Deletion, but not heterozygosity, of eNOS raises blood pressure and aggravates nephropathy in BTBR ob/ob mice.

INTRODUCTION: ob/ob mice are a leptin-deficient type 2 diabetes mellitus model, which, on a BTBR background, mimics glomerular pathophysiology of diabetic nephropathy (DN). Since leptin deficiency reduces blood pressure (BP), and endothelial nitric oxide synthase (eNOS) lowers BP and is kidney protective, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. METHODS: Six experimental groups included littermate male and female BTBR ob/ob or wild-type for ob (control) as well as wild-type (WT), heterozygote (HET) or knockout (KO) for eNOS. Systolic BP (by automated tail-cuff) and GFR (by FITC sinistrin plasma kinetics) were determined in awake mice at 27-30 weeks of age followed by molecular and histological kidney analyses. RESULTS: Male and female ob/ob WT presented hyperglycemia and larger body and kidney weight, GFR, glomerular injury, and urine albumin to creatinine ratio (UACR) despite modestly lower BP vs control WT. These effects were associated with higher tubular injury score and renal mRNA expression of NGAL only in males, whereas female ob/ob WT unexpectedly had lower KIM-1 and COL1A1 expression vs control WT, indicating sex differences. HET for eNOS did not consistently alter BP or renal outcome in control or ob/ob. In comparison, eNOS KO increased BP (15-25 mmHg) and worsened renal markers of injury, inflammation and fibrosis, GFR, UACR, and survival rates, as observed in control and, more pronounced, in ob/ob mice and independent of sex. CONCLUSIONS: Deletion, but not heterozygosity, of eNOS raises blood pressure and aggravates nephropathy in BTBR ob/ob mice.

SGLT2 inhibitor dapagliflozin protects the kidney in a murine model of Balkan nephropathy.

Proximal tubular uptake of aristolochic acid (AA) forms aristolactam (AL)-DNA adducts, which cause a p53/p21-mediated DNA damage response and acute tubular injury. Recurrent AA exposure causes kidney function loss and fibrosis in humans (Balkan endemic nephropathy) and mice and is a model of (acute kidney injury) AKI to chronic kidney disease (CKD) transition. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. C57BL/6J mice (15-wk-old) were administered vehicle or AA every 3 days for 3 wk (10 and 3 mg/kg ip in females and males, respectively). Dapagliflozin (dapa, 0.01 g/kg diet) or vehicle was initiated 7 days prior to AA injections. All dapa effects were sex independent, including a robust glycosuria. Dapa lowered urinary kidney-injury molecule 1 (KIM-1) and albumin (both normalized to creatinine) after the last AA injection and kidney mRNA expression of early DNA damage response markers (p53 and p21) 3 wk later at the study end. Dapa also attenuated AA-induced increases in plasma creatinine as well as AA-induced up-regulation of renal pro-senescence, pro-inflammatory and pro-fibrotic genes, and kidney collagen staining. When assessed 1 day after a single AA injection, dapa pretreatment attenuated AL-DNA adduct formation by 10 and 20% in kidney and liver, respectively, associated with reduced p21 expression. Initiating dapa application after the last AA injection also improved kidney outcome but in a less robust manner. In conclusion, the first evidence is presented that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.NEW & NOTEWORTHY Recurrent exposure to aristolochic acid (AA) causes kidney function loss and fibrosis in mice and in humans, e.g., in the form of the endemic Balkan nephropathy. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. Here we provide the first evidence in a murine model that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.

Aristolochic acid-induced nephropathy is attenuated in mice lacking the neutral amino acid transporter B0AT1 (Slc6a19).

