Modulation of single-nephron GFR in the db/db mouse model of type 2 diabetes mellitus. II. Effects of renal mass reduction

This study examines for the first time the effects of uninephrectomy (Nx) on modulation of whole kidney glomerular filtration rate (GFR), single-nephron GFR (SNGFR), and progression of diabetic nephropathy in the db/db mouse model of type 2 diabetes mellitus. To characterize SNGFR and tubuloglomerular feedback (TGF) responses to Nx and chronic neuronal nitric oxide synthase inhibition in the db/db mouse, we studied the effects of Nx on whole kidney GFR, SNGFR, and TGF characteristics in db/db and wild-type (WT) mice after Nx or sham Nx. We also documented progression of glomerular changes over a 6-mo period. Whole kidney GFR and SNGFR were significantly higher in db/db Nx than db/db sham mice, without change in proximal tubule reabsorptive rates. The TGF responses, determined as proximal-distal SNGFR differences, were brisk: 12.1 +/- 1.0 vs. 8.4 +/- 0.6 nl/min in WT sham (P < 0.05), 15.7 +/- 1.0 vs. 12.0 +/- 1.0 nl/min in WT Nx (P < 0.05), and 17.8 +/- 1.3 vs. 14.3 +/- 1.0 nl/min in db/db Nx (P < 0.05) mice. Chronic ingestion of the neuronal nitric oxide synthase inhibitor S-methylthiocitrulline for 2-3 wk after Nx had no effect on SNGFR or the TGF response. These studies show further elevations in whole kidney GFR and SNGFR in these hyperglycemic morbidly obese db/db mice, with an intact TGF system after Nx. In addition, in the db/db Nx mice, 4-6 mo after Nx, there was an exacerbation of the lesions of diabetic nephropathy, as quantified by a significant increase in the ratio of mesangial surface area to total glomerular surface area.     

Ginseng berry reduces blood glucose and body weight in db/db mice.

In this study, we observed anti-diabetic and anti-obesity effects of Panax ginseng berry in adult C57BL/Ks db/db mice and their lean littermates. Animals received daily intraperitoneal injections of Panax ginseng berry extract at 150 mg/kg body wt. for 12 consecutive days. On Day 5, the extract-treated db/db mice had significantly lower fasting blood glucose levels as compared to vehicle-treated mice (180.5+/-10.2 mg/dl vs. 226.0+/-15.3 mg/dl, P < 0.01). On day 12, the extract-treated db/db mice were normoglycemic (134.3+/-7.3 mg/dl) as compared to vehicle-treated mice (254.8+/-24.1 mg/dl; P < 0.01). Fasting blood glucose levels of lean mice did not decrease significantly after treatment with extract. After 12 days of treatment with the extract, glucose tolerance increased significantly, and overall blood glucose exposure calculated as area under the curve (AUC) decreased 53.4% (P < 0.01) in db/db mice. Furthermore, db/db mice treated with extract (150 mg/kg body wt.) showed weight loss from 51.0+/-1.9 g on Day 0, to 46.6+/-1.7 g on Day 5, and to 45.2+/-1.4 g on Day 12 (P < 0.05 and P < 0.01 compared to Day 0, respectively). The body weight of lean littermates also decreased at the same dose of extract. These data suggest that Panax ginseng berry extract may have therapeutic value in treating diabetic and obese patients.     

Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.     

Structural and functional analysis of pancreatic islets preserved by pioglitazone in db/db mice

