre + Tmem27 Y/Flox mice (n=6) displayed significantly higher systolic BP (SBP) at baseline (120.0 ± 2.5 vs 131.6 ± 2.9 mm Hg; p = 0.014) and after HSD (135.3 ± 2.6 vs 151.5 ± 5.2 mm Hg; p = 0.019). Renal blood flow was not different between groups, at baseline nor after HSD. Conclusion: Collectrin in the PT plays an important role in blood pressure homeostasis and response to sodium intake, independent of renal blood flow. Increasing proximal tubular collectrin activity may be a novel therapeutic strategy for the treatment of hypertension and salt-sensitivity.

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P < 1.25×10−8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension., Author summary Hypertension is a global health problem which affects disproportionally people of African descent. We conducted a genome-wide association study of blood pressure in 31,968 Africans and African Americans to identify genes conferring susceptibility to increased blood pressure. This research identified three novel genomic regions associated with blood pressure which have not been previously reported in studies of other race/ethnicity. Using experimental models, we also showed an altered expression of these genes in kidney tissue in hypertension. These findings provide new evidence for genes influencing hypertension risk and supports the need to study diverse ancestry populations in order to identify biologic factors contributing to hypertension.

Oxidative stress is acknowledged to play a role in kidney disease progression. Genetic variants that affect the capacity to handle oxidative stress may therefore influence the outcome of kidney disease. We examined whether genetic variants of the GSTM1 gene, a member of a superfamily of glutathione S-transferases, influence the course of kidney disease progression in participants of the African American Study of Kidney Disease (AASK) trial. Groups with and without the common GSTM1 null allele, GSTM1(0), differed significantly in the time to a glomerular filtration rate (GFR) event or dialysis ( P = 0.04) and in the time to GFR event, dialysis, or death ( P = 0.02). The hazard ratios (HR) for the time to a GFR event or dialysis in those with two or one null allele relative to those possessing none were 1.88 [95% confidence interval (CI), 1.07 to 3.30, P = 0.03] and 1.68 (95% CI, 1.00 to 2.84, P < 0.05), respectively. For the time to GFR event, dialysis, or death, the HR for two null alleles was 2.06 (95% CI, 1.20 to 3.55, P = 0.01) and for one null allele 1.70 (95% CI, 1.02 to 2.81, P = 0.04). We demonstrated that GSTM1 directly regulates intracellular levels of 4-hydroxynonenal (4-HNE) in vascular smooth muscle cells. Furthermore, we showed that renal 4-HNE levels and GSTM1 are both increased after reduction of renal mass (RRM) in the mouse. We conclude that GSTM1 is normally upregulated in chronic kidney disease (CKD) in a protective response to increased oxidative stress. A genetic variant that results in loss of GSTM1 activity may be deleterious in CKD.

Collectrin ( Tmem27 ) is transmembrane glycoprotein with homology to ACE-2, but lacks any catalytic domain. It plays a key role as a chaperone of amino acid transporters, and is abundantly expressed in the kidney in the proximal tubules and collecting duct. Deletion of collectrin in the mouse results in hypertension (HTN) at baseline and augmented salt-sensitivity that are associated with decreased renal nitric oxide and increased superoxide levels. During high salt diet, renal expression of collectrin is upregulated, suggesting an adaptive homeostatic response to salt loading. Here, we queried whether the expression of collectrin is regulated by angiotensin II (Ang II). Wild-type 129S6 mice were made hypertensive with Ang II osmotic minipump @ 600 ng/kg/min x 2 weeks, and were compared to age-matched untreated WT 129 mice. Shown in Fig. 1 , renal mRNA expression of collectrin is significantly reduced after 2 weeks of Ang II (Panel A). Immunostaining shows collectrin protein level is also significantly diminished to near undetectable level (Panel B). We show for the first time that Ang II regulates the expression of collectrin, suggesting that the action of Ang II on blood pressure may be mediated, in part, through the downregulation of collectrin. Further studies are needed to determine the effect of AT 1 and AT 2 receptor signaling on renal expression of collectrin during Ang II-HTN in vivo.