B0AT1 (Slc6a19) mediates absorption of neutral amino acids in the small intestine and in the kidneys, where it is primarily expressed in early proximal tubules (S1-S2). To determine the role of B0AT1 in nephropathy induced by aristolochic acid (AA), which targets the proximal tubule, littermate female B0AT1-deficient (Slc6a19-/-), heterozygous (Slc6a19+/-), and wild-type (WT) mice were administered AA (10 mg/kg ip) or vehicle every 3 days for 3 wk, and analyses were performed after the last injection or 3 wk later. Vehicle-treated mice lacking Slc6a19 showed normal body and kidney weight and plasma creatinine versus WT mice. The urinary glucose-to-creatinine ratio (UGCR) and urinary albumin-to-creatinine ratio (UACR) were two to four times higher in vehicle-treated Slc6a19-/- versus WT mice, associated with lesser expression of early proximal transporters Na+-glucose cotransporter 2 and megalin, respectively. AA caused tubular injury independently of B0AT1, including robust increases in cortical mRNA expression of p53, p21, and hepatitis A virus cellular receptor 1 (Havcr1), downregulation of related proximal tubule amino acid transporters B0AT2 (Slc6a15), B0AT3 (Slc6a18), and Slc7a9, and modest histological tubular damage and a rise in plasma creatinine. Absence of B0AT1, however, attenuated AA-induced cortical upregulation of mRNA markers of senescence (p16), inflammation [lipocalin 2 (Lcn2), C-C motif chemokine ligand 2 (Ccl2), and C-C motif chemokine receptor 2 (Ccr2)], and fibrosis [tissue inhibitor of metallopeptidase 1 (Timp1), transforming growth factor-ß1 (Tgfb1), and collagen type I-α1 (Col1a1)], associated with lesser fibrosis staining, lesser suppression of proximal tubular organic anion transporter 1, restoration of Na+-glucose cotransporter 2 expression, and prevention of the AA-induced fivefold increase in the urinary albumin-to-creatinine ratio observed in WT mice. The data suggest that proximal tubular B0AT1 is important for the physiology of renal glucose and albumin retention but potentially deleterious for the kidney response following AA-induced kidney injury.NEW & NOTEWORTHY Based on insights from studies manipulating glucose transport, the hypothesis has been proposed that inhibiting intestinal uptake or renal reabsorption of energy substrates has unique therapeutic potential to improve metabolic disease and kidney outcome in response to injury. The present study takes this idea to B0AT1, the major transporter for neutral amino acids in the intestine and kidney, and shows that its absence attenuates aristolochic acid-induced nephropathy.

Age-related external anal sphincter muscle dysfunction and fibrosis: possible role of Wnt/ß-catenin signaling pathways.

Studies show an age-related increase in the prevalence of anal incontinence and sphincter muscle atrophy. The Wnt/ß-catenin signaling pathway has been recently recognized as the major molecular pathway involved in age-related skeletal muscle atrophy and fibrosis. The goals of our study were to 1) evaluate the impact of normal aging on external anal sphincter (EAS) muscle length-tension (L-T) function and morphology and 2) specifically examine the role of Wnt signaling pathways in anal sphincter muscle fibrosis. New Zealand White female rabbits [6 young (6 mo of age) and 6 old (36 mo of age)] were anesthetized, and anal canal pressure was measured to determine the L-T function of EAS. Animals were killed at the end of the study, and the anal canal was harvested and processed for histochemical studies (Masson trichrome stain for muscle/connective tissue) as well as for molecular markers for fibrosis and atrophy [collagen I, ß-catenin, transforming growth factor-ß (TGF-ß), atrogin-1, and muscle-specific RING finger protein-1 (MuRF-1)]. The L-T was significantly impaired in older animals compared with young animals. Anal canal sections stained with trichrome showed a significant decrease in the muscle content (52% in old compared with 70% in young) and an increase in the connective tissue/collagen content in the old animals. An increased protein and mRNA expression of all the fibrosis markers was seen in the older animals. Aging EAS muscle exhibits impairment of function and increase in connective tissue. Upregulation of atrophy and profibrogenic proteins with aging may be the reason for the age-related decrease in anal sphincter muscle thickness and function.NEW & NOTEWORTHY Our studies using a female rabbit model show age-related alterations in the structure and function of the external anal sphincter (EAS) muscle. We used endoluminal ultrasound to measure age-related changes in EAS muscle thickness. We employed Western blot and quantitative PCR to demonstrate age-related changes in the levels of important fibrogenic as well as atrophy markers. Our findings may have significant clinical implications, i.e., use of specific antagonists to prevent age-related EAS muscle dysfunction.