To evaluate preventive effects of pioglitazone on pancreatic beta-cell damage in C57BL/KsJ db/db mice, an obese diabetic animal model, the pancreatic islets were compared morphologically between pioglitazone-treated (100 mg/kg daily po) and untreated db/db mice (n = 7 for each) after a 12-wk intervention (6-18 wk of age). The fasting blood glucose level was significantly improved by the treatment with pioglitazone (260 +/- 12 vs. 554 +/- 62 mg/dl, P < 0.05). The islet mass in the pancreas was significantly greater in pioglitazone-treated mice than in untreated mice (10.2 +/- 1.1 vs. 4.6 +/- 0.2 mg, P < 0.01). Subsequently, biochemical and physiological analyses of the beta-cell function were employed using pioglitazone-treated and untreated db/db mice (n = 6 for each) and pioglitazone-treated and untreated db/+ mice (n = 6 for each). After 2 wk of treatment (10-12 wk of age), the plasma levels of triglyceride and free fatty acid were significantly decreased, whereas the plasma adiponectin level increased significantly compared with the untreated group (65.2 +/- 18.0 vs. 18.3 +/- 1.3 microg/ml, P < 0.05). Pioglitazone significantly reduced the triglyceride content in the islets (43.3 +/- 3.6 vs. 65.6 +/- 7.6 ng/islet, P < 0.05) with improved glucose-stimulated insulin secretion. Pioglitazone showed no significant effects on the biochemical and physiological parameters in db/+ mice. The present study first demonstrated that pioglitazone prevents beta-cell damage in an early stage of the disease progression in db/db mice morphologically and physiologically. Our results suggest that pioglitazone improves glucolipotoxicity by increasing insulin sensitivity and reducing fat accumulation in the pancreatic islets.     

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.     

p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats

Angiotensin II (ANG II) infusion increases renal superoxide (O(2)(-)) and enhances renal vasoconstriction via macula densa (MD) regulation of tubuloglomerular feedback, but the mechanism is unclear. We targeted the p22(phox) subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) with small-interfering RNA (siRNA) to reduce NADPH oxidase activity and blood pressure response to ANG II in rats. We compared single nephron glomerular filtration rate (SNGFR) in samples collected from the proximal tubule (PT), which interrupts delivery to the MD, and from the distal tubule (DT), which maintains delivery to the MD, to assess MD regulation of GFR. SNGFR was measured in control and ANG II-infused rats (200 ng.kg(-1).min(-1) for 7 days) 2 days after intravenous injection of vehicle or siRNA directed to p22(phox) to test the hypothesis that p22(phox) mediates MD regulation of SNGFR during ANG II. The regulation of SNGFR by MD, determined by PT SNGFR-DT SNGFR, was not altered by siRNA in control rats (control + vehicle, 13 +/- 1, n = 8; control + siRNA, 12 +/- 2 nl/min, n = 8; not significant) but was reduced by siRNA in ANG II-treated rats (ANG II + vehicle, 13 +/- 2, n = 7; ANG II + siRNA, 7 +/- 1 nl/min, n = 8; P < 0.05). We conclude that p22(phox) and NADPH oxidase regulate the SNGFR during ANG II infusion via MD-dependent mechanisms.     

Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration

Secretin is a vasoactive peptide capable of acting on transmembrane volume fluxes. We measured nephron filtration (SNGFR) and resorption during secretin microinjection (MIJ) into the tubular lumen or microperfusion (MP) into peritubular capillaries. In 24 rat nephrons, SNGFR, measured by collections from the distal tubule, rose from 25+/- 4 to 61+/-8 nl/min during MIJ of saline containing secretin 10(-9) M into the last convolution of the proximal tubule (LP). Percent and absolute resorptions rose from 70 to 90% and from 20+/-4 to 56+/-8 nl/min, respectively. During MIJ of secretin, 3 x 10(-)(9) M into the first convolution of the proximal tubule, SNGFR, measured at LP, rose from 32+/-4 to 61+/-8 nl/min, percent and absolute reabsorptions from 52+/-4 to 78+/-3% and from 16+/-2 to 50+/-7 nl/min, respectively (n = 30). During MP of secretin, 1.5x10(-9) M, SNGFR fell from 39+/-6 to 15+/-4, resorption from 19+/-4 to 9+/-2 nl/min, while percent resorption rose from 43+/-6 to 59+/-5% (n = 15). While all MIJ and MP changes were significant (P<0.001), paired pre- versus post-MIJ and MP values were not. Secretin is a powerful vasoconstrictor when perfused into peritubular capillary blood, unlike systemic and intra-arterial injections. When injected into the tubular lumen, it up-regulates SNGFR and increases reabsorption directly.     

Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice

Control db/+ and diabetic db/db mice at 6 and 12 wk of age were subjected to echocardiography to determine whether contractile function was reduced in vivo and restored in transgenic db/db-human glucose transporter 4 (hGLUT4) mice (12 wk old) in which cardiac metabolism has been normalized. Systolic function was unchanged in 6-wk-old db/db mice, but fractional shortening and velocity of circumferential fiber shortening were reduced in 12-wk-old db/db mice (43.8 +/- 2.1% and 8.3 +/- 0.5 circs/s, respectively) relative to db/+ control mice (59.5 +/- 2.3% and 11.8 +/- 0.4 circs/s, respectively). Doppler flow measurements were unchanged in 6-wk-old db/db mice. The ratio of E and A transmitral flows was reduced from 3.56 +/- 0.29 in db/+ mice to 2.40 +/- 0.20 in 12-wk-old db/db mice, indicating diastolic dysfunction. Thus a diabetic cardiomyopathy with systolic and diastolic dysfunction was evident in 12-wk-old diabetic db/db mice. Cardiac function was normalized in transgenic db/db-hGLUT4 mice, indicating that altered cardiac metabolism can produce contractile dysfunction in diabetic db/db hearts.     

Acute saline expansion increases nephron filtration and distal flow rate but maintains tubuloglomerular feedback responsiveness: role of adenosine A(1) receptors

Temporal adaptation of tubuloglomerular feedback (TGF) permits readjustment of the relationship of nephron filtration rate [single nephron glomerular filtration rate (SNGFR)] and early distal tubular flow rate (V(ED)) while maintaining TGF responsiveness. We used closed-loop assessment of TGF in hydropenia and after acute saline volume expansion (SE; 10% body wt over 1 h) to determine whether 1) temporal adaptation of TGF occurs, 2) adenosine A(1) receptors (A(1)R) mediate TGF responsiveness, and 3) inhibition of TGF affects SNGFR, V(ED), or urinary excretion under these conditions. SNGFR was evaluated in Fromter-Wistar rats by micropuncture in 1) early distal tubules (ambient flow at macula densa), 2) recollected from early distal tubules while 12 nl/min isotonic fluid was added to late proximal tubule (increased flow to macula densa), and 3) from proximal tubules of same nephrons (zero flow to macula densa). SE increased both ambient SNGFR and V(ED) compared with hydropenia, whereas TGF responsiveness (proximal-distal difference in SNGFR, distal SNGFR response to adding fluid to proximal tubule) was maintained, demonstrating TGF adaptation. A(1)R blockade completely inhibited TGF responsiveness during SE and made V(ED) more susceptible to perturbation in proximal tubular flow, but did not alter ambient SNGFR or V(ED). Greater urinary excretion of fluid and Na(+) with A(1)R blockade may reflect additional effects on the distal nephron in hydropenia and SE. In conclusion, A(1)R-independent mechanisms adjust SNGFR and V(ED) to higher values after SE, which facilitates fluid and Na(+) excretion. Concurrently, TGF adapts and stabilizes early distal delivery at the new setpoint in an A(1)R-dependent mechanism.     

Adenosine A2A receptor activation attenuates tubuloglomerular feedback responses by stimulation of endothelial nitric oxide synthase