Adenosine A(1) receptors are inhibitory G-protein coupled receptors that presynaptically regulate neurotransmitter release, but their role in self-regulating adenosine release is not known. In this study, we examined the modulation of evoked adenosine and dopamine efflux by A(1) receptors and studied whether D(1) receptors mediate these effects. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the simultaneous detection of adenosine and dopamine efflux on a subsecond time scale. Short electrical stimulation trains delivered to the substantia nigra (60 pulses, 60 Hz) were used to evoke dopamine and adenosine release in the striatum. The adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 1 mg/kg, i.p.) decreased both adenosine and dopamine efflux, although the effect for adenosine occurred more quickly than for dopamine. The A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 6 mg/kg, i.p.) increased stimulated adenosine release. The effects of CPA were partially attenuated by the dopamine D(1) receptor antagonist SCH-23390. Thus, A(1) and D(1) receptors have a synergistic interaction that modulates both stimulated adenosine and dopamine. The decrease in adenosine is not a downstream effect of lowered dopamine release, as decreasing dopamine synthesis and release with α-methyl-p-tyrosine or increasing release with haloperidol had no effect on adenosine release. This study shows that A(1) receptors have some characteristics of an autoreceptor, including self-regulation of adenosine release.

[This corrects the article DOI: 10.1371/journal.pgen.1006728.].

Background: Early and non-invasive biomarkers of kidney damage are needed to identify hypertensive patients at risk for kidney damage. Urinary micropaticles (UMPs) have gained significant attention as potential novel biomarkers for kidney damage, and have already been identified in pre-albuminuric diabetic glomerular injury. These vesicles are less than 1 micron in size and carry markers of the parent cell. We hypothesized that podocyte derived UMPs are elevated in angiotensin II-induced hypertension (HTN) Methods: Primary podocytes were isolated and grown in culture. Wild-type mice were treated with AII (400ng/kg/min) via mini-osmotic pumps. Untreated WT mice served as controls. 24 hour urines were collected after 5 days of AII treatment. Enumeration and phenotyping of MPs was done of podocyte culture supernatant and urine. Podocalyxin (Pcal), podoplanin (Ppla) and annexin 5 (AV) were used as surface markers. Result: Pcal and Ppla positive MPs as well as AV positive and negative MPs were detectable in supernatant from primary podocyte cultures. Compared to untreated controls (n=3), AII treated mice (n=2) had an increase in systolic blood pressure (SBP) by 33 mmHG (p=0.02). Despite similar urinary albumin/creatinine ratios between groups, there was a trend of higher levels of total numbers of Ppla and Pcal positive MPs in hypertensive mice compared to untreated (Figure 1). In addition, Annexin negative but Ppla and Pcal positive MPs were also numerically higher in hypertensive mice. In conclusion, podocyte derived UMPs are detectable in AII HTN. These findings need to be confirmed in a larger group of animals. UMPs can be potential marker for kidney end-organ damage in HTN.