Wnt-ß Catenin Signaling Pathway: A Major Player in the Injury Induced Fibrosis and Dysfunction of the External Anal Sphincter.

Wnt-ß catenin is an important signaling pathway in the genesis of fibrosis in many organ systems. Our goal was to examine the role of Wnt pathway in the external anal sphincter (EAS) injury-related fibrosis and muscle dysfunction. New Zealand White female rabbits were subjected to surgical EAS myotomy and administered local injections of either a Wnt antagonist (sFRP-2; daily for 7 days) or saline. Anal canal pressure and EAS length-tension (L-T) were measured for 15 weeks after which the animals were sacrificed. Anal canal was harvested and processed for histochemical studies (Masson trichrome stain), molecular markers of fibrosis (collagen and transforming growth factor-ß) and immunostaining for ß catenin. Surgical myotomy of the EAS resulted in significant impairment in anal canal pressure and EAS muscle L-T function. Following myotomy, the EAS muscle was replaced with fibrous tissue. Immunostaining revealed ß catenin activation and molecular studies revealed 1.5-2 fold increase in the levels of markers of fibrosis. Local injection of sFRP-2 attenuated the ß catenin activation and fibrosis. EAS muscle content and function was significantly improved following sFRP-2 treatment. Our studies suggest that upregulation of Wnt signaling is an important molecular mechanism of injury related EAS muscle fibrosis and sphincter dysfunction.

Sildenafil increases the force of right atrial contractions in vitro via the NO-guanylyl cyclase pathway involving ß-adrenoceptor linked mechanisms.

Sildenafil, a drug used in the treatment of erectile dysfunction, is a phosphodiesterase 5A inhibitor that increases cyclic guanosine monophosphate (cGMP) levels. In addition to its vascular actions, sildenafil is also known to alter cardiac functions. This study was undertaken to elucidate the effect of sildenafil on cardiac contractility and the underlying mechanisms. The experiments were conducted on spontaneously-beating right atria isolated from adult rats. The effect of sildenafil on the isometric contractions in vitro was examined in the absence or presence of antagonists. Sildenafil (0.001-10 microM) produced a concentration-dependent increase in the atrial force of contraction without altering the atrial rate, even up to 10 microM. A concentration as low as 0.001 microM produced a significant increase (16%) in force and the increase was about 50% at 10 microM. Pretreatment with methylene blue (a guanylyl cyclase inhibitor) or N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) blocked the force changes induced by sildenafil. Sildenafil-induced increase in force of contraction was also blocked by propranolol (a beta-adrenoceptor antagonist) and diltiazem (an L-type Ca(2+) channel antagonist). The present results demonstrate that sildenafil increases the atrial force of contraction involving cGMP-beta-adrenoceptor-Ca(2+) channel-dependent mechanisms.

Cardiac dysrhythmia produced by Mesobuthus tamulus venom involves NO-dependent G-Cyclase signaling pathway.

Role of G-protein coupled pathways in modulating the cardiotoxic effects produced by Indian red scorpion (Mesobuthus tamulus) venom were examined. The isometric contractions of spontaneously beating or paced (3.5 Hz) rat right atrial preparations in vitro were recorded. The cumulative concentration (0.01-3.0 microg/ml)-response of venom on spontaneously beating atria exhibited a marked decrease in rate (by 55%) and an increase in force (by 92%) only at a higher concentration (3.0 microg/ml). The venom-induced decrease in rate and increase in force were sensitive to atropine, N-omega-nitro-L-arginine methylester (NO synthase inhibitor) and methylene blue (guanylyl cyclase inhibitor). Further, nifedipine, a Ca(2+) channel antagonist, blocked the force changes but not the rate changes induced by venom. In the paced atrium, on the other hand, a concentration-dependent decrease in force was observed, and at 3 microg/ml, the decrease was 50%. Pretreatment with nifedipine, but not with methylene blue, significantly attenuated the venom-induced force changes in paced atrium. The observations of this study demonstrate that the venom-induced atrial dysrhythmia is mediated through the muscarinic receptor-dependent NO-G-cyclase cell-signaling pathways.