Adenosine A(2) receptors have been suggested to modulate tubuloglomerular feedback (TGF) responses by counteracting adenosine A(1) receptor-mediated vasoconstriction, but the mechanisms are unclear. We tested the hypothesis that A(2A) receptor activation blunts TGF by release of nitric oxide in the juxtaglomerular apparatus (JGA). Maximal TGF responses were measured in male Sprague-Dawley rats as changes in proximal stop-flow pressure (ΔP(SF)) in response to increased perfusion of the loop of Henle (0 to 40 nl/min) with artificial tubular fluid (ATF). The maximal TGF response was studied after 5 min intratubular perfusion (10 nl/min) with ATF or ATF + A(2A) receptor agonist (CGS-21680; 10(-7) mol/l). The interaction with nitric oxide synthase (NOS) isoforms was tested by perfusion with a nonselective NOS inhibitor [N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME); 10(-3) mol/l] or a selective neuronal NOS (nNOS) inhibitor [N(ω)-propyl-L-arginine (L-NPA); 10(-6) mol/l] alone, and with the A(2A) agonist. Blood pressure, urine flow, and P(SF) at 0 nl/min were similar among the groups. The maximal TGF response (ΔP(SF)) with ATF alone (12.3 ± 0.6 mmHg) was attenuated by selective A(2A) stimulation (9.5 ± 0.4 mmHg). L-NAME enhanced maximal TGF responses (18.9 ± 0.4 mmHg) significantly more than L-NPA (15.2 ± 0.7 mmHg). Stimulation of A(2A) receptors did not influence maximal TGF response during nonselective NOS inhibition (19.0 ± 0.4) but attenuated responses during nNOS inhibition (10.3 ± 0.4 mmHg). In conclusion, adenosine A(2A) receptor activation attenuated TGF responses by stimulation of endothelial NOS (eNOS), presumably in the afferent arteriole. Moreover, NO derived from both eNOS and nNOS in the JGA may blunt TGF responses.     

Renal hemodynamic response to L-arginine in uncomplicated, type 1 diabetes mellitus: the role of buffering anions and tubuloglomerular feedback

According to the "tubulocentric" hypothesis of the glomerular hyperfiltration of diabetes mellitus (DM), tubuloglomerular feedback (TGF) is the critical determinant of the related renal hemodynamic dysfunction. To examine the role of TGF in human type 1 DM, 12 salt-replete healthy (C) and 11 uncomplicated DM individuals underwent measurements of glomerular filtration rate (GFR), renal blood flow (RBF), and lithium-derived absolute "distal" sodium delivery (DDNa). Measurements were made during two 3-h infusions of 0.012 mmol·kg(-1)·min(-1) l-arginine (ARG) buffered with either equimolar HCl (ARG.HCl) or citric acid (ARG.CITR). Our hypothesis was that changes in TGF signaling would be directionally opposite ARG.HCl vs. ARG.CITR according to the effects of the ARG-buffering anion on DDNa. Similar changes in C and DM followed ARG.CITR, with declines in DDNa (-0.26 ± 0.07 mmol/min C vs. -0.31 ± 0.07 mmol/min DM) and increases in RBF (+299 ± 25 vs. +319 ± 29 ml·min(-1)·1.73 m(-2)) and GFR (+6.6 ± 0.8 vs. +11.6 ± 1.2 ml·min(-1)·1.73 m(-2)). In contrast, with ARG.HCl, DDNa rose in both groups (P = 0.001), but the response was 73% greater in DM (+1.50 ± 0.15 mmol/min C vs. +2.59 ± 0.22 mmol/min DM, P = 0.001). RBF also increased (P = 0.001, +219 ± 20 ml·min(-1)·1.73 m(-2) C, +105 ± 14 DM), but ΔRBF after ARG.HCl was lower vs. ARG.CITR in both groups (P = 0.001). After ARG.HCl, ΔRBF also was 50% lower in DM vs. C (P = 0.001) and GFR, unchanged in C, declined in DM (-7.4 ± 0.9 ml·min(-1)·1.73 m(-2), P = 0.02 vs. C). After ARG.HCl, unlike ARG.CITR, DDNa increased in C and DM, associated with less ΔRBF and ΔGFR vs. ARG.CITR. This suggests that the renal hemodynamic response to ARG is influenced substantially by the opposite actions of HCl vs. CITR on DDNa and TGF. In DM, the association of ARG.HCl-induced exaggerated ΔDDNa, blunted ΔRBF, and the decline in GFR vs. C shows an enhanced TGF dependence of renal vasodilatation to ARG, in agreement with a critical role of TGF in DM-related renal hemodynamic dysfunction.     

