Skeletal muscle and kidney crosstalk in chronic kidney disease

The functioning of complex organisms requires a constant and delicate balance of processes both between and within cells, tissues, and organ systems. There is growing appreciation for the role of signalling crosstalk connecting different organ systems of the body, even from tissues traditionally classified as “inert” in terms of their capacity to produce chemical signals that can act on other organ systems. Many of these secreted molecules have been shown to contribute to, or exacerbate, a variety of functions and diseases in other organ systems, even if the two organs are not functionally linked. For example, there is a strong association with skeletal muscle atrophy and dysfunction in patients with chronic kidney disease (CKD). Identification of molecules produced and secreted by skeletal muscle has existed for some time, and there is emerging evidence that skeletal muscle may directly affect kidney function. Conversely, factors produced and secreted by the kidneys in various models of CKD have been shown to contribute to reduced muscle functionality. This review will focus on crosstalk in both directions between skeletal muscle and the kidneys. The emphasis will be on direct interaction between these organs using examples of secreted factors that are produced by the muscle or kidneys (including activin A, myostatin, microRNA’s, irisin and mitsugumin 53),often under pathophysiological conditions. Our understanding of how the kidneys and skeletal muscle interact with each other is key to elucidating the pathophysiology processes that drive health and disease

Nedd4-2-dependent ubiquitination potentiates the inhibition of human NHE3 by cholera toxin and enteropathogenic Escherichia coli

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Naþ/Hþ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Naþ/ Hþ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans

Atypical cannabinoid ligands O-1602 and O-1918 administered chronically in diet-induced obesity

Atypical cannabinoid compounds O-1602 and O-1918 are ligands for the putative cannabinoid receptors G protein-coupled receptor 55 and G protein-coupled receptor 18. The role of O-1602 and O-1918 in attenuating obesity and obesity-related pathologies is unknown. Therefore, we aimed to determine the role that either compound had on body weight and body composition, renal and hepatic function in diet-induced obesity. Male Sprague-Dawley rats were fed a high-fat diet (40% digestible energy from lipids) or a standard chow diet for 10 weeks. In a separate cohort, male Sprague-Dawley rats were fed a high-fat diet for 9 weeks and then injected daily with 5 mg/kg O-1602, 1 mg/kg O-1918 or vehicle (0.9% saline/0.75% Tween 80) for a further 6 weeks. Our data demonstrated that high-fat feeding upregulates whole kidney G protein receptor 55 expression. In diet-induced obesity, we also demonstrated O-1602 reduces body weight, body fat and improves albuminuria. Despite this, treatment with O-1602 resulted in gross morphological changes in the liver and kidney. Treatment with O-1918 improved albuminuria, but did not alter body weight or fat composition. In addition, treatment with O-1918 also upregulated circulation of pro-inflammatory cytokines including IL-1α, IL-2, IL-17α, IL-18 and RANTES as well as plasma AST. Thus O-1602 and O-1918 appear not to be suitable treatments for obesity and related comorbidities, due to their effects on organ morphology and pro-inflammatory signaling in obesity

CB1 Ligand AM251 induces weight loss and fat reduction in addition to increased systemic inflammation in diet-induced obesity

Diet-induced obesity (DIO) reduces fatty acid oxidation in skeletal muscle and decreases circulating levels of adiponectin. Endocannabinoid signaling is overactive in obesity, with some effects abated by antagonism of cannabinoid receptor 1 (CB1). This research aimed to determine if treatment with the global CB1 antagonist/inverse agonist, AM251, in high-fat diet (HFD) fed rats influenced adiponectin signaling in skeletal muscle and a “browning” of white adipose tissue (WAT) defined by UCP1 expression levels. Male Sprague Dawley rats consumed an HFD (21% fat) for 9 weeks before receiving daily intraperitoneal injections with vehicle or AM251 (3 mg/kg) for 6 weeks. mRNA expression of genes involved in metabolic functions were measured in skeletal muscle and adipose tissue, and blood was harvested for the measurement of hormones and cytokines. Muscle citrate synthase activity was also measured. AM251 treatment decreased fat pad weight (epididymal, peri-renal, brown), and plasma levels of leptin, glucagon, ghrelin, and GLP-1, and increased PAI-1 along with a range of pro-inflammatory and anti-inflammatory cytokines; however, AM251 did not alter plasma adiponectin levels, skeletal muscle citrate synthase activity or mRNA expression of the genes measured in muscle. AM251 treatment had no effect on white fat UCP1 expression levels. AM251 decreased fat pad mass, altered plasma hormone levels, but did not induce browning of WAT defined by UCP1 mRNA levels or alter gene expression in muscle treated acutely with adiponectin, demonstrating the complexity of the endocannabinoid system and metabolism. The CB1 ligand AM251 increased systemic inflammation suggesting limitations on its use in metabolic disorders

