Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues that play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter (UT)-B, UT-A1/A3, or all UTs leads to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UTA1–knockout mouse model. Phenotypically, daily urine output in UT-A1–knockout mice was nearly 3-fold that of WT mice and 82% of all-UT–knockout mice, and the UT-A1–knockout mice had significantly lower urine osmolality than WT mice. After 24-h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1–knockout mice were unable to increase urine-concentrating ability. Compared with all-UT–knockout mice, the UT-A1–knockout mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UTA1 represents a promising diuretic target.
It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice.
These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent 22Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.
P2X purinergic receptors, activated by extracellular ATP, mediate a number of cardiac cellular effects and may be important under pathophysiological conditions. The objective of the present study was to characterize the P2X receptor-mediated ionic current and determine its role in heart failure using the calsequestrin (CSQ) model of cardiomyopathy. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and CSQ mice. The P2X agonist 2-methylthio-ATP (2-meSATP) induced an inward current that was greater in magnitude in CSQ than in WT ventricular cells. The novel agonist, MRS-2339, an N-methanocarba derivative of 2-chloro-AMP relatively resistant to nucleotidase, induced a current in the CSQ myocyte similar to that by 2-meSATP. When administered via a miniosmotic pump (Alzet), it significantly increased longevity compared with vehicle-injected mice (log rank test, P = 0.02). The improvement in survival was associated with decreases in the heart weight-to-body weight ratio and in cardiac myocyte cross-sectional area [MRS-2339-treated mice: 281 ± 15.4 (SE) μm2, n = 6 mice vs. vehicle-treated mice: 358 ± 27.8 μm2, n = 6 mice, P < 0.05]. MRS-2339 had no vasodilator effect in mouse aorta ring preparations, indicating that its salutary effect in heart failure is not because of any vascular unloading. The cardiac P2X current is upregulated in the CSQ heart failure myocytes. Chronic administration of a nucleotidase-resistant agonist confers a beneficial effect in the CSQ model of heart failure, apparently via an activation of the cardiac P2X receptor. Cardiac P2X receptors represent a novel and potentially important therapeutic target for the treatment of heart failure.
Dendritic cells are key players in regulating immunity. These cells both activate and inhibit the immune response depending on their cellular environment. Their response to hyperglycemia, a condition common amongst diabetics wherein glucose is abnormally elevated, remains to be elucidated. In this study, the phenotype and immune response of dendritic cells exposed to hyperglycemia were characterized in vitro and in vivo using the streptozotocin-induced diabetes model. Dendritic cells were shown to be sensitive to hyperglycemia both during and after differentiation from bone marrow precursor cells. Dendritic cell behavior under hyperglycemic conditions was found to vary by phenotype, among which, tolerogenic dendritic cells were particularly sensitive. Expression of the costimulatory molecule CD86 was found to reliably increase when dendritic cells were exposed to hyperglycemia. Additionally, hydrogel-based delivery of the anti-inflammatory molecule interleukin-10 was shown to partially inhibit these effects in vivo.
by
Chuanfu Li;
Fang Hua;
Tuanzhu Ha;
Krishna Singh;
Chen Lu;
John Kalbfleisch;
Kevin F. Breuel;
Tiffany Ford;
Race L. Kao;
Ming Gao;
Tammy R. Ozment;
David L. Williams
Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p < 0.01) and 50% survived > 30 days (p < 0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box-1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.
by
Sunil Yeruva;
Giriprakash Chodisetti;
Min Luo;
Mingmin Chen;
Ayhan Cinar;
Lisa Ludolph;
Maria Luennemann;
Julia Goldstein;
Anurag Kumar Singh;
Brigitte Riederer;
Oliver Bachmann;
Andre Bleich;
Markus Gereke;
Dunja Bruder;
Susan Hagen;
Peijian He;
Chang-Hyon Yun;
Ursula Seidler
A dysfunction of the Na<sup>+</sup>/H<sup>+</sup> exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.