Collectrin ( Tmem27 ), an ACE-2 homologue and a chaperone of amino acid transporters, is expressed in the endothelium and renal epithelia, including the proximal tubules and collecting duct. We previously reported that collectrin-deficient (KO) mice exhibit hypertension (HTN) at baseline and augmented salt-sensitivity associated with impaired endothelial-dependent relaxation and a rightward shift of the pressure-natriuresis relationship. To determine the effect of renal vs. extra-renal collectrin on blood pressure (BP) regulation, we performed renal cross-transplantation studies (at the Duke O’Brien Center), then treated with normal salt (NSD) and high salt (HSD) diets, in the following groups (n = 3-5/group): 1) wild-type (WT) mice with WT kidney (WT WT), 2) collectrin KO mice with WT kidney (WT KO), and 3) WT mice with collectrin KO kidney (KO WT). Collectrin KO WT group displayed a trend towards higher baseline systolic BP (SBP, mmHg); however, under HSD, collectrin KO WT had significantly higher SBP (WT WT 148.9, WT KO 151.6, KO WT 168.6, p = 0.037). To determine the renal endothelial and epithelial contribution of collectrin in BP regulation, we assessed renal blood flow (RBF) by contrast-enhanced ultrasound and renal sodium transporters and channels by immunoblotting. KO mice displayed significantly reduced total, cortical and medullary RBF under both NSD and HSD (n ≥ 11 each, p ≤ 0.01). Moreover, at baseline, compared to WT mice (n = 4), KO mice (n = 5) displayed significantly elevated abundance of NHE3 (p = 0.002), activated NCC (NCC-pS71) (p = 0.004) and ENaC alpha (p = 0.03). We next conducted a candidate gene association study in the HyperGen cohort (1,270 white participants, 50% men), using intronic SNPs in the ACE2 , TMEM27 and CA5BP1 genes on the X chromosome. Only 2 SNPs, rs6629114 and rs41492646, both of TMEM27 and in perfect linkage disequilibrium (r 2 =1.0), were associated with diastolic BP (DBP). Meta-analysis of men and women showed this was statistically significant, (p=0.04 for both), with stronger effects in men (p=0.028). In conclusion, deletion of collectrin alters renal hemodynamics and epithelial sodium handling to favor sodium reabsorption. Collectrin is associated with DBP in human subjects.

The immune system plays a major role in animal models of hypertension (HTN) and end-organ damage. However, few studies have assessed the role of lymphoid organs in the pathogenesis of HTN. We have shown previously that prior splenectomy (SPLX) significantly alters tissue inflammation; however the effect of SPLX on HTN remains unclear. Therefore, the objective of the current study is to determine whether prior SPLX influences the development of HTN in 2 different mouse models. Mice underwent SPLX or sham surgery 7 days prior to the induction of HTN using angiotensin-II (AngII, 400ng/kg*min s.c.) or nitric oxide synthase inhibition using L-NAME (30mg/kg*d in drinking water). Systolic blood pressure (SBP) was measured by tail-cuff manometer daily and mice were euthanized 14 days after induction of HTN. Heart weight/body weight (H/BW) ratios were calculated and kidney leukocyte infiltration was analyzed by flow cytometry. Mice with prior SPLX+AngII had significantly lower (P=0.03) SBP at both week 1 (148±7) and week 2 (135±7) as compared to Sham+AngII (174 and 173±7mmHg, n=5). Similarly, SPLX+AngII mice had significantly smaller (P=0.007) H/BW (4.3±0.3) as compared to Sham+AngII treated mice (5.2±0.4mg/g BW). Interestingly, no difference was observed in renal CD45+ (9.8±3 vs 10.6±3x105 cells/g, P=0.64) or CD3+ T-cell infiltration (8.8±0.2 vs 9.6±0.1x104 cells/g , P=0.64) between the Sham+AngII and SPLX+AngII treated mice, respectively (n=4/5). Furthermore, SPLX did not appear to influence the development of L-NAME HTN. SPLX+L-NAME mice had similar (P=0.84) SBP (145±4mmHg) as the Sham+L-NAME group (146±4mmHg, n=6) after 2 weeks. Relative heart weights were also similar (P=0.45) between SPLX+L-NAME (4.9±0.2) and Sham+L-NAME treated mice (4.8±0.3mg/g BW). Our data suggests that the full pressor response to AngII is dependent on the spleen. However, the effect of the spleen appears to be independent of renal inflammation. Moreover, the protective effect of the spleen is specific to AngII-dependent HTN and does not appear to be generalizable to all mouse models of hypertension. Further studies are needed to understand the physiological link between lymphoid organs (such as the spleen), renal inflammation, and the development of chronic HTN.

