Podocyte Density and Albuminuria in Aging Diabetic Ins2± Mice with or Without Adenosine A1 Receptor Signaling.

AIM OF STUDY: To investigate podocyte density in aging diabetic Ins2± and Ins2±, A1AR-/- mouse models in C57Bl/6 background. METHODS: Ins2± mice and especially Ins2±, adenosine A1 receptor knockout mice (Ins2±, A1AR-/-) are mouse models with a phenotype of diabetic nephropathy. Aged mice (at ~40 weeks) were assessed for glomerular filtration barrier function by measuring albuminuria, glomerular filtration, glomerular damage by electron microscopy, and podocyte numbers by Wilms Tumor protein (WT-1) staining. RESULTS: Compared to healthy wild-type mice, both diabetic mouse models developed diabetic nephropathy, including hyperfiltration (p<0.01) and albuminuria (p<0.05). Typical diabetic structural glomerular and podocyte damage was visualized by electron microscopy. Podocyte count per glomerular area (podocyte density) was significantly decreased in both diabetic mouse models (p<0.01). In contrast, no significant correlation was detected between albuminuria and absolute podocyte count per glomerulus. CONCLUSION: The amount of albuminuria as marker of diabetic nephropathy does not correlate with the podocytes density; however, a relative podocyte deficiency became evident with an increase in glomerular area in the diabetic animals, suggesting a relative podocytopenia.

High-resolution vascular tissue characterization in mice using 55MHz ultrasound hybrid imaging.

Ultrasound and Duplex ultrasonography in particular are routinely used to diagnose cardiovascular disease (CVD), which is the leading cause of morbidity and mortality worldwide. However, these techniques may not be able to characterize vascular tissue compositional changes due to CVD. This work describes an ultrasound-based hybrid imaging technique that can be used for vascular tissue characterization and the diagnosis of atherosclerosis. Ultrasound radiofrequency (RF) data were acquired and processed in time, frequency, and wavelet domains to extract six parameters including time integrated backscatter (T(IB)), time variance (T(var)), time entropy (T(E)), frequency integrated backscatter (F(IB)), wavelet root mean square value (W(rms)), and wavelet integrated backscatter (W(IB)). Each parameter was used to reconstruct an image co-registered to morphological B-scan. The combined set of hybrid images were used to characterize vascular tissue in vitro and in vivo using three mouse models including control (C57BL/6), and atherosclerotic apolipoprotein E-knockout (APOE-KO) and APOE/A(1) adenosine receptor double knockout (DKO) mice. The technique was tested using high-frequency ultrasound including single-element (center frequency=55 MHz) and commercial array (center frequency=40 MHz) systems providing superior spatial resolutions of 24 µm and 40 µm, respectively. Atherosclerotic vascular lesions in the APOE-KO mouse exhibited the highest values (contrast) of -10.11±1.92 dB, -12.13±2.13 dB, -7.54±1.45 dB, -5.10±1.06 dB, -5.25±0.94 dB, and -10.23±2.12 dB in T(IB), T(var), T(E), F(IB), W(rms), W(IB) hybrid images (n=10, p<0.05), respectively. Control segments of normal vascular tissue showed the lowest values of -20.20±2.71 dB, -22.54±4.54 dB, -14.94±2.05 dB, -9.64±1.34 dB, -10.20±1.27 dB, and -19.36±3.24 dB in same hybrid images (n=6, p<0.05). Results from both histology and optical images showed good agreement with ultrasound findings within a maximum error of 3.6% in lesion estimation. This study demonstrated the feasibility of a high-resolution hybrid imaging technique to diagnose atherosclerosis and characterize plaque components in mouse. In the future, it can be easily implemented on commercial ultrasound systems and eventually translated into clinics as a screening tool for atherosclerosis and the assessment of vulnerable plaques.

Impaired glucose tolerance in the absence of adenosine A1 receptor signaling.

