We examined the impact of sex on high fat diet induced renal alterations in Dahl salt sensitive and Sprague Dawley rats. In Dahl rats, high fat diet (60% kcal from fat for 24–26 weeks starting at weaning) significantly and equally increased blood pressure in males and females when compared to rats fed a control diet (10% kcal from fat). Male Dahl rats on high fat diet exhibited progressive renal histological injury, and moderately increased renal macrophage infiltration at 10 and 24 weeks of feeding when compared to males on control diet. Female Dahl rats had lower grade renal injury and less macrophage infiltration (except at 17 weeks) than males regardless of diet. Male Dahl rats on both diets showed progressively increasing numbers of renal T-cells, a pattern not observed in females. High fat diet per se did not significantly affect renal T-cell number. Male Dahl rats had lower renal T-reg cell ratio than females at 24 weeks. Renal macrophage and T-cell infiltrations were highly correlated to final MAP levels in males but not in females. Sprague Dawley rats fed high fat diet were normotensive without significant renal injury/inflammation after 24 weeks of feeding. In summary, high fat diet feeding fails to increase arterial blood pressure in Sprague Dawley rats, but strongly promotes hypertension in both male and female Dahl salt sensitive rats. Only Dahl males, however, exhibited blood pressure-associated renal inflammation and injury. Maintenance of T-reg ratio may protect against hypertension associated renal injury/inflammation but not high fat diet-induced hypertension.

-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon. Am J Physiol Gastrointest Liver Physiol 311: G210 -G220, 2016. First published June 10, 2016; doi:10.1152/ajpgi.00085.2016.-We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high-fat diet (HFD). We used wild-type (WT) mice and mice lacking the ␤1-subunit of the BK channel (BK␤1 Ϫ/Ϫ ). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons, and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-L-arginine (NLA) was less effective at inhibiting relaxations in HFD compared with control diet (CD) WT mice (11 vs. 37%, P Ͻ 0.05). SMCs from HFD WT mice had depolarized membrane potentials (Ϫ47 Ϯ 2 mV) and continuous action potential firing compared with CD WT mice (Ϫ53 Ϯ 2 mV, P Ͻ 0.05), which showed rhythmic firing. SMCs from HFD or CD fed BK␤1 Ϫ/Ϫ mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BK␤1 Ϫ/Ϫ mice. Migrating motor complexes were disrupted in colons from BK␤1 Ϫ/Ϫ mice and HFD WT mice. BK channel ␣-subunit protein and ␤1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD-induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability, and colonic motility by promoting oxidative stress, loss of nitrergic neurons, and SMC BK channel dysfunction. enteric nervous system; large conductance calcium-activated K ϩ channel; gastrointestinal motility; obesity A HIGH-FAT DIET (HFD) can cause obesity (35) that leads to an increased risk for the development of type 2 diabetes (13,17,33), heart disease (29, 33), and arthritis (25). Obesity is also associated with gastrointestinal (GI) dysmotility (15,35,41,63). GI motility is controlled largely by interactions between enteric neurons, interstitial cells of Cajal (ICCs), and smooth muscle cells (SMCs) but how obesity alters the function of these cells is poorly understood.A HFD is associated with increased oxidative stress and altered function of myenteric neurons in rodent models of obesity (11,60,61). The neuropathological changes that cause GI dysmotility vary among animal models and location in the GI tract (11). Inhibitory motor neurons that express nitric oxide synthase (NOS) are most susceptible to damage caused by obesity (3, 10, 11). These neurons are crucial for coordination of SMC relaxation (23, 41). Thus the disruption of nitrergic neuron function is a mechanism that contributes to HFDinduced GI dysmotility.Enteric motorneurons regulate propulsive motility patterns while GI SMCs have rhythmic electrical activ...