Nitric oxide (NO), a cell messenger for activating soluble guanylyl cyclase, is produced by activation of the enzyme NO synthase (NOS) in a wide variety of tissues, including the central nervous system. We have previously demonstrated that inhibition of NOS decreased the minimum alveolar anesthesia concentration (MAC) for isoflurane anesthesia. Moving more distally in the NOS-guanylyl cyclase signaling pathway, we investigated the effects of the specific soluble guanylyl cyclase inhibitor ODQ, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, on anesthetic requirements. The effect of ODQ on the MAC of isoflurane anesthesia was investigated in Sprague-Dawley rats while concurrently monitoring the their arterial blood pressure and heart rate. After determining control MAC, ODQ 20-500 mg/kg was administered intraperitoneally 30 min before re-determining MAC in the presence of the soluble guanylyl cyclase inhibitor. In one series, the effect of 250 mg/kg of ODQ on neuronal cyclase guanosine monophosphate production was determined by microdialysis. ODQ produced a statistically significant, dose-dependent decrease from isoflurane control MAC (maximal effect 52.4% +/- 2.7%). No ceiling effect was observed over the dose-range studied. This reduction in isoflurane MAC was not accompanied by changes in either heart rate or blood pressure. Inhibition of the NOS-guanylyl cyclase signaling pathway decreased the MAC for isoflurane, which suggests that inhibition of this pathway may play a role in the anesthetic state. The MAC reduction by the soluble guanylyl cyclase inhibitor ODQ was devoid of any significant hemodynamic effects. The current findings, along with the previous observations that structurally distinct NOS inhibitors and the nonspecific soluble guanylyl cyclase inhibitor methylene blue decrease the MAC for volatile anesthetics, support that this is an effect specific to the NOS-guanylyl cyclase signaling pathway.

Introduction: We previously reported a locus on mouse chromosome 11 (chrom 11) linked to development of hypertension (HTN) after sub-total nephrectomy (Nx). To begin fine mapping, we used the 129S6 (129) susceptible strain and the C57BL/6 (B6) resistant strain to generate 4 reciprocal congenic lines carrying ~ 2-LOD intervals of the linked locus for recombinant progeny testing. The intervals are defined by microsatellite markers 1-5 and markers 1-6, corresponding to regions defined by markers D11Mit2 and D11Mit177, and D11Mit2 and D11Mit285, respectively. Through brother-sister matings, the 4 lines are as follows: 1) 129 background, homozygous for B6 segment defined by markers 1-5 (129 Chrom11:1-5ofB6 ), 2) 129 background, homozygous for 129 segment defined by markers 1-5 (129 Chrom11:1-5of129 ), 3) B6 background, homozygous for 129 segment defined by markers 1-6 (B6 Chrom11:1-6of129 ) and 4) B6 background, homozygous for B6 segment defined by markers 1-6 (B6 Chrom11:1-6ofB6 ). Methods: Sub-total Nx was performed on male and female congenic mice at ~ 8 weeks of age. Beginning 4 weeks after surgery, using tail cuff manometer, mice were trained for 2 weeks, after which systolic blood pressure (SBP) was recorded daily for 2 weeks. Results: Table 1 summarizes the average SBP of the congenic lines. Conclusion: These findings confirm the effect of this chrom 11 region is dependent on the 129 genetic background, and suggest that interactions between this 129 locus and other loci in the 129 genome are necessary for the development of HTN after nephron mass reduction. Moreover, the influence of this region on chrom 11 appears to be dependent on sex.

