Central role for melanocortin-4 receptors in offspring hypertension arising from maternal obesity.

Melanocortin-4 receptor (Mc4r)-expressing neurons in the autonomic nervous system, particularly in the paraventricular nucleus of the hypothalamus (PVH), play an essential role in blood pressure (BP) control. Mc4r-deficient (Mc4rKO) mice are severely obese but lack obesity-related hypertension; they also show a reduced pressor response to salt loading. We have previously reported that lean juvenile offspring born to diet-induced obese rats (OffOb) exhibit sympathetic-mediated hypertension, and we proposed a role for postnatally raised leptin in its etiology. Here, we test the hypothesis that neonatal hyperleptinemia due to maternal obesity induces persistent changes in the central melanocortin system, thereby contributing to offspring hypertension. Working on the OffOb paradigm in both sexes and using transgenic technology to restore Mc4r in the PVH of Mc4rKO (Mc4rPVH) mice, we have now shown that these mice develop higher BP than Mc4rKO or WT mice. We have also found that experimental hyperleptinemia induced in the neonatal period in Mc4rPVH and WT mice, but not in the Mc4rKO mice, leads to heightened BP and severe renal dysfunction. Thus, Mc4r in the PVH appears to be required for early-life programming of hypertension arising from either maternal obesity or neonatal hyperleptinemia. Early-life exposure of the PVH to maternal obesity through postnatal elevation of leptin may have long-term consequences for cardiovascular health.

Impact of Early Postnatal NSAID Treatment on Nephrogenesis in Wistar Rats.

BACKGROUND: Prematurely born children with patent ductus arteriosus are treated with ibuprofen or indomethacin, which may inhibit kidney development. We determined whether clinical doses affected kidney development and function, with or without extrauterine growth retardation. METHODS: Wistar rats were cross-fostered in normal food (NF) or food restricted (FR) litters at postnatal day (PND) 2. On PND 3 to 4, three doses of 0.9% NaCl, 0.1 mg/kg indomethacin, or 10 mg/kg ibuprofen were administered via intraperitoneal injection with 12-hr intervals. Kidneys were evaluated for apoptosis, proliferation, and gene expression at PND 8; stereological assessment of nephron number at PND 35; and clinical pathology and neutrophil gelatinase-associated lipocalin at 4 and 9 months. Blood pressure was measured at the ages of 4, 6, and 9 months. RESULTS: NF and FR bodyweight differed from PND 3 onwards, ranging from 16.5 g at weaning (p < 0.001) to 39 g at necropsy (p = 0.019). Kidney proliferation/apoptosis ratios were 7:1 and 3:1 (p = 0.001), respectively and different expression of Wnt4 (0.7x), Oat1 (1.3x), Nphs1 (1.7x), and Aqp4 (1.3x) was noted (but its biological relevance doubted). Nephron numbers were decreased by 12% (p = 0.109) in the ibuprofen-NF group and 7.5% (p = 0.237) in FR groups. This coincided with a tendency to increased neutrophil gelatinase-associated lipocalin at 9 months. No differences were noted in electrolytes, creatinine, or urea clearance. No valid blood pressure results could be obtained. CONCLUSION: A clinical Ibuprofen dose showed potential to inhibit kidney development in neonatal rats. FR did not modulate these effects.

Prostaglandin D2 regulates joint inflammation and destruction in murine collagen-induced arthritis.