OBJECTIVE: The role of adenosine (ADO) in the regulation of glucose homeostasis is not clear. In the current study, we used A1-ADO receptor (A1AR)-deficient mice to investigate the role of ADO/A1AR signaling for glucose homeostasis. RESEARCH DESIGN AND METHODS: After weaning, A1AR(-/-) and wild-type mice received either a standard diet (12 kcal% fat) or high-fat diet (HFD; 45 kcal% fat). Body weight, fasting plasma glucose, plasma insulin, and intraperitoneal glucose tolerance tests were performed in 8-week-old mice and again after 12-20 weeks of subsequent observation. Body composition was quantified by magnetic resonance imaging and epididymal fat-pad weights. Glucose metabolism was investigated by hyperinsulinemic-euglycemic clamp studies. To describe pathophysiological mechanisms, adipokines and Akt phosphorylation were measured. RESULTS: A1AR(-/-) mice were significantly heavier than wild-type mice because of an increased fat mass. Fasting plasma glucose and insulin were significantly higher in A1AR(-/-) mice after weaning and remained higher in adulthood. An intraperitoneal glucose challenge disclosed a significantly slower glucose clearance in A1AR(-/-) mice. An HFD enhanced this phenotype in A1AR(-/-) mice and unmasked a dysfunctional insulin secretory mechanism. Insulin sensitivity was significantly impaired in A1AR(-/-) mice on the standard diet shortly after weaning. Clamp studies detected a significant decrease of net glucose uptake in A1AR(-/-) mice and a reduced glucose uptake in muscle and white adipose tissue. Effects were not triggered by leptin deficiency but involved a decreased Akt phosphorylation. CONCLUSIONS: ADO/A1AR signaling contributes importantly to insulin-controlled glucose homeostasis and insulin sensitivity in C57BL/6 mice and is involved in the metabolic regulation of adipose tissue.

Adenosine mediated desensitization of cAMP signaling enhances T-cell responses.

Adenosine has long been regarded as a crucial anti-inflammatory agent that protects the host from excessive damage. It has been reported to play an important role in suppressing immune activation, particularly that of T cells. However, it is a general observation that induction of T-cell activation is an efficient event despite the high adenosine levels that are often present in the affected host due to injury or stress. We report here that prior to antigenic stimulation via TCR/CD3, exposure of T cells to adenosine desensitizes adenosine receptors, so as to create a window of time where the T cells are insensitive to this ubiquitous suppressor. T cells from mice that were pre-exposed to this manipulation showed stronger responses to antigenic stimulation; therefore, the P1 adenosine receptor desensitization demonstrated an adjuvant-like effect. Our results suggest that adenosine receptor desensitization may be a mechanism for T cells to escape the general suppression during early points of T-cell activation and may emerge as a potential alternative for vaccine adjuvants.

Vascular PPARgamma controls circadian variation in blood pressure and heart rate through Bmal1.

Thiazolidinediones (TZDs) are PPARgamma activators that exhibit vasculoprotective properties. To determine the vascular function of PPARgamma, we analyzed Tie2Cre/flox and SM22Cre/flox mice. Unexpectedly, both knockout strains exhibited a significant reduction of circadian variations in blood pressure and heart rate in parallel with diminished variations in urinary norepinephrine/epinephrine excretion and impaired rhythmicity of the canonical clock genes, including Bmal1. PPARgamma expression in the aorta exhibited a robust rhythmicity with a more than 20-fold change during the light/dark cycle. Rosiglitazone treatment induced aortic expression of Bmal1 mRNA, and ChIP and promoter assays revealed that Bmal1 is a direct PPARgamma target gene. These studies have uncovered a role for vascular PPARgamma as a peripheral factor participating in regulation of cardiovascular rhythms.

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Nitric oxide stimulates COX-2 expression in cultured collecting duct cells through MAP kinases and superoxide but not cGMP.

Collecting ducts are a major site of renal production and action of both prostaglandins and nitric oxide. Experiments were undertaken to examine whether nitric oxide regulates cyclooxygenase (COX)-2 expression and PGE(2) release in cultured collecting duct cells. In mIMCD-K2 cells, sodium nitroprusside (SNP) in the 50- to 800-microM range induced a marked dose- and time-dependent increase in COX-2 protein levels, determined by immunoblotting, and the induction was detectable at 4 h. This was preceded by induction of COX-2 mRNA as determined by real-time-RT-PCR. The COX-2 induction was accompanied by a significant rise in PGE(2) release as determined by enzyme immunoassay. S-nitroso-N-acetylpenicillamine (SNAP) had a similar stimulatory effect on COX-2 expression and PGE(2) release. 8-bromo-cGMP (200 microM) had no effect on COX-2 expression. The SNP-stimulated COX-2 expression was not affected by the guanylyl cyclase inhibitor methylene blue or the protein kinase G inhibitor KT-5823 (2.0 microM). In contrast, the SNP-stimulated COX-2 expression was significantly reduced by either the Erk1/2 inhibitor PD-98059 or the P38 inhibitor SB-203580 and was abolished by combination of the two kinase inhibitors. The stimulation was also significantly blocked by the SOD mimetic tempol. Thus we conclude that NO stimulates COX-2 expression in collecting duct cells through mechanisms involving MAP kinase and superoxide, but not cGMP.