Collectrin (Tmem27), an ACE-2 homologue and a chaperone of amino acid transporters, is expressed in the endothelium throughout the vasculature and in the renal epithelia, including the proximal tubules and collecting duct. Its renal expression is upregulated after reduction of nephron mass and during salt-sensitive hypertension (SSH). These observations raise the question of how collectrin might be functionally integrated in the ACE family in the context of blood pressure (BP) regulation. We previously reported that, on a permissive genetic background, collectrin-deficient (KO) mice have baseline hypertension (HTN) that is ameliorated by supplementation with L-arginine (L-Arg). Furthermore, they have augmented SSH that is corrected by TEMPOL, a superoxide dismutase mimetic. These phenotypes are associated with impaired cellular uptake of L-Arg, impaired endothelial-dependent relaxation, decreased levels of endothelial nitric oxide synthase dimerization (eNOS), and a rightward shift of the pressure-natriuresis relationship. Here, we demonstrate that the activity or dimerization of neuronal NOS (nNOS) in the renal medulla is also significantly decreased in KO mice. To determine the effect of renal versus extra-renal collectrin on BP regulation, we performed renal cross-transplantation (XTP) studies (through a service provided by the Duke O’Brien Center), under normal and high salt conditions, in the following groups: 1) wild-type (WT) mice with WT kidney (WT -> WT), 2) collectrin KO mice with WT kidney (WT -> KO), and 3) WT mice with collectrin KO kidney (KO -> WT). BP was measured by radiotelemetry. Collectrin KO -> WT group displayed a trend towards higher baseline systolic BP (SBP, mm Hg), but did not reach significance (WT -> WT 137.3, WT -> KO 133.5, KO -> WT 142.4, p = not significant, n = 3-5 per group). By paired analysis, all groups had significantly higher SBP on high salt diet (HSD), compared to normal salt diet. However, collectrin KO -> WT group had a statistically significantly higher SBP under HSD (WT -> WT 148.9, WT -> KO 151.6, KO -> WT 168.6, p= 0.037, one way ANOVA). In conclusion, collectrin may regulate BP by influencing renal hemodynamics and/or epithelial sodium handling through its central role in mediating cellular L-Arg uptake.

Background— Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. Methods and Results— Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates l -arginine uptake and plasma membrane levels of CAT1 and y + LAT1 amino acid transporters in endothelial cells. Treatment with l -arginine modestly lowers blood pressure of collectrin knockout mice. Conclusions— Collectrin is a consequential link between the transport of l -arginine and endothelial nitric oxide synthase uncoupling in hypertension.

GSTM1 gene encodes an enzyme that belongs to a superfamily of Glutathione-S-transferases that metabolize xenobiotic, and a broad range of reactive oxygen species and electrophilic compounds formed as secondary metabolites during the oxidative stress. There is a general consensus that oxidative stress contributes to development of hypertension (HTN). We hypothesized that Gstm1 may influence blood pressure (BP) regulation through its central role in modulating redox homeostasis. Therefore the objective of this study was to determine the response of Gstm1 knockout (KO) mice to 2 models of hypertension that are mediated in part by oxidative stress: 1) high salt model, and 2) angiotensin II model. Methods: Gstm1-/- (KO) mice were generated on the 129S6 background through conventional gene targeting strategy. Hypertension was induced using high salt diet (HSD, 6% NaCl) for 2 weeks or by infusion of angiotensin II (Ang II) via mini-osmotic pump at dose 1000 ng/kg/min for 3 weeks. Systolic BP (SBP) at baseline and under high salt condition was measured using radio-telemetry. Tail-cuff manometry was used to measure SBP in Ang II induced hypertension. Urinary isoprostane (index of oxidative stress) was measured using ELISA assay. For all studies, n ≥ 4 per group. Results: By radiotelemetry, Gstm1 KO mice display a modest but significantly higher baseline SBP compared to their wild type (WT) littermates: KO: 138.8 ± 1.3; WT: 132.1 ± 1.1, p < 0.01. On high salt diet, both WT and KO mice displayed salt sensitivity, and the difference in SBP was maintained between the two genotypes (KO: 148.7 ± 1.6; WT: 143.8 ± 1.3, p < 0.05). Urinary isoprostane (ng/100 mg of body weight) is significantly higher in KO mice than WT mice at baseline as well as during HSD (baseline: KO 15.1 ± 2.9; WT: 8.0 ± 0.8, p < 0.04; HSD: KO 27.2 ± 2.0; WT: 21.0 ± 1.3, p = 0.04). Angiotensin II infusion augments the difference in SBP between WT and KO mice (185.0 ± 3.1; WT mice 175.4 ± 1.4, p < 0.05). Conclusion: Loss of Gstm1 results in elevated baseline blood pressure and augmented Ang II induced hypertension. The altered blood pressure homeostasis from deficiency in GSTM1 enzyme may be mediated by a decreased capacity to handle oxidative stress.

There is a general consensus that oxidative stress is a factor in the progression of chronic kidney disease (CKD). Hence, genetic variants that affect the capacity to handle oxidative stress may influence the outcomes of CKD. One important class of enzymes that has evolved to combat the damaging effects of reactive oxygen species is the glutathione-S-transferases. In particular, the μ class isoform 1 (GSTM1) has emerged as a potential modifier of multiple chronic diseases in humans. Approximately 30%-50% of humans are completely deficient of the GSTM1 enzyme because of homozygous inheritance of the GSTM1 null allele, GSTM1(0). We have identified the GSTM1 gene as a modifier of disease progression in hypertensive nephrosclerosis (HN). In an ancillary study of the African American (AA) Study of Hypertension and Kidney Disease (AASK) Trial, we reported that participants carrying one ( 1/0 ) or two ( 0/0 ) null alleles had 1.7- and 2-fold higher risk of the composite outcome of a 50% decline in the glomerular filtration rate (GFR), dialysis, or death relative to those with two active alleles. Here, the objective of our study was to determine the consequence of deletion of Gstm1 on the course of chronic kidney disease induced by reduction of renal mass (RRM) model in mice. We generated Gstm1-/- (KO) mice on the 129S6 background through conventional gene targeting strategy. By radiotelemetry, Gstm1 KO mice displayed a modest but significantly higher baseline systolic blood pressure (SBP) compared to their wild type (WT, Gtsm1 +/+ ) littermates: KO (n = 5): 138.8 ± 1.3; WT (n = 5): 132.1 ± 1.1, p < 0.01. Baseline urinary isoprostane (ng/100 mg of body weight) was significantly higher in KO mice than WT mice (15.1 ± 2.9; WT: 8.0 ± 0.8, p < 0.04). Four weeks after sub-total nephrectomy, Gstm1 KO mice developed significantly more severe hypertension than WT mice. The average SBP over a 2 week recording by radiotelemetry was 154.0 ± 3.2 mm Hg in KO mice (n = 5), and 142.3 ± 4.2 in WT mice (n = 3), p < 0.01. The effects of deletion of Gstm1 on kidney function and histopathology are under investigation. In conclusion, loss of GSMT1 increases oxidative stress and exaggerates hypertension in the murine model of chronic kidney disease.