OBJECTIVE: Prostaglandin D2 (PGD2) may exert proinflammatory or antiinflammatory effects in different biologic systems. Although this prostanoid and the enzymes responsible for its synthesis are up-regulated by interleukin-1ß (IL-1ß) in human chondrocytes in vitro, the role of PGD2 in arthritis remains unclear. This study was undertaken to investigate the role of PGD2 in the inflammatory response and in joint destruction during the development of collagen-induced arthritis (CIA) in mice. METHODS: PGD2 and cytokine levels in mice with CIA were determined by enzyme-linked immunosorbent assay. Expression of hematopoietic PGD synthase (h-PGDS), lipocalin-type PGD synthase (l-PGDS), and DP1 and DP2 receptors was analyzed by immunohistochemical methods. PGE2 levels were determined by radioimmunoassay. RESULTS: The arthritic process up-regulated the expression of h-PGDS, l-PGDS, DP1, and DP2 in articular tissue. PGD2 was produced in the joint during the early phase of arthritis, and serum PGD2 levels increased progressively throughout the arthritic process, reaching a maximum during the late stages of CIA. Treatment of arthritic mice with the DP1 antagonist MK0524 soon after the onset of disease increased the incidence and severity of CIA as well as the local levels of IL-1ß, CXCL-1, and PGE2, whereas IL-10 levels were reduced. The administration of the DP2 antagonist CAY10595 did not modify the severity of arthritis. The injection of PGD2 into the paw, as well as the administration of the DP1 agonist BW245C, significantly lowered the incidence of CIA, the inflammatory response, and joint damage. CONCLUSION: Our findings indicate that PGD2 is produced in articular tissue during the development of CIA and plays an antiinflammatory role, acting through the DP1 receptor.

Downregulation of the inflammatory response by CORM-3 results in protective effects in a model of postmenopausal arthritis.

CO-releasing molecules (CORMs) are a new class of drugs able to release small amounts of CO in biological systems. We have shown previously that one of these molecules, CORM-3, exerts anti-inflammatory effects in animal models. The aim of this study was to assess the effects of CORM-3 on bone metabolism in a model of postmenopausal rheumatoid arthritis osteoporosis. Ovariectomy was followed by collagen-induced arthritis in female DBA-1/J mice. Animals showing arthritis on day 22 after immunization were then randomized into control and treatment groups. CORM-3 was administered at 10 mg/kg, intraperitoneally, once a day. Alendronate was administered at 100 µg/kg, orally, once a day. On days 36 and 50 after immunization, animals were killed and tissues analyzed. The arthritic score was significantly reduced by CORM-3 but not by alendronate treatment. Histopathological analyses indicated that both compounds reduced cellular infiltration and cartilage degradation. Local bone erosion and reduction in TNFα levels were seen for CORM-3 on day 50 and for alendronate on day 36. Serum levels of COMP, IL-6, MMP-3, CTX-I, alkaline phosphatase, and osteocalcin were decreased by both treatments, whereas TNFα levels were reduced by CORM-3 and TRAP-5b by alendronate. Micro-computed tomographic analysis showed protective effects on trabecular bone, which were more prominent for CORM-3 on day 36 and for alendronate on day 50. Our results suggest that CORMs represent a novel anti-inflammatory strategy to counteract joint bone erosion with partial protective effects on systemic bone loss in postmenopausal rheumatoid arthritis.

Extravascular renal denervation ameliorates juvenile hypertension and renal damage resulting from experimental hyperleptinemia in rats.

BACKGROUND: Material obesity in rodents is associated with neonatal hyperleptinemia and hypertension of sympathetic origin in adult offspring. Previously, we reported that experimentally induced hyperleptinemia in rat pups results in adulthood hypertension. Here, we addressed the hypothesis that experimental neonatal hyperleptinemia, through renal nerve activation, adversely affects adult renal function. METHOD: Sprague-Dawley male and female pups were treated with neonatal leptin (3 mg/kg, intraperitoneal) or neonatal saline, twice daily from postnatal day 9-14. Juvenile (1 month) neonatal leptin and neonatal saline rats were subjected to either bilateral renal denervation, unilateral renal denervation or Sham surgery. Arterial pressure was telemetrically monitored. RESULTS: Juvenile neonatal leptin rats with intact renal nerves demonstrated increased mean arterial pressure (MAP) accompanied by local renin-angiotensin system overactivity and reduced glomerular filtration rate. Bilateral renal denervation in rats protected against neonatal leptin-induced MAP, renal renin-angiotensin system and impaired glomerular filtration rate. A two-fold increase in sympathetically mediated tubulointerstitial damage in young adult (2 months) neonatal leptin females, was suppressed by unilateral renal denervation, independent of MAP. Neonatal leptin rats also demonstrated increases in urinary protein, neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1. Raised blood pressure was associated with increased salt sensitivity and with sustained renal dysfunction in adulthood. CONCLUSION: We propose that neonatal hyperleptinemia programmes long-term renal structural and functional damage, through renal sympathetic nerve activation.