Association between endothelin-1 and collagen deposition in db/db diabetic mouse kidneys

Endothelin-1 has been implicated in diabetic kidney injury, but there are few firm data establishing the temporal and spatial expression of kidney endothelin-1 in diabetes. We performed an immunohistochemical and histopathological analysis to determine endothelin-1 peptide expression in the kidneys of diabetic db/db mice and non-diabetic db/m controls. Diabetic mice were studied at 8 weeks, before histological damage is evident, and again at 16 weeks, when significant glomerular injury has occurred. Urinary endothelin-1 was 6.2- and 3.6-fold higher in 8- and 16-week diabetic mice compared to age-matched controls (P<0.01 db/db vs. db/m). Compared to non-diabetic kidneys, immunoreactive endothelin-1 was first elevated 2.5-fold (P=0.02) in the tubulointerstitial compartment at 8-week and remained high (3.8-fold, P<0.01) at 16 weeks. In contrast, glomerular endothelin-1 was elevated 3.2-fold (P=0.03) only in 16-week diabetic mice. Glomerular and tubulointerstitial endothelin-1 were unchanged in 8- and 16-week non-diabetic mice. Elevated endothelin-1 in diabetic mice associated temporally and spatially with collagen deposition, especially in the tubulointerstitial compartment. The localization of kidney endothelin-1 is consistent with a role for this peptide in renal fibrogenesis. These results also highlight the potential role of ET-1 in the pathogenesis of early tubulointerstitial changes in diabetes.     

Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice

The hypothesis that adenosine acting on adenosine A1 receptors (A1R) regulates several renal functions and mediates tubuloglomerular feedback (TGF) was examined using A1R knockout mice. We anesthetized knockout, wild-type, and heterozygous mice and measured glomerular filtration rate, TGF response using the stop-flow pressure (P(sf)) technique, and plasma renin concentration. The A1R knockout mice had an increased blood pressure compared with wild-type and heterozygote mice. Glomerular filtration rate was similar in all genotypes. Proximal tubular P(sf) was decreased from 36.7 +/- 1.2 to 25.3 +/- 1.6 mmHg in the A1R+/+ mice and from 38.1 +/- 1.0 to 27.4 +/- 1.1 mmHg in A1R+/- mice in response to an increase in tubular flow rate from 0 to 35 nl/min. This response was abolished in the homozygous A1R-/- mice (from 39.1 +/- 4.1 to 39.2 +/- 4.5 mmHg). Plasma renin activity was significantly greater in the A1R knockout mice [74.2 +/- 14.3 milli-Goldblatt units (mGU)/ml] mice compared with the wild-type and A1R+/- mice (36.3 +/- 8.5 and 34.1 +/- 9.6 mGU/ml), respectively. The results demonstrate that adenosine acting on A1R is required for TGF and modulates renin release.     

烟酰胺核糖抑制db/db小鼠糖尿病心肌病的作用及机制研究

目的:探讨烟酰胺核糖(NR)对2型糖尿病小鼠心肌病的治疗作用及其机制。方法:2型糖尿病模型db/db鼠和及其严格对照小鼠db/+小鼠,将小鼠分为Con (db/+)组,DM (db/db)组,DM+NR组。采用超声测小鼠心脏功能,western-blot及免疫组化测SIRT1表达含量,DHE染色、MDA含量和MnSOD活性检测反映氧化应激水平。结果:与对照组相比,db/db小鼠心脏功能显著下降(LVEF:42.3±7.2vs 73.7±10.2, P0.01;LVFS:22.1±4.2vs 42.7±6.9, P0.01),SIRT1表达量显著下调(P0.01)。NR喂养提高SIRT1表达量(P0.01),并有效改善db/db小鼠心脏功能(LVEF:53.1±8.1vs 42.3±7.2, P0.01;LVFS:33.4±6.9vs 22.1±4.2, P0.01)。同时,NR喂养显著降低了db/db小鼠心肌组织的凋亡水平和氧化应激水平(P0.05)。结论:NR有效改善了db/db小鼠的心功能障碍,降低了db/db小鼠的心肌凋亡水平和氧化应激水平,这些作用的发挥可能与NR增加SIRT1的表达量有关。     

Increased pulmonary responses to acute ozone exposure in obese db/db mice

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Displacement-encoded and manganese-enhanced cardiac MRI reveal that nNOS, not eNOS, plays a dominant role in modulating contraction and calcium influx in the mammalian heart