The MYH9 gene encodes the nonmuscle myosin heavy chain IIA. Recent genome-wide association studies (GWAS) demonstrated that intronic variants of the MYH9 gene are associated with non-diabetic kidney disease and hypertension-attributed ESRD in African-Americans. However, the causal functional variants of MYH9 have remained elusive. Rare autosomal-dominant missense mutations in MYH9 cause macrothrombocytopenia, and are occasionally associated with development of nephropathy. The E1841K mutation is the most common MYH9 missense mutation and has been associated with nephropathy in some carriers. To determine the role of the MYH9 E1841K mutation in the development of kidney disease, we studied a mouse line carrying the E1841K mutation. On a mixed genetic background (129S6 and C57BL/6), there was no difference in systolic blood pressure (SBP) between wild-type (WT, -/-) and mice with the E1841K mutation (designated as E1841K (+) : +/- and/or +/+) at baseline or after high salt diet (HSD,6 % NaCl). There was also no difference in albumin/creatinine (A/C ratio) at baseline or after HSD between groups. We next assessed the effects of the E1841K mutation in response to angiotensin II (Ang II) infusion @1000 ng/kg/min for 28 days via mini-osmotic pump. Despite similar increases in SBP after Ang II (WT 197 ± 4 mm Hg, n=15; E1841K +/- 197 ± 3 mm Hg, n=9; E1841K +/+ 199 ± 5 mm Hg; n=6), E1841K(+) mice displayed severe proteinuria in a dose-dependent manner (A/C ratio (ug/mg): WT 806±94, E1841K +/- 1341±293, E1841K +/+ 2209± 366, p=0.001, ANOVA ). By light microscopy, WT mice had only mild mesangial expansion, whereas E1841K +/+ mice had focal segmental glomerulosclerosis and large amounts of proteinaceous casts. By electron microscopy, WT mice had intact podocytes, whereas, E1841K +/+ mice displayed severe podocyte foot process effacement/fusion. Treatment of E1841K (+) mice with candesartan, an angiotensin type 1 receptor (AT 1 R) inhibitor, significantly prevented Ang II-induced hypertension and proteinuria ( E1841K (+) (n=6; +/- (n=4) and +/+ (n=2)): SBP = 138 ± 4 mm Hg, A/C ratio = 111 ± 47 ug/mg; p ≤ 0.0001 vs. untreated), and ameliorated podocyte foot process effacement in E1841K +/+ mice. In conclusion, the MYH9 E1841K mutation predisposes to AT 1 R mediated podocyte injury.

Collectrin, an ACE-2 homologue, plays a major role in amino acid transport in renal epithelial cells. In the rodent kidney, the expression of collectrin is upregulated after reduction of nephron mass and during salt-sensitive hypertension. These observations raise the question of how collectrin might be functionally integrated in the ACE family in the context of blood pressure (BP) regulation. We previously reported that, on a susceptible genetic background, collectrin-deficient (KO) mice have elevated baseline systolic blood pressure (SBP) and impaired endothelium-dependent relaxation (EDR) of thoracodorsal arteries (TDA). Treatment with L-NAME, a nitric oxide synthase (NOS) inhibitor, normalized their BP and EDR of TDA. Here, to determine whether collectrin plays a role in salt-sensitivity (SS), we fed mice a high salt diet (HSD) for 2 weeks. By radiotelemetry, KO mice displayed augmented salt-sensitivity (SS) (Normal salt (NS): Wild-type (WT) 133 ± 1 mm Hg, KO 141 ± 3 mm Hg; HSD: WT 141 ± 2 mm Hg, KO 155 ± 3 mm Hg; change in SBP: WT 7.8 ± 1.4 mm Hg, KO 14.4 ± 1.5 mm Hg; p ≤ 0.02), and lower urinary sodium excretion (UNa) during the first 3 days of HSD (mean UNa over 3 days: KO: 1980 ± 193 μmol/24h, WT: 2650 ± 98 μmol/24h, p = 0.006, ANOVA; n ≥ 6). The impaired pressure natriuresis in KO mice was associated with altered urinary excretion of 8-isoprostane F 2 α (WT 3.0 ± 0.67 ng/mg, KO 7.7 ± 1.5 ng/mg creatinine, p = 0.008) and nitrates/nitrites (WT 10.2 ± 1.4 nmol/day, KO 6.2 ± 0.61 nmol/day, p = 0.03, n ≥ 4 each). We previously reported that KO mice have increased state of endothelial NOS (eNOS) uncoupling. Here, by immunostaining, we found that collectrin is expressed in endothelial cells (ECs). We next queried whether collectrin affects L-arginine (L-Arg) transport in ECs. After isolation from the pulmonary artery, primary ECs from KO mice displayed impaired [ 3 H]L-Arg uptake (WT 2.00 ± 0.19, KO 0.90 ± 0.06 pmol/mg protein/min, p=0.01) To establish direct cause and effect, we over-expressed collectrin in primary human coronary ECs which resulted in increased [ 3 H]L-Arg uptake (Control 4.20 ± 0.32; Overexpression 6.60 ± 0.59 pmol/mg protein/min, p < 0.003). In conclusion, collectrin may regulate BP and SS by modulating the balance of NO and SO through its role in L-Arg transport in ECs.