Developmental Programming of Obesity and Liver Metabolism by Maternal Perinatal Nutrition Involves the Melanocortin System.

Maternal obesity predisposes offspring to metabolic dysfunction and Non-Alcoholic Fatty Liver Disease (NAFLD). Melanocortin-4 receptor (Mc4r)-deficient mouse models exhibit obesity during adulthood. Here, we aim to determine the influence of the Mc4r gene on the liver of mice subjected to perinatal diet-induced obesity. Female mice heterozygous for Mc4r fed an obesogenic or a control diet for 5 weeks were mated with heterozygous males, with the same diet continued throughout pregnancy and lactation, generating four offspring groups: control wild type (C_wt), control knockout (C_KO), obese wild type (Ob_wt), and obese knockout (Ob_KO). At 21 days, offspring were genotyped, weaned onto a control diet, and sacrificed at 6 months old. Offspring phenotypic characteristics, plasma biochemical profile, liver histology, and hepatic gene expression were analyzed. Mc4r_ko offspring showed higher body, liver and adipose tissue weights respect to the wild type animals. Histological examination showed mild hepatic steatosis in offspring group C_KO. The expression of hepatic genes involved in regulating inflammation, fibrosis, and immune cell infiltration were upregulated by the absence of the Mc4r gene. These results demonstrate that maternal obesogenic feeding during the perinatal period programs offspring obesity development with involvement of the Mc4r system.

Human Alpha-1-Antitrypsin (hAAT) therapy reduces renal dysfunction and acute tubular necrosis in a murine model of bilateral kidney ischemia-reperfusion injury.

Several lines of evidence have demonstrated the anti-inflammatory and cytoprotective effects of alpha-1-antitrypsin (AAT), the major serum serine protease inhibitor. The aim of the present study was to investigate the effects of human AAT (hAAT) monotherapy during the early and recovery phase of ischemia-induced acute kidney injury. Mild renal ischemia-reperfusion (I/R) injury was induced in male C57Bl/6 mice by bilateral clamping of the renal artery and vein for 20 min. hAAT (80 mg/kg, Prolastin®) was administered daily intraperitoneally (i.p.) from day -1 until day 7 after surgery. Control animals received the same amount of human serum albumin (hAlb). Plasma, urine and kidneys were collected at 2h, 1, 2, 3, 8 and 15 days after reperfusion for histological and biochemical analysis. hAAT partially preserved renal function and tubular integrity after induction of bilateral kidney I/R injury, which was accompanied with reduced renal influx of macrophages and a significant decrease of neutrophil gelatinase-associated lipocalin (NGAL) protein levels in urine and plasma. During the recovery phase, hAAT significantly decreased kidney injury molecule-1 (KIM-1) protein levels in urine but showed no significant effect on renal fibrosis. Although the observed effect size of hAAT administration was limited and therefore the clinical relevance of our findings should be evaluated carefully, these data support the potential of this natural protein to ameliorate ischemic and inflammatory conditions.

The Effects of Early Postnatal Diuretics Treatment on Kidney Development and Long-Term Kidney Function in Wistar Rats.

BACKGROUND: Diuretics are administered to neonates to control fluid balance. We studied whether clinical doses affected kidney development and function and whether extrauterine growth retardation (EUGR) could be a modulator. METHODS: Wistar rats were cross-fostered in normal food or food restricted litters at postnatal day (PND) 2 and treated daily with 0.9% NaCl, 5 mg/kg furosemide or 5 mg/kg hydrochlorothiazide (HCTZ) up to PND 8. Kidneys were evaluated on proliferation, apoptosis and a set of mRNA target genes at PND 8, glomerular- and glomerular generation count at PND 35, clinical pathology parameters at 3- and 9 months, neutrophil gelatinase-associated lipocalin at PND 8, 3 and 6 months, monthly blood pressure from 3 months onward and histopathology at study end. RESULTS: Treatment with furosemide or HCTZ did not have relevant effects on measured parameters. EUGR resulted in lower body weight from day 3 onwards (-29% at weaning; p < 0.001, -10% at necropsy; p < 0.001), less glomerular generations (4.4 ± 0.32 vs. 5.0 ± 0.423; p = 0.025, males only), decreased glomerular numbers (27,861 ± 3,468 vs. 30,527 ± 4,096; p = 0.026), higher creatinine clearance (0.84 ± 0.1 vs. 0.77 ± 0.09 ml/min/kg; p = 0.047) at 3 months and lower plasma creatinine (25.7 ± 1.8 vs. 27.5 ± 2.8 µmol/l; p = 0.043) at 9 months. CONCLUSION: Furosemide and HCTZ did not influence kidney development or function when administered in a clinically relevant dose to rat pups at a stage of ongoing nephrogenesis. EUGR led to impaired kidney development but did not modify furosemide or HCTZ findings.

Potentially inappropriate prescribing in institutionalised older patients in Spain: the STOPP-START criteria compared with the Beers criteria.

OBJECTIVE: The aims of this study were to identify potentially inappropriate prescribing using the Beers and STOPP criteria. The START criteria were applied to detect prescription omission in the geriatric population. We compared the utility of these criteria in institutionalised older people. METHODS: Descriptive study reviewing the medication and clinical records of 81 residents (aged 65 years and more) by pharmacists in a nursing home in the Lleida region (Spain). RESULTS: The mean patients''age was 84 (SD=8) years, with an average of 5 drugs per resident (total prescriptions: 416 medicines). The Beers criteria identified potentially inappropriate medication use in 25% of patients and 48% of patients used at least 1 inappropriate medication according to STOPP criteria. The most frequent potentially inappropriate medications for both criteria were long-acting benzodiazepines and NSAIDs. START detected 58 potential prescribing omissions in 44% of patients. Calcium-vitamin D supplementation in osteoporosis was the most frequent rule (15%), but omissions corresponding to the cardiovascular system implied 23% of patients. CONCLUSIONS: The STOPP-START criteria reveal that potentially inappropriate prescribing (PIP) is a highly prevalent problem among Spanish nursing home residents, and a statistically significant positive correlation was found between the number of medicines prescribed and the number of PIP detected in this study. The STOPP criteria detect a larger number of PI medications in this geriatric population than the Beers criteria. The prescribing omissions detected by the START criteria are relevant and require intervention. Pharmacists' review of medications may help identify potentially inappropriate prescribing and, through an interdisciplinary approach, working with physicians may improve prescribing practices among geriatric residents of nursing homes.

Heme oxygenase-1 regulates the progression of K/BxN serum transfer arthritis.

BACKGROUND: Heme oxygenase-1 (HO-1) is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice. METHODOLOGY/PRINCIPAL FINDINGS: Arthritis was induced in C57/Black-6 xFVB (HO-1(+/+), HO-1(+/-) and HO-1(-/-)) mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1(+/-) and HO-1(-/-) groups compared with HO-1(+/+). The inflammatory response was aggravated in HO-1(+/-) mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1(+/-) group showed proteoglycan depletion significantly higher than HO-1(+/+) mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1(-/-) mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1(+/+) or HO-1(+/-) mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1(+/-) animals. CONCLUSION/SIGNIFICANCE: Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.

Up-regulation of the inflammatory response by ovariectomy in collagen-induced arthritis. effects of tin protoporphyrin IX.