Exploration of the altar painting “Sacra Conversazione” by Bernardino Licinio from the church of St. Francis in Krk

Slika Sacra Conversazione Bernardina Licinija iz crkve Sv. Franje Asiškog u Krku izrađena je tehnikom masne tempere na dasci. Velike dimenzije slike u kombinaciji sa smještajem na visini uz teška oštećenja nosioca i slikanih slojeva, zahtijevala su izvođenje konzervatorsko-restauratorskih istraživanja in situ. Tom je prilikom prvi put u Hrvatskoj izvan radionice, na terenu, primijenjena vizualizacija oltarne pale velikog formata računalnom radiografjom. Omogućila je uvid u stanje nosioca i slikanih slojeva, smanjila rizik demontaže i transporta te usmjerila daljnje konzervatorsko-restauratorske radove.The altar pala Sacra Conversazione, by Bernardino Licinio (Venice, 1485/1489–c. 1550), is located on the main altar of the Church of St. Francis in the town of Krk. It was painted in 1531 in tempera grassa on board. In contrast to the later Venetian Sacra Conversazione by the same artist, painted in 1535, the Krk pala is almost completely unknown in professional literature; it is mentioned only in some older literature, with a short iconographic description. Due to the painting’s large dimensions (280x195 cm) and weight, its location high on the main altar, and the significant damage to the painted layers, its wooden base and the support system, the conservation-restoration explorations were carried out in situ, at the church altar. This significantly reduced the possibility of additional damage being incurred during dismantling and transport. The artefact was filmed in the visible, UV and IR parts of the spectrum, and under slanted light. Samples of pigment and binding material were analysed in a laboratory, and the order of layers was established. A particularly interesting phase of the conservation-restoration exploration was the visualization of the artefact by computer radiography. This method, created and developed for medical purposes, is being applied ever more often in restoration for exploring and documenting artefacts. Prior to this occasion, it had not been used in Croatia for in situ explorations. The visualization of the Krk pala by computer radiography made it possible to assess the condition of the original painted layer, wooden base and support system, which is normally hidden by the large wall at the back of the altar. The computer radiography uncovered grave and extensive damage to the painting base and the painted layers, but which can be remedied and reconstructed. Exploratory attempts to remove the “secondary” coats from the surface of the original painted layer suggested that several interventions had already been made on the artefact, two of them rather substantial, and they included reduction of the altar painting’s format. The explorations carried out thus far have provided the basic information on this artefact, which will be supplemented by further restoration procedures, and archival and art-historical research

Renal Cannabinoid Receptor Expression and Function: Their Role in Obesity and Diabetes

Obesity and diabetes are clearly established independent risk factors for renal disease. Therapeutic targets have been investigated for their role in treating obesity and diabetic associated renal damage. The endocannabinoid system is an important endogenous lipid signalling system known to mediate glucose and lipid metabolism, inflammation and energy storage. Specifically, diabetes mellitus and obesity induces alterations in the expression of cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2) and putative cannabinoid receptor G-protein coupled receptor 55 (GPR55) in a tissue specific manner. Renal expression and function of these receptors, particularly within the pathophysiological context of obesity and diabetes related renal damage is poorly understood. The research presented in this thesis examines the renal expression and function of CB1, CB2 and GPR55. The significant aim of this PhD candidature was to examine the expression of cannabinoid receptors in the kidney in obese and diabetic conditions. Subsequent studies sought to evaluate the actions of selective manipulation of the receptors by synthetic compounds on markers of renal damage and structure in an animal model of diet induced obesity (DIO)

Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo

Cannabinoid receptor 2 expression in proximal tubule cells exposed to elevated glucose and albumin

Introduction. Hyperglycaemia plays a significant role in the aetiology of diabetic nephropathy. Compounded with this, renal tubule cells are also exposed to high levels of albumin in the filtrate, which is caused by damage to the glomerulus. We have previously identified that the cannabinoid receptor 2 (CB2) is present in proximal tubule cells. In renal tubules, hypertrophy is an early indicator of diabetic nephropathy, and previously we have shown that activation of CB2 reduces tubular hypertrophy (Jenkin et al., 2010). Despite this, to date, there has been little investigation into changes in the expression of the CB2 in the proximal tubule under diabetic conditions. The aim of this research was to quantify the expression of endocannabinoid receptor CB2 in proximal tubule cells exposed to high levels of glucose and albumin. Method. Human kidney (HK2) cells were treated with one of four protocols; a control medium containing physiological normal levels of glucose (5 mM) and no albumin; a high glucose (25 mM) medium with no albumin; a high albumin (1 mg/ml) medium with normal glucose levels (5 mM); or a combination medium composed of high glucose and high albumin. HK2 cells were incubated for 4, 6, 18 or 24 h. Following treatment, mRNA was extracted and DNAse treated. The mRNA was reverse transcribed and the level of the endocannabinoid receptor CB2 mRNA was assessed by real time PCR. Protein expression for the CB2 receptor was investigated using Western blot analysis. Band density was quantified using Image Lab software, and treatment groups were normalized to control treatment. Values were expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA. Results. High levels of glucose did not significantly alter CB2 mRNA expression in HK2 cells. High albumin treatment alone and in combination with high glucose did result in a significant reduction in CB2 receptor mRNA expression at 6 and 18 h compared to control treatment (P<0.05, n = 9). CB2 protein expression was reduced compared to control treatment at 6 and 24 h treatment periods again in high albumin treatment alone and in combination with high glucose (P<0.05, n = 4). Conclusion. We have demonstrated that exposure to elevated levels of albumin alone and in combination with glucose significantly reduces mRNA and protein levels of the CB2 receptor in a proximal tubule cell line. High glucose alone did not affect CB2 expression in the proximal tubule. Given that the reduction in the CB2 receptor expression occurred only in treatments containing elevated albumin, this indicates that CB2 may play a protective role in the advanced stages of diabetic nephropathy when the proximal tubule is exposed to elevated albumin levels in the filtrate following glomerular damage due to hyperglycaemia. Further investigation may indicate that this receptor could provide a novel target for the treatment and prevention of diabetic nephropathy

The expression of human NHE3 in mouse intestine leads to increased diarrheal symptoms

Diarrheal disease is a frequent cause of emergency room visits and often results from altered ion transport across the gut epithelium by infectious agents. The Na+/H+ Exchanger 3 (NHE3) is responsible for the majority of intestinal electroneutral sodium absorption and is associated with many diarrheal diseases. While mice/rabbits have been used to investigate the mechanisms of diarrhea, they are less prone to develop diarrhea than humans. Recently, we have shown that human NHE3, but not mouse/rabbit NHE3s, interacts with the ubiquitin E3 ligase Nedd4-2. We hypothesize that this property of human NHE3 contributes to the increased severity of diarrhea. To investigate this hypothesis, we generated transgenic mice expressing human NHE3 in the intestine (hNHE3int) and Caco/2bb cells transfected with human or rabbit NHE3. The regulation of human and non-human NHE3 in response to forskolin (FSK) or cholera toxin (CTX) was investigated by measuring NHE3 activity and ubiquitination. We found that FSK significantly increased human NHE3 ubiquitination and the extent of inhibition of human NHE3 activity by FSK was greater than rabbit NHE3. Nedd4-2 knockdown blunted the inhibitory effect on human NHE3 but not rabbit. Consistently, inhibition of intestinal NHE3 by FSK was greater in hNHE3int than WT mice. In addition, treatment with CTX led to significantly higher water accumulation in the small intestine of hNHE3int compared to WT mice. These findings demonstrate that human and non-human NHE3s are differentially regulated, suggesting that the characteristics of human NHE3 regulation may contribute to increased diarrhea severity in humans