Objective Reduced expression or increased degradation of BK (large conductance Ca2+-activated K+) channel β1-subunits has been associated with increased vascular tone and hypertension in some metabolic diseases. The contribution of BK channel function to control of blood pressure (BP), heart rate (HR) and vascular function/structure was determined in wild-type and BK channel β1-subunit knockout mice fed a high-fat or control diet. Methods and Results After 24 weeks of high-fat diet, wild-type and BK β1-knockout mice were obese, diabetic, but normotensive. High-fat-BK β1-knockout mice had decreased HR while high-fat-wild-type mice had increased HR compared with mice on the control diet. Ganglion blockade caused a greater fall in BP and HR in high-fat mice than in mice on the control diet. β1-adrenergic receptor blockade reduced BP and HR equally in all groups. α1-adrenergic receptor blockade decreased BP in high-fat-BK β1-knockout mice only. Echocardiographic evaluation revealed left ventricular hypertrophy in high-fat-BK β1-knockout mice. Although under anesthesia, mice on a high-fat had higher absolute stroke volume and cardiac output, these measures were similar to control mice when adjusted for body weight. Mesenteric arteries from high-fat-BK β1-knockout mice had higher norepinephrine reactivity, greater wall thickness and collagen accumulation than high-fat-wild-type mesenteric arteries. Compared with control-wild-type mesenteric arteries, high-fat-wild-type mesenteric arteries had blunted contractile responses to a BK channel blocker, although BK α-subunit (protein) and β1-subunit (mRNA) expression were unchanged. Conclusions BK channel deficiency promotes increased sympathetic control of blood pressure, and vascular dysfunction, remodeling and fibrosis, but does not cause hypertension in high-fat fed mice.

Large conductance Ca2+‐activated K+ (BK) channels consist of pore‐forming α‐ and accessory β‐subunits. There are four β‐subunit subtypes (β1–β4), BK β1‐subunit is specific for smooth muscle cells (SMC). Reduced BK β1‐subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1‐subunit reduces channel activity and increases SMC contractility. Several anti‐BK β1‐subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1‐subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1‐subunit enriched tissues (mesenteric arteries and colons) and non‐SM tissue (cortex of kidney) from wild‐type (WT) and BK β1‐KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1‐KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1‐subunit. The absence of BK β1‐subunit mRNA expression in arteries, colons, and kidneys from BK β1‐KO mice was confirmed by RT‐PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1‐subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses.

We determined the contribution of vascular large conductance Ca2+-activated K+ (BK) and L-type Ca2+ channel dysregulation to exaggerated mortality in cecal ligation/puncture (CLP)-induced septic BK channel β1-subunit knockout (BK β1-KO, smooth muscle specific) mice. CLP-induced hemodynamic changes and mortality were assessed over 7 days in wild-type (WT) and BK β1-KO mice that were either untreated, given volume resuscitation (saline), or saline + calcium channel blocker nicardipine. Some mice were euthanized 24 h post-CLP to measure tissue injury and vascular and immune responses. CLP-induced hypotension was similar in untreated WT and BK β1-KO mice, but BK β1-KO mice died sooner. At 24 h post-CLP (mortality latency in BK β1-KO mice), untreated CLP-BK β1-KO mice showed more severe hypothermia, lower tissue perfusion, polymorphonuclear neutrophil infiltration-independent severe intestinal necrosis, and higher serum cytokine levels than CLP-WT mice. Saline resuscitation improved survival in CLP-WT but not CLP-BK β1-KO mice. Saline + nicardipine-treated CLP-BK β1-KO mice exhibited longer survival times, higher tissue perfusion, less intestinal injury, and lower cytokines versus untreated CLP-BK β1-KO mice. These improvements were absent in treated CLP-WT mice, although saline + nicardipine improved blood pressure similarly in both septic mice. At 24 h post-CLP, BK and L-type Ca2+ channel functions in vitro were maintained in mesenteric arteries from WT mice. Mesenteric arteries from BK β1-KO mice had blunted BK/enhanced L-type Ca2+ channel function. We conclude that vascular BK channel deficiency exaggerates mortality in septic BK β1-KO mice by activating L-type Ca2+ channels leading to blood pressure-independent tissue ischemia.