The Per2luc mouse model developed by Takahashi laboratory is one of the most powerful models to study circadian rhythms in real time. In this study, we report that photoreceptors degenerate in male Per2luc mice during aging. Young (2.5- to 5-month-old) and aged (11- to 13.5-month-old) homozygous male Per2luc mice and C57BL/6J mice were used for this study. Retina structure and function were investigated via spectral domain optical coherence tomography (SD-OCT), fundus imaging, and electroretinography (ERG). Zonula occludens-1 (ZO-1) immunofluorescence was used to analyze the retinal pigment epithelium (RPE) morphology. Fundus examination revealed no difference between young Per2luc and wild-type (WT) mice. However, the fundus of aged Per2luc mice showed white deposits, suggestive of age-related drusen-like formation or microglia, which were absent in age-matched WT mice. No differences in retinal structure and function were observed between young Per2luc and WT mice. However, with age, Per2luc mice showed a significant reduction in total retinal thickness with respect to C57BL/6J mice. The reduction was mostly confined to the photoreceptor layer. Consistent with these results, we observed a significant decrease in the amplitude of a- and b-waves of the ERG in aged Per2luc mice. Analysis of the RPE morphology revealed that in aged Per2luc mice there was an increase in compactness and eccentricity with a decrease in solidity with respect to the values observed in WT, pointing toward signs of aging in the RPE of Per2luc mice. Our data demonstrate that homozygous Per2luc mice show photoreceptor degeneration during aging and a premature aging of the RPE.
Post-traumatic stress disorder (PTSD) is characterized by a heightened emotional and physiological state and an impaired ability to suppress or extinguish traumatic fear memories. Exaggerated physiological responses may contribute to increased cardiovascular disease (CVD) risk in this population, but whether treatment for PTSD can offset CVD risk remains unknown. To further evaluate physiological correlates of fear learning, we used a novel pre-clinical conditioned cardiovascular testing paradigm and examined the effects of Pavlovian fear conditioning and extinction training on mean arterial pressure (MAP) and heart rate (HR) responses. We hypothesized that a fear conditioned cardiovascular response could be detected in a novel context and attenuated by extinction training. In a novel context, fear conditioned mice exhibited marginal increases in MAP (∼3 mmHg) and decreases in HR (∼20 bpm) during CS presentation. In a home cage context, the CS elicited significant increases in both HR (100 bpm) and MAP (20 mmHg). Following extinction training, the MAP response was suppressed while CS-dependent HR responses were variable. These pre-clinical data suggest that extinction learning attenuates the acute MAP responses to conditioned stimuli over time, and that MAP and HR responses may extinguish at different rates. These results suggest that in mouse models of fear learning, conditioned cardiovascular responses are modified by extinction training. Understanding these processes in pre-clinical disease models and in humans with PTSD may be important for identifying interventions that facilitate fear extinction and attenuate hyper-physiological responses, potentially leading to improvements in the efficacy of exposure therapy and PTSD–CVD comorbidity outcomes.
Diabetic neuropathy (DN), the most common complication of diabetes, frequently leads to foot ulcers and may progress to limb amputations. Despite continuous increase in incidence, there is no clinical therapy to effectively treat DN. Pathogenetically, DN is characterized by reduced vascularity in peripheral nerves and deficiency in angiogenic and neurotrophic factors. We will briefly review the pathogenetic mechanism of DN and address the effects and the mechanisms of cell therapies for DN. To reverse the changes of DN, studies have attempted to deliver neurotrophic or angiogenic factors for treatment in the form of protein or gene therapy; however, the effects turned out to be very modest if not ineffective. Recent studies have demonstrated that bone marrow (BM)-derived cells such as mononuclear cells or endothelial progenitor cells (EPCs) can effectively treat various cardiovascular diseases through their paracrine effects. As BM-derived cells include multiple angiogenic and neurotrophic cytokines, these cells were used for treating experimental DN and found to reverse manifestations of DN. Particularly, EPCs were shown to exert favorable therapeutic effects through enhanced neural neovascularization and neuro-protective effects. These findings clearly indicate that DN is a complex disorder with pathogenetic involvement of both vascular and neural components. Studies have shown that cell therapies targeting both vascular and neural elements are shown to be advantageous in treating DN.
by
Ayesha Sultan;
Min Luo;
Qin Yu;
Brigitte Riederer;
Weiliang Xia;
Mingmin Chen;
Simone Lissner;
Johannes E. Gessner;
Mark Donowitz;
Chris Yun;
Hugo deJonge;
Georg Lamprecht;
Ursula Seidler
Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na + /H + exchanger NHE3 is regulated by the Na + /H + Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ('lipid rafts') were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO 2 /HCO 3 - mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.