Adenosine is an endogenous byproduct of metabolism that regulates cerebral blood flow and modulates neurotransmission. Four receptors, with affinities ranging from nanomolar to micromolar, mediate the effects of adenosine. Real-time measurements are needed to understand the extracellular adenosine concentrations available to activate these receptors. In this study, we measured the subsecond time course of adenosine efflux in the caudate-putamen of anesthetized rats after a 1 s, high-frequency stimulation of dopamine neurons in the substantia nigra. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for simultaneous detection of adenosine and dopamine, which have different oxidation potentials. While dopamine was immediately released after electrical stimulation, adenosine accumulation was slightly delayed and cleared in about 15 seconds. The concentration of adenosine measured after electrical stimulation was 0.94 +/- 0.09 microM. An adenosine kinase inhibitor, adenosine transport inhibitor, and a histamine synthetic precursor were used to pharmacologically confirm the identity of the measured substance as adenosine. Adenosine efflux was also correlated with increases in oxygen, which occur because of changes in cerebral blood flow. This study shows that extracellular adenosine transiently increases after short bursts of neuronal activity in concentrations that can activate receptors.

Background. Glutamate transporters [also named excitatory amino acid transporters (EAATs)] bind and take up extracellular glutamate, a major excitatory neurotransmitter, and can regulate glutamatergic neurotransmission in synapses. As anaesthesia is proposed to be induced by enhancing inhibitory neurotransmission, inhibiting excitatory neurotransmission, or both we hypothesize that inhibition of EAAT activity can increase the anaesthetic requirement. Methods. The minimum alveolar concentration (MAC, the anaesthetic concentration required to suppress movement in response to noxious stimulation in 50% of subjects) for isoflurane was determined in adult male Sprague–Dawley rats after intrathecal administration of EAAT inhibitors. Results. Application of DL-threo-b-benzyloxyaspartate, a selective EAAT inhibitor, dose- and time-dependently increased the MAC for isoflurane. The MAC was 109 (1)% and 116 (4)% of the baseline, respectively, for 0.2 and 0.4 mmol of DL-threo-b-benzyloxyaspartate 15 min after the injection of the drug (n=5, P

We have recently discovered a novel class of compounds that have oral general anesthetic activity, potent anticonvulsant activity, and minimal hemodynamic effects. The 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide (1) demonstrated potent ability to reduce the minimum alveolar concentration (MAC) of isoflurane, with no effects on heart rate or blood pressure at therapeutic concentrations. Analogue 1 also had potent oral anticonvulsant activity against maximal electroshock (MES) and subcutaneous metrazol (scMET) models with a therapeutic index of 10 for MES activity. In this study, we further synthesized nine new racemic analogues and evaluated these compounds for effects on isoflurane MAC reduction and blood pressure. Preliminary data demonstrate potent reduction in the isoflurane MAC for two new compounds. Current mechanistic studies were unrevealing for effects on voltage-gated ion channels as a putative mechanism. Liposomal partitioning studies using (19)F NMR reveal that the aromatic region partitions into the core of the lipid. This partitioning correlated with general anesthetic activity of this class of compounds. Further, compound 1 was used at a concentration of 1 mM and slightly enhanced GABA(A) current in hippocampal neurons at 10 microM. Altogether, 3,3,3-trifluoro-2-hydroxy-2-phenyl-propionamide exhibited excellent oral general anesthetic activity and appears devoid of significant side effects (i.e., alterations in blood pressure or heart rate).