Hypertonic induction of COX-2 in collecting duct cells by reactive oxygen species of mitochondrial origin.

Our previous studies have documented MAPK mediation of the hypertonicity-induced stimulation of COX-2 expression in cultured renal medullary epithelial cells. The present study extends this observation by examining the role of reactive oxygen species (ROSs). ROS levels, determined using dichlorodihydrofluorescence diacetate and cytochrome c, were rapidly and significantly increased following exposure of mIMCD-K2 cells to media made hypertonic by adding NaCl. Hypertonic treatment (550 mosmol/kg) for 16 h induced a 5.6-fold increase in COX-2 protein levels and comparable increases in prostaglandin E(2) release, both of which were completely abolished by the NADPH oxidase inhibitor diphenyleneiodonium (25-50 microM). The general antioxidant N-acetyl-l-cysteine (6 mM), and the superoxide dismutase mimetic TEMPO (2.0 mm) reduced COX-2 levels by 75.6 and 79.8%, respectively. Exposure of mIMCD-K2 cells to exogenous O(2)(-.) generated by the xanthine/xanthine oxidase system mimicked the effect of hypertonicity on COX-2 expression and prostaglandin E(2) release. The increases in phosphorylation of ERK1/2 and p38 were detected 20 min following the hypertonic treatment and were both prevented by N-acetyl-l-cysteine. The increases in ROSs in response to hypertonic treatment were completely blocked by any one of the mitochondrial inhibitors tested, such as rotenone, thenoyltrifluoroacetone, or carbonyl cyanide m-chlorophenylhydrazone, associated with remarkable inhibition of COX-2 expression. In contrast, the increases in ROSs were not significantly altered in IMCD cells deficient in either gp91(phox) or p47(phox), nor were the increases in COX-2 expression. We conclude that ROSs derived from mitochondria, but not NADPH oxidase, mediate the hypertonicity-induced phosphorylation of MAPK and the stimulation of COX-2 expression.

Influence of genetic background and gender on hypertension and renal failure in COX-2-deficient mice.

The present study was undertaken to determine whether the severity of renal failure or hypertension in homozygous cyclooxygenase (COX)-2-deficient (COX-2-/-) mice affected by genetic background or gender. COX-2 deletion was introduced into three congenic genetic backgrounds, 129/Sv (129/COX-2-/-), C57/BL6 (C57/COX-2-/-), and BALB/c (BALB/COX-2-/-), by backcrossing the original mixed-background knockout mice with the respective inbred strains for 9 or 10 generations. Evaluation of the severity of hypertension and renal failure was performed in knockout and wild-type mice at the age of 2.5-3.5 mo. Blood pressure measured by tail-cuff plethysmography was significantly elevated in the male 129/COX-2-/- mice (165.8 +/- 9.2 vs. 116 +/- 5.1 mmHg, P < 0.05), and to a much lesser extent in the female 129/COX-2-/- mice (127.4 +/- 3.3 vs. 102.4 +/- 3.3), whereas it was unchanged in the C57- or BALB/COX-2-/- mice regardless of gender. Urinary excretion of albumin, determined by EIA, was remarkably increased in the 129/COX-2-/- (16.4 +/- 4.1 vs. 0.16 +/- 0.043 mg albumin/mg creatinine, P < 0.001), and to a lesser extent in the male C57/COX-2-/- mice (0.595 +/- 0.416 vs. 0.068 +/- 0.019). Albumin excretion was not elevated in the male BALB/COX-2-/- or in female COX-2-/- mice on any of the three genetic backgrounds. Histological analysis showed abundant protein casts, dilated tubules, and infiltration of inflammatory cells in the male 129/COX-2-/- mice, but not in COX-2-/- mice in other strains or gender. However, the presence of small glomeruli in the nephrogenic zone was observed in all strains of COX-2 knockout mice, regardless of genetic background and gender. Therefore, we conclude that the severity of hypertension and renal failure in COX-2-deficient mice is influenced by genetic background and gender, whereas the incomplete maturation of outer cortical nephrons appears to be independent of genetic background effects.

Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E(2).

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/- mice on a mixed genetic background and in COX-2 -/- mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of PGE(2) on nNOS expression. In a cultured macula densa cell line, PGE(2) significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/- mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/- mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE(2) on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation.