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

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

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

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

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

Megalin binds to NHERF1 and NHERF2 scaffold proteins

Albumin endocytosis in the renal proximal tubule is regulated by the scavenger receptor megalin, as well as a number of transmembrane and accessory proteins. Previously we have demonstrated an essential role for the scaffold proteins NHERF1 and NHERF2 in albumin uptake. NHERF1 and NHERF2 are PDZ domain containing proteins that interact with specific sequences that form a PDZ binding domain (S/TXΦ) in the C-terminus of transmembrane proteins. Interestingly, megalin contains a functional PDZ binding domain (SDV), however an interaction between meaglin and the scaffold proteins NHERF1 and NHERF2 has not been investigated. In this study we will investigate if there is an interaction between megalin and NHERF1 and NHERF2, and then characterize the specific domains required for this interaction. Initially, immunoprecipitation experiments were performed using anti-megalin, anti-NHERF1 and anti-NHERF2 antibodies that were incubated with rat kidney lysate. The immunoprecipitates were analysed by Western blot analysis using the anti-NHERF1 and anti-NHERF2 and anti-megalin antibodies, respectively. These studies clearly indicated that megalin bound to NHERF1 and NHERF2 in vivo. To determine which domains in NHERF1 and NHERF2 were required for this interaction, GST fusion proteins were generated as described previously. These fusion proteins included the full length NHERF proteins as well as their 2 PDZ domains (PDZ1 and PDZ2) and C-terminal ezrin binding domain. Incubation with rat kidney lysate and analysis by Western blot analysis indicated that megalin bound to PDZ2 of NHERF1 and PDZ2 and the C-terminal ezrin binding domain of NHERF2. Megalin binds to NHE3, an exchanger that that is also essential for albumin endocytosis. Interestingly, NHE3 also binds to in NHERF1 and NHERF2 using the same PDZ binding domains. We also investigated the distribution of megalin and NHERF1 and NHERF2 in the opossum kidney (OK) proximal tubule cell line. OK cells are a well characterized model of albumin endocytosis that contains a large number of essential endogenous proteins including megalin, NHE3, NHERF1 and NHERF2. Confocal analysis of OK cells demonstrated that the distribution of megalin was predominantly apical with some cytosolic localization. Importantly, NHERF1 had a strong apical localization which overlapped with megalin. Further, as previously described NHERF2 was predominantly cytosolic, and this protein co-localized with megalin in this region. Therefore, we have described for the first time an interaction between megalin and the scaffold proteins NHERF1 and NHERF2. As the NHERF proteins have been shown to be required for the formation of macromolecular complexes in other cell systems, further investigation should determine if they play a role for the complex formation required for the regulation of megalin medicated albumin endocytosis in proximal tubule cells

Human and non-human intestinal NHE3 : human NHE3 demonstrates increased susceptibility to inhibition and unique regulation by ubiquitin

Introduction: The Na+/H+ Exchanger 3 (NHE3) is responsible for the majority of the electroneutral sodium absorption occurring in the intestine. As such, NHE3 has a major role in controlling electrolyte and fluid balance and is a frequent target of inhibition in many diarrheal diseases. While mice and 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 non-human NHE3s, interacts with the ubiquitin E3 ligase Nedd4-2. We hypothesize that this property of human NHE3 contributes to the increased severity of diarrhea. Method and Materials: To investigate this hypothesis, we determined human and non-human NHE3 activities and ubiquitination levels in response to the NHE3 inhibitors forskolin (FSK), cholera toxin (CTX) or Enteropathogenic E.coli (EPEC). In vitro, we generated Caco-2/bbe cells transfected with human or rabbit NHE3, and in vivo we generated transgenic mice expressing human NHE3 in the intestine (hNHE3int). For in vitro experiments, inhibitor treatments of 30 and 90 min were directly applied to cells diluted with normal growth media. NHE3 activity was measured by Na+ dependent intracellular pH recovery. NHE3 ubiquitination was evaluated by immunoprecipitation of NHE3 followed by western blot of ubiquitin. Nedd4-2 was knocked down in cells via electroporation. For in vivo experiments, mice were anesthetised with a ketamine/xylazine cocktail and a 2 - 5 cm section of ileum was tied off and injected with inhibitor treatment or Hanks Buffered Saline Solution (HBSS) vehicle buffer. Mice were allowed to recover for 5h post-injection before cervical dislocation was performed. Closed loops were removed, measured and weighed, and villi were dissected and used for NHE3 activity analysis. Results: In vitro, we found that 10uM CTX significantly increased human NHE3 ubiquitination. Both CTX (1-10 uM) and EPEC treatments induced significantly more inhibition of human NHE3 activity in Caco-2/bbe cells than what was observed in rabbit NHE3 transfected cells. Nedd4-2 knockdown blunted the inhibitory effect on human NHE3, demonstrating the importance of Nedd4-2 in regulating human NHE3. In vivo, NHE3 knockout mice (NHE3-/-) have previously been shown to display symptoms of diarrhea. However, our model of hNHE3int mice did not show any signs of diarrhea, indicating that the transgenic hNHE3 is functional. In anesthetised hNHE3int mice, we found that both 5h closed-loop intestinal treatment with inhibitors EPEC (2 ×108 CFU) and CTX (10ug) significantly increased water accumulation in the small intestine and significantly reduced NHE3 activity compared to wild type mice. Conclusion: 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