We have studied the influence of ovariectomy on the inflammatory response and bone metabolism on CIA as a model of postmenopausal arthritis as well as the effects of tin protoporphyrin IX (SnPP), a heme oxygenase inhibitor. Ovariectomy in non-arthritic mice produced increased serum PGD2 levels and up-regulated the expression of COX-2, h-PGDS, l-PGDS, and HO-1 in the joints. In CIA, ovariectomy potentiated the inflammatory response with higher levels of serum IL-6 and MMP-3, local PGD2 and MMP-3 as well as trabecular bone erosion. In OVX-CIA, SnPP decreased the serum levels of IL-6, MMP-3, and PGD2; down-regulated TNFα, COX-2, hPGDS, PGD2, PGE2, and MMP-3 in joint tissues; and also decreased focal bone loss in the inflamed joint. Ovariectomy up-regulates inflammatory mediators in non-arthritic and in arthritic animals. In the OVX-CIA model, SnPP exerts anti-inflammatory effects which are not associated with the prevention of systemic bone loss.

Deficiency of Nrf2 accelerates the effector phase of arthritis and aggravates joint disease.

AIMS: Although oxidative stress participates in the etiopathogenesis of rheumatoid arthritis, its importance in this inflammatory disease has not been fully elucidated. In this study, we analyzed the relevance of the transcription factor Nrf2, master regulator of redox homeostasis, in the effector phase of an animal model of rheumatoid arthritis, using the transfer of serum from K/BxN transgenic mice to Nrf2(-/-) mice. RESULTS: Nrf2 deficiency accelerated the incidence of arthritis, and animals showed a widespread disease affecting both front and hind paws. Therefore, the inflammatory response was enhanced, with increased migration of leukocytes and joint destruction in front paws. We observed an increased production of tumor necrosis factor-α, interleukin-6, and CXCL-1 in the joint, with small changes in eicosanoid levels. Serum levels of CXCL-1 and receptor activator for nuclear factor κB ligand were enhanced and osteocalcin decreased in arthritic Nrf2(-/-) mice. The expression of cyclooxygenase-2, inducible nitric oxide synthase, and peroxynitrite in the joints was higher in Nrf2 deficiency, whereas heme oxygenase-1 was downregulated. INNOVATION: Nrf2 may be a therapeutic target for arthritis. CONCLUSION: Our results support a protective role of Nrf2 against joint inflammation and degeneration in arthritis.

The CO-releasing molecule CORM-3 protects against articular degradation in the K/BxN serum transfer arthritis model.

Carbon monoxide-releasing molecules can counteract inflammatory responses. The aim of this study was to investigate whether tricarbonylchloro(glycinate)ruthenium (II) (CORM-3) is able to control the effector phase of experimental arthritis. Arthritis was induced in C57Black-6 mice by an intraperitoneal injection of serum from arthritic K/BxN mice. CORM-3 was administered intraperitoneally at 10 mg/kg/day (5 mg/kg twice a day) from days 0 to 10 and animals were sacrificed on day 11. Serum levels of osteocalcin and prostanoids were measured by enzyme-linked immunosorbent assay and radioimmunoassay. Gene expression was determined by real-time PCR. Histological analysis was performed and protein expression was examined by immunohistochemistry. Treatment with CORM-3 reduced the macroscopic score in hind paws, the migration of inflammatory cells and erosion of cartilage and bone. CORM-3 increased the levels of osteocalcin in the serum and reduced PGD2 levels, whereas PGE2 and 6-ketoPGF1alpha were not affected. In synovial tissues, we also observed a significant reduction in gene expression of interleukin-1beta, receptor activator of nuclear factor kappaB ligand (RANKL), matrix metalloproteinase (MMP)-9 and MMP-13. CORM-3 induced HO-1 expression in joint tissues but inhibited high mobility group box 1 (HMGB1), hematopoietic-prostaglandin D2 synthase (H-PGDS) and lipocalin-type prostaglandin D2 synthase (L-PGDS), as well as RANKL and intercellular adhesion molecule-1. COX-2 expression was not affected by CORM-3 treatment. We have shown that CORM-3 decreases the inflammatory response and protects against the degradation of cartilage and bone in the arthritic mice. Pharmacological CO delivery represents a novel strategy to regulate the effector phase of arthritis.