Involvement of phospholipase A2 pathway for the Indian red scorpion venom-induced augmentation of cardiopulmonary reflexes elicited by phenyldiguanide.

The present study was conducted to examine the role of phospholipase A(2) and prostaglandins in Indian red scorpion (Mesobuthus tamulus; MBT) venom-induced augmentation of cardiopulmonary reflexes elicited by phenyldiguanide (PDG). Trachea, femoral artery and jugular vein were cannulated in urethane anesthetized adult albino rats. The effect of jugular venous injection of PDG on ECG, BP and respiratory activity were recorded. Injection of PDG (10 microg/kg) evoked tachypnea/apnea, bradycardia and hypotension lasting for 60s. After injecting MBT venom (100 microg/kg) for 30 min, the PDG evoked reflex responses were augmented by two times and increased the pulmonary water content in envenomed animals, significantly. The venom-induced augmentation of PDG reflex and the increase in pulmonary water content were blocked in animals pretreated with B(2) kinin receptor antagonist (Hoe 140; 2.32 microg/kg). These responses induced by venom were also blocked by a phospholipase A(2) antagonist (PACOCF(3); 1 mg/kg) and a prostaglandin synthase inhibitor (indomethacin; 10 mg/kg). The observations indicate that the venom-induced responses (augmentation of PDG reflex response and increased pulmonary water content) involve PLA(2)-prostaglandin pathway that is triggered by B(2) kinin receptors to sensitize the receptors located on the vagal C-fibres.

Bradycardia induced by Mesobuthus tamulus scorpion venom involves muscarinic receptor-G-protein-coupled cell signaling pathways.

Indian red scorpion (Mesobuthus tamulus; MBT) envenomation produces various cardio-respiratory abnormalities including cardiac dysrhythmias. The underlying cell signaling pathways for the cardiac dysrhythmias produced by MBT venom are not known. The present study was therefore conducted to delineate the second messenger signaling pathways involved in MBT venom-induced atrial rhythm changes. The effects of venom and various antagonists were examined on spontaneously beating rat right atrial preparations in vitro. The MBT-venom produced an increase (35%), a decrease (45%) and again an increase (50%) in rate at 0.03, 0.3 and 3.0 microg/ml of venom, respectively. On the other hand, force of contraction exhibited a concentration-dependent rise (up to 40%) at all concentrations of venom. Pretreatment with atropine (0.3 microM) blocked the decrease in atrial rate at 0.3 microg/ml concentration of venom while no such blockade was seen in force of contraction. Submaximal concentration of ACh (0.1 nM) decreased the atrial rate by 25%. In the presence of MBT venom (0.3 microg/ml), ACh-induced fall in atrial rate was enhanced. The venom-induced fall in atrial rate and augmentation of ACh response were blocked by pertussis toxin (PTx; a Gi-inhibitor) or methylene blue (a G-cyclase inhibitor). The results indicate that the decrease in atrial rate produced by venom is mediated muscarinic by receptors via Gi-guanylyl cyclase mediated cell signaling pathways.

Estrogen modulates in vitro atrial bradycardia induced by Indian red scorpion venom via G-protein coupled mechanisms.

Role of estrogen on cardiac dysrhythmia produced by Indian red scorpion (Mesobuthus tamulus) venom was examined using rat right atrial preparations in vitro. In females, the M. tamulus venom produced an increase, a decrease and an increase in rate at 0.03, 0.3 and 3 microg/ml of venom, respectively, producing N-shaped response curve, whereas no such response pattern was observed in males. Force of contraction in females was increased at all the concentrations of the venom, while in males the increase was seen only at 3 microg/ml. Castration of male rats did not alter the venom response to female type, while 'estrogenisation of castrated male rats' (pseudofemales) produced a response similar to females. Tamoxifen reversed the venom-induced responses both in females and pseudofemales. Further in females, the venom action at 0.3 microg/ml was blocked by atropine. Response at this concentration was also blocked by pertussis toxin and methylene blue. Results suggest that the cholinergic component of venom response is modulated by estrogen receptors via G(i)-protein-guanylyl cyclase mechanism.