Acupuncture in drug and alcohol withdrawal at the Community Residential Withdrawal Unit, Footscray Hospital, Melbourne

Background: Acupuncture has been offered as an adjunct therapy in drug and alcohol withdrawal at the Community Residential Withdrawal Unit (CRWU), Western Hospital, Footscray, since 1996. Anecdotal reports from staff and clients indicate that acupuncture is a useful treatment approach, and, to investigate more thoroughly, a collaborative study was undertaken in 2007. Aims: To identify and explore client and staff perceptions of the benefits/limitations of acupuncture in the CRWU program. Design: Semi-structured interviews were used to capture data that would provide understanding of client and staff experiences of acupuncture. The data were analysed qualitatively to identify major themes. Participant selection criteria: Consenting in-patient clients at CRWU aged 18 years or over who had acupuncture during the period of the study, plus all clinical staff at CRWU who consented to participate in the study. Data analysis: Client and staff interview data were analysed using thematic content analysis to identify major themes and insights that related to the aims of the study. A comparative analysis of client and staff views, based on the two sets of data, was also undertaken to explore convergences and divergences of views. Results: The study found that there was a strong consensus amongst clients and staff interviewed that acupuncture was a beneficial therapy that had a relaxing effect with various ‘flow-on’ benefits such as decrease in anxiety and reduction of pain. Conclusion: Drug and alcohol treatment guidelines support the view that matching treatment approaches to individuals is critical to the success of returning clients to the community. It is also acknowledged that a combination of treatment regimes is a best-practice approach. This study revealed that staff and clients at CRWU believe that acupuncture is a beneficial non-pharmacotherapeutic approach in the treatment of drug and alcohol dependency

Elevated CB1 and GPR55 cannabinoid receptor expression in proximal tubule cells and whole kidney exposed to diabetic conditions

Background: Hyperglycemia has been implicated in the etiology of diabetic nephropathy, which typically leads to elevated albumin levels in the urine as a result of glomerular and tubular dysfunction. A number of molecular targets involved in the pathophysiology of this disease have been identified. Specifically, diabetes mellitus induces an upregulation in the cannabinoid receptor 1 (CB1) and putative cannabinoid receptor g-protein coupled receptor 55 (GPR55) in a tissue specific manner. To date, there has been little investigation into changes in the expression of the CB1 and GPR55 receptors in the kidney and specifically the proximal tubule under diabetic conditions. Method: Human kidney (HK2) proximal tubule cells were incubated with one of four treatments; a control media containing physiological normal levels of glucose (5 mM) and no albumin, a high glucose (25 mM) media with no albumin, a high albumin (1 mg/ml) media with normal glucose levels (5 mM), or a combination media composed of high glucose and high albumin. HK2 cells were incubated for 4, 6, 18 or 24 hours. Following treatment, protein lysate and mRNA was extracted and mRNA was DNAse treated. The mRNA was reverse transcribed and the level of CB1 and GPR55 expression was assessed by ‘real-time’ PCR. Protein expression was assessed by western blot analysis. Further, we characterized whole kidney protein expression of these receptors in Sprague Dawley rats with streptozotocin (STZ) induced diabetes. Protein expression for the CB1 and GPR55 receptors were investigated using Western blot analysis. Results: In whole kidney, CB1 protein expression was significantly increased in diabetic compared to non-diabetic animals. In vitro proximal tubule cells exposed to elevated high albumin alone significantly increased CB1 protein expression at 4 hours. Albumin treatment in conjunction with high glucose also lead to significantly elevated CB1 mRNA (6 hour) and protein (4 hours) expression. No alterations to GPR55 expression was found in whole kidney. Specifically in proximal tubule cells high glucose appears to be driving an increase in GPR55 expression. GPR55 mRNA was elevated at 6 and 24 hour treatments with high glucose and high glucose combined with high albumin media. However, only high glucose alone induced significant increases in GPR55 protein expression (6 hour). Conclusions: We have demonstrated that in vivo the CB1 receptor is upregulated in whole kidney of diabetic animals. Further, an in vitro model of diabetic nephropathy leads to increases to both CB1 and GPR55 receptor expression specifically within the proximal tubule cells. Here we have demonstrated that both CB1 and GPR55 are significantly increased in the renal system under diabetic conditions. Additional investigation may indicate that these receptors may provide useful physiological targets for the treatment and prevention of diabetic nephropathy