Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension.
Background: Graduate medical education varies in different countries. There is a general consensus in training methods, including residency and fellowship training systems. The graduate medical education system in western countries including the UK and the USA has been shown to be successful. The new graduate medical education training system in China was recently established and is still evolving and being implemented nationally. Summary: This paper reviews the history of nephrology training programs in the USA, the role of the Accreditation Council for Graduate Medical Education (ACGME) in establishing and enforcing guidelines and curriculum for specialty training programs, the fellowship application and Match system for the recruitment of prospective fellows, and the quality control of fellowship training programs through rigorous evaluation and In-Training examination. This review specifically discusses the nephrology subspecialty fellowship and ACGME-accredited training programs in nephrology. The authors also provide several critical suggestions on the newly established postgraduate medical education training system in China, particularly in nephrology, based on experiences from successful US nephrology fellowship practices. Key Messages: The ACGME-accredited nephrology fellowship program has been shown to be effective and successful, which could provide an insight into the newly established graduate medical education training system in China. The authors are optimistic that reforms in Chinese medical training systems will be successful in the near future.
Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
Mortality and morbidity are increased in patients with muscle atrophy resulting from catabolic diseases such as diabetes. At present there is no pharmacological treatment that successfully reverses muscle wasting from catabolic conditions. We hypothesized that acupuncture plus low frequency electric stimulation (Acu-LFES) would mimic the impact of exercise and prevent diabetes-induced muscle loss. Streptozotocin (STZ) was used to induce diabetes in mice. The mice were then treated with Acu-LFES for 15 minutes daily for 14 days. Acupuncture points were selected according to the WHO Standard Acupuncture Nomenclature guide. The needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20Hz and 1mA. Acu-LFES prevented soleus and EDL muscle weight loss and increased hind-limb muscle grip function in diabetic mice. Muscle regeneration capacity was significantly increased by Acu-LFES. The expression of Pax7, MyoD, myogenin and embryo myosin heavy chain (eMyHC) was significantly decreased in diabetic muscle vs. control muscle. The suppressed levels in diabetic muscle were reversed by Acu-LFES. The IGF-1 signaling pathway was also upregulated by Acu-LFES. Phosphorylation of Akt, mTOR and p70S6K were downregulated by diabetes leading to a decline in muscle mass, however, Acu-LFES countered the diabetes-induced decline. In addition, microRNA-1 and -206 were increased by Acu-LFES after 24 days of treatment. We conclude that Acu-LFES is effective in counteracting diabetes-induced skeletal muscle atrophy by increasing IGF-1 and its stimulation of muscle regeneration.
The pharmacokinetics of the tricarbonyl core radiopharmaceutical 99mTc(CO)3-nitrilotriacetic acid (99mTc(CO)3(NTA)) in rats and subjects with normal renal function are comparable to those of 131I-o-iodohippuran (131I-OIH), the radiopharmaceutical gold standard for the measurement of effective renal plasma flow. Our objective was to compare the pharmacokinetics of these 2 tracers in subjects with renal failure.
Methods: 99mTc (CO)3(NTA) was prepared with commercially available NTA and a commercially available labeling kit and isolated by reversed-phase high-performance liquid chromatography. Approximately 74 MBq (2.0 mCi) of 99mTc(CO)3(NTA) were coinjected with approximately 11.1 MBq (300 µCi) of 131I-OIH in 8 subjects with stage 3–4 renal failure; simultaneous images were obtained for 24 min, followed by an anterior image over the gallbladder and abdomen. Plasma clearances were determined from 10 blood samples obtained 3–180 min after injection using the single-injection, 2-compartment model. Plasma protein binding, red cell uptake, and percentage injected dose in the urine at 30 and 180 min were determined.
Results: There was no difference in the plasma clearances of 99mTc(CO)3(NTA) and 131I-OIH (177 ± 63 vs. 171 ± 66 mL/min/1.73 m2, respectively) (P = 0.41). The plasma protein binding and red cell uptake of 99mTc(CO)3(NTA) were 35% ± 7% and 6% ± 3%, respectively; both values were significantly lower than the plasma protein binding (71% ± 5%) and red cell uptake (16% ± 2%) of 131I-OIH (P < 0.001). There was no significant difference in the percentage injected dose in the urine at 30 min (P = 0.24) and at 3 h (P = 0.82); for comparison, the percentage dose in the urine at 3 h was 77% ± 9% for 99mTc(CO)3(NTA) and 78% ± 11% for 131I-OIH. Image quality with 99mTc(CO)3(NTA) was excellent and no activity was identified in the gallbladder or intestine.
Conclusion: Results in patients with renal failure show the clearance and rate of urine excretion of 99mTc(CO)3(NTA) to be equivalent to that of 131I-OIH.
Impaired bone formation is one of the major causes of low bone mass and skeletal fragility that occurs in osteoporosis. However, the mechanisms underlying the defects in bone formation are not well understood. Here, we report that big conductance calcium-activated potassium channels (BKs) are required for bone formation and osteoblast function both in vivo and in vitro. By 15 weeks of age, BK knockout (BKO) mice exhibited a decline in bone mineral density and trabecular bone volume of the tibiae and lumbar vertebrae, which were associated with impaired bone formation and osteoblast activity. Mechanistically, BK ablation in bone and bone marrow mesenchymal stem cells (BMSCs) of BKO mice inhibited integrin signaling. Furthermore, the binding of α subunit of BK with integrin β1 protein in osteoblasts was confirmed, and FAK-ERK1/2 signaling was proved to be involved by genetic modification of KCNMA1 (which encodes the α subunit of BK) in ROS17/2.8 osteoblast cells. These findings indicated that BK regulates bone formation by promoting osteoblast differentiation via integrin pathway, which provided novel insight into ion transporter crosstalk with the extracellular matrix in osteoblast regulation and revealed a new potential strategy for intervention in correcting bone formation defects.
Background: Muscle wasting is a common complication of chronic kidney disease (CKD) that is associated with higher mortality. Although the mechanisms of myofibre loss in CKD has been widely studied, the contribution of muscle precursor cell (MPC) senescence remains poorly understood. Senescent MPCs no longer proliferate and can produce proinflammatory factors or cytokines. In this study, we tested the hypothesis that the senescence associated secretory phenotype (SASP) of MPCs contributes to CKD-induced muscle atrophy and weakness. Methods: CKD was induced in mice by 5/6th nephrectomy. Kidney function, muscle size, and function were measured, and markers of atrophy, inflammation, and senescence were evaluated using immunohistochemistry, immunoblots, or qPCR. To study the impact of senescence, a senolytics cocktail of dasatinib + quercetin (D&Q) was given orally to mice for 8 weeks. To investigate CKD-induced senescence at the cellular level, primary MPCs were incubated with serum from CKD or control subjects. The roles of specific proteins in MPC senescence were studied using adenoviral transduction, siRNA, and plasmid transfection. Results: In the hindlimb muscles of CKD mice, (i) the senescence biomarker SA-β-gal was sharply increased (~30-fold); (ii) the DNA damage response marker γ-H2AX was increased 1.9-fold; and (iii) the senescence pathway markers p21 and p16INK4a were increased 1.99-fold and 2.82-fold, respectively (all values, P < 0.05), whereas p53 was unchanged. γ-H2AX, p21, and p16INK4A were negatively correlated at P < 0.05 with gastrocnemius weight, suggesting a causal relationship with muscle atrophy. Administration of the senolytics cocktail to CKD mice for 8 weeks eliminated the disease-related elevation of p21, p16INK4a, and γ-H2AX, abolished positive SA-β-gal, and depressed the high levels of the SASP cytokines, TNF-α, IL-6, IL-1β, and IFN (all values, P < 0.05). Skeletal muscle weight, myofibre cross-sectional area, and grip function were improved in CKD mice receiving D&Q. Markers of protein degradation, inflammation, and MPCs dysfunction were also attenuated by D&Q treatment compared with the vehicle treatment in 5/6th nephrectomy mice (all values, P < 0.05). Uraemic serum induced senescence in cultured MPCs. Overexpression of FoxO1a in MPCs increased the number of p21+ senescent cells, and p21 siRNA prevented uraemic serum-induced senescence (P < 0.05). Conclusions: Senescent MPCs are likely to contribute to the development of muscle wasting during CKD by producing inflammatory cytokines. Limiting senescence with senolytics ameliorated muscle wasting and improved muscle strength in vivo and restored cultured MPC functions. These results suggest potential new therapeutic targets to improve muscle health and function in CKD.
Background: Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) prevents muscle atrophy by attenuation of protein degradation in mice. The current study examines the impact of Acu/LFES on protein synthesis. Method: C57/BL6 mice received Acu/LFES treatment on hindlimb for 30 min once. Acu/LFES points were selected by WHO Standard Acupuncture Nomenclature and electric stimulation applied using an SDZ-II Electronic acupuncture instrument. Muscle protein synthesis was measured by the surface-sensing of translation (SUnSET) assay. Exosomes were isolated using serial centrifugation and concentration and size of the collected exosomes were measured using a NanoSight instrument. The mature microRNA library in serum exosomes was validated using a High Sensitivity DNA chip. Results: Protein synthesis was enhanced in the both hindlimb and forelimb muscles. Blocking exosome secretion with GW4869 decreased the Acu/LFES-induced increases in protein synthesis. MicroRNA-deep sequencing demonstrated that four members of the Let-7 miRNA family were significantly decreased in serum exosomes. Real time qPCR further verified Acu/LFES-mediated decreases of let-7c-5p in serum exosomes and skeletal muscles. In cultured C2C12 myotubes, inhibition of let-7c not only increased protein synthesis, but also enhanced protein abundance of Igf1 and Igf1 receptors. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibits Igf1. Conclusion: Acu/LFES on hindlimb decreases let-7-5p leading to upregulation of the Igf1 signaling and increasing protein synthesis in both hindlimb and forelimb skeletal muscles. This provides a new understanding of how the electrical acupuncture treatment can positively influence muscle health.
Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α<inf>1c</inf> subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β<inf>3</inf> subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.