Within cardiomyocytes, endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are thought to modulate L-type calcium channel (LTCC) function and sarcoplasmic reticulum calcium cycling, respectively. However, divergent results from mostly invasive prior studies suggest more complex roles. To elucidate the roles of nNOS and eNOS in vivo, we applied noninvasive cardiac MRI to study wild-type (WT), eNOS(-/-), and nNOS(-/-) mice. An in vivo index of LTCC flux (LTCCI) was measured at baseline (Bsl), dobutamine (Dob), and dobutamine + carbacholamine (Dob + CCh) using manganese-enhanced MRI. Displacement-encoded MRI assessed contractile function by measuring circumferential strain (E(cc)) and systolic (dE(cc)/dt) and diastolic (dE(cc)/dt(diastolic)) strain rates at Bsl, Dob, and Dob + CCh. Bsl LTCCI was highest in nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)) and increased only in WT and eNOS(-/-) mice with Dob (P < 0.05 vs. Bsl). LTCCI decreased significantly from Dob levels with Dob + CCh in all mice. Contractile function, as assessed by E(cc), was similar in all mice at Bsl. With Dob, E(cc) increased significantly in WT and eNOS(-/-) but not nNOS(-/-) mice (P < 0.05 vs. WT and eNOS(-/-)). With Dob + CCh, E(cc) returned to baseline levels in all mice. Systolic blood pressure, measured via tail plethysmography, was highest in eNOS(-/-) mice (P < 0.05 vs. WT and nNOS(-/-)). Mice deficient in nNOS demonstrate increased Bsl LTCC function and an attenuated contractile reserve to Dob, whereas eNOS(-/-) mice demonstrate normal LTCC and contractile function under all conditions. These results suggest that nNOS, not eNOS, plays the dominant role in modulating Ca(2+) cycling in the heart.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.     

Type 2 diabetic patients may have a mild form of an injury response: a clinical research center study

Patients with type 2 diabetes (DM) demonstrate inadequate insulin release, elevated gluconeogenesis, and diminished nonoxidative glucose disposal. Similar metabolic changes occur during systemic injury caused by infection, trauma, or cancer. Described here are metabolic changes occurring in 16 DM and 11 lung cancer patients (CA) and 13 normal volunteers (NV). After a 10-h overnight fast, all subjects had fasting hormone and substrate concentrations determined, along with rates of glucose production, leucine appearance (LA), and leucine oxidation (LO). Fasting insulin (data not shown) and C-peptide concentrations were elevated in DM and CA compared with weight-matched NV (0.72 +/- 0.09 and 0.64 +/- 0.08 vs. 0.51 +/- 0.03 mg/l, P < 0.05). C-reactive protein concentration was elevated in CA compared with DM and NV (23.3 +/- 6.0 vs. 4.2 +/- 1.4 and 2.1 +/- 0.5 mg/l, P < 0.01). All counterregulatory hormones were normal except for serum cortisol (11.4 +/- 1.0 and 12.1 +/- 1.0 vs. 8.9 +/- 0.7 microg/dl, DM and CA vs. NL, respectively, P < 0.05). Glucose production was increased in DM and CA compared with NV (4.22 +/- 0.6 and 3.53 +/- 0.3 vs. 2.76 +/- 0.2 mg x kg lean body wt(-1) x min(-1), P < 0.01). LO and LA were increased in DM and CA compared with NV (LO: 27.3 +/- 1.5 and 19.7 +/- 1.5 vs. 12.5 +/- 1.1 mmol x kg lean body wt(-1) x min(-1), P < 0.05; LA: 91.9 +/- 6.6 and 90.7 +/- 7.0 vs. 79.1 +/- 6.0 mmol. kg lean body wt(-1) x min(-1), P < 0.01). DM share similar metabolic derangements with CA. The increase in LA may be secondary to an increased glucose production where amino acids are mobilized to provide the liver with adequate substrate to make glucose. The increase in glucose production may also be part of the injury response, or it may represent a form of insulin resistance that exists in both the DM and (non-DM) CA patients.