Hidden secret in hepatitis B viral X protein mutation and hypoxia-inducible factor-1α in hepatocarcinoma cancer

Hepatitis B type virus (HBV) is an old hepato oncogenic and hepatitis agent. Hepatitis B viral X protein (HBx)-induced malignant transformation requires the excess amounts of ATP level, inducing the extremely oxygen-deprived condition in the cancer tissues and vessels. To adapt, cells go to shift the hypoxic responsive state by altered hypoxia-responsive molecules such as HIF-1. In addition, tumors avoid or suppress immune recognition in the energy-deprived condition. The hypoxia-inducible factor-1α (HIF-1α) regulates MAP1, histone deacetylase and MAPK pathway. In the hypoxia, the HIF-1α interacts with HIF-1β, allowing DNA binding at the hypoxia response elements (HREs), while HBx binds with the nHLH/PAS domain of HIF-1α, preventing pVHL and HIF-1α binding capacity and degradation of HIF-1α protein. Recent work of Liu et al. [2013] demonstrated that HBx in hepatocellular carcinoma (HCC) tissues contained mutations, affecting the HBx transactivation capacity and C-terminal HBx mutation. In the HCC tissues, the HBx C-terminal mutation and HIF-1α expression were related and the different C-terminal mutations of HBx exhibit the different functionality of HIF-1α. The C-terminal region of amino acids 119-140 was important for the stability and transactivation, and the point mutations K130M/V131I enhance the functionality of HIF-1α, while C-terminal truncation diminish the HIF-1α function.     

Renin Lineage Cells Repopulate the Glomerular Mesangium after Injury

Mesangial cell injury has a major role in many CKDs. Because renin-positive precursor cells give rise to mesangial cells during nephrogenesis, this study tested the hypothesis that the same phenomenon contributes to glomerular regeneration after murine experimental mesangial injury. Mesangiolysis was induced by administration of an anti-mesangial cell serum in combination with LPS. In enhanced green fluorescent protein–reporter mice with constitutively labeled renin lineage cells, the size of the enhanced green fluorescent protein–positive area in the glomerular tufts increased after mesangial injury. Furthermore, we generated a novel Tet-on inducible triple-transgenic LacZ reporter line that allowed selective labeling of renin cells along renal afferent arterioles of adult mice. Although no intraglomerular LacZ expression was detected in healthy mice, about two-thirds of the glomerular tufts became LacZ positive during the regenerative phase after severe mesangial injury. Intraglomerular renin descendant LacZ-expressing cells colocalized with mesangial cell markers α8-integrin and PDGF receptor-β but not with endothelial, podocyte, or parietal epithelial cell markers. In contrast with LacZ-positive cells in the afferent arterioles, LacZ-positive cells in the glomerular tuft did not express renin. These data demonstrate that extraglomerular renin lineage cells represent a major source of repopulating cells for reconstitution of the intraglomerular mesangium after injury.     

Adult Renal Mesenchymal Stem Cell–Like Cells Contribute to Juxtaglomerular Cell Recruitment

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44⁺ mesenchymal stem cell (MSC)–like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44⁺ MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.The renin-angiotensin-aldosterone system plays a key physiologic role in the regulation of BP, electrolyte homeostasis, and kidney development. The first and rate-limiting enzyme is renin, an aspartyl protease synthesized and secreted into the circulation by highly specialized endocrine cells named juxtaglomerular (JG) cells that line the afferent arteriole of the kidney. Despite the physiologic importance of these cells, the molecular factors responsible for the development and maintenance of the JG cell identity remain unclear.^1,2 Moreover, in response to specific pathophysiologic conditions that demand a significant renin response (i.e., chronic ischemia, prolonged adrenergic activation, and sodium depletion), the number of renin-expressing cells in the adult kidney increases dramatically. It has been suggested that this increase is potentially due to the transdifferentiation of existing arterial smooth muscle cells.^2–4 Still, the complete cellular and genetic mechanisms underlying this phenomenon, known as JG cell recruitment remain poorly understood.Our group recently demonstrated that liver X receptor-α (LXRα) agonists and/or cyclic AMP (cAMP) upregulated renin expression in both murine and human bone marrow–derived mesenchymal stem cells (MSCs) in vitro. Stimulation of cultured murine MSCs that overexpress LXRα also resulted in their differentiation into JG-like cells expressing renin and α-smooth muscle actin.^5,6 These MSC-derived JG-like cells contained renin in secretory granules and were capable of releasing renin.⁵ Interestingly, accumulating evidence suggests that stem cells/progenitor cells reside in the adult kidney (Supplemental Table 1),^7–10 although their physiologic importance is not yet clear.On the basis of the preceding findings, we investigated whether adult kidney stem or progenitor cells have the capacity to differentiate into JG-like cells or contribute to JG cell recruitment. For this we performed in vitro and in vivo differentiation assays as well as lineage tracing using genetic models.     

Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C_m) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C_m significantly and enhanced outward current. Indomethacin, PLA₂ inhibition, and an antagonist of prostaglandin transport impaired the C_m and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2^{−/ −} and AQP1^{−/ −} mice, hypotonicity increased neither C_m nor outward current, but 0.1-μM PGE₂ increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C_m and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA₂/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.Juxtaglomerular (JG) granular cells in the terminal part of the renal afferent glomerular arterioles are the only cells in the organism that synthesize preprorenin, process it to active renin, and store active renin in mature secretory granules. The rate of renin granule exocytosis determines the level of activation of the renin-angiotensin-aldosterone system. Renin secretion from most^1,2 but not all^3,4 in vitro preparations displays a uniquely high sensitivity to changes in extracellular osmolality such that a moderate reduction in the extracellular osmolality leads to rapid increases in renin secretion. The sensing and transduction events for renin release in response to osmotic perturbations are not known. At the glomerular tuft, the extracellular osmolality may vary depending on sodium chloride (NaCl)transport rate by the adjacent macula densa and thick ascending limb cells, which are relatively water impermeable.^5,6 JG cell capacitance (C_m), an index of cell surface area, increased when extracellular osmolality was decreased by 5 to 10% at the single cell level.⁷ This observation shows exocytotic release of renin in response to decreases in extracellular osmolality.⁷ Introduction of a pipette solution with slightly increased osmolality to the cell cytoplasm is sufficient to initiate exocytosis of renin in JG cells.⁷ This indicates that cell swelling, and not granule swelling, is involved in the response and shows that sensing and transduction of the initial change in osmolality is not dependent on an extracellular receptor for the agent used (e.g., sucrose or mannitol), as shown recently to be the case for succinate.⁴ The estimated number of granules recruited for exocytosis by a hypotonic extracellular challenge corresponded closely to the number that fused after receptor-dependent activation of cAMP formation.⁷ The existing data predict an involvement of water fluxes across the JG cell membrane, but aquaporin water channels have not been demonstrated in JG cells. Marked cell swelling normally initiates a regulatory volume decrease response whereby the cell, through coordinated activation of ion and organic osmolyte efflux, regains cell volume.⁸ A distinct role for phospholipase A2 (PLA₂) and prostaglandin E2(PGE₂) EP2 receptors in swelling-induced activation of regulatory processes in single cells has been demonstrated.⁹ Prostaglandin E₂ and PGI₂ enhance outward current and renin secretion from JG cells.¹⁰ In the study presented here, we hypothesized that JG cells respond to a decrease in extracellular osmolality by water uptake, Cyclooxygenase (COX)-dependent prostaglandin formation and renin release. The hypotheses were tested using single JG cells subjected to whole-cell patch-clamp analysis and primary cultures enriched in JG cells from rats, wild-type mice, and mice with targeted deletions of COX-2 and aquaporin-1 (AQP1).     

Hypoxia-Inducible Factor-2α Limits Natural Killer T Cell Cytotoxicity in Renal Ischemia/Reperfusion Injury

Natural killer T (NKT) cells are the major early-acting immune cell type and fundamental immune modulators in ischemia-reperfusion injury (IRI). Because lymphocytes are exposed to various oxygen tensions under pathophysiologic conditions, we hypothesize that hypoxia-inducible factors (HIFs) have roles in NKT cell activation, and thus determine the final outcome of renal IRI. In this study, we used Lck-Cre transgenic mice to specifically disrupt HIF-2α in T/NKT cells and found that HIF-2α knockout led to upregulated Fas ligand expression on peripheral NKT cells, but not on conventional T cells. HIF-2α knockout promoted infiltration of NKT cells into ischemic kidneys and exacerbated IRI, which could be mitigated by in vivo NK1.1⁺ cell depletion or Fas ligand blockade. Compared with wild-type NKT cells, HIF-2α^−/− NKT cells adoptively transferred to Rag1-knockout mice elicited more severe renal injury, and these mice were not protected by {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, an adenosine A2A receptor agonist. Mechanistically, hypoxia-induced expression of adenosine A2A receptor in NKT cells and {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680-induced cAMP production in thymocytes were HIF-2α-dependent. Hydrogen peroxide-induced Fas ligand expression on thymic wild-type NKT cells was significantly attenuated by {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 treatment, but this effect was lost in HIF-2α^−/− NKT cells. Finally, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 and LPS, an inducer of HIF-2α in endothelium, synergistically reduced renal IRI substantially, but this effect was absent in Mx1-Cre-induced global HIF-2α-knockout mice. Taken together, our results reveal a hypoxia/HIF-2α/adenosine A2A receptor axis that restricts NKT cell activation when confronted with oxidative stress and thus protects against renal IRI.     

Stimulation of Renin Secretion by Angiotensin II Blockade is Gsα-Dependent

Angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) presumably stimulate renin secretion by interrupting angiotensin II feedback inhibition. The increase in cytosolic calcium caused by activation of Gq-coupled AT1 receptors may mediate the renin-inhibitory effect of angiotensin II at the cellular level, implying that ACEI and ARB may work by reducing intracellular calcium. Here, we investigated whether angiotensin II blockade acts predominantly through Gs-mediated stimulation of adenylyl cyclase (AC) by testing the effect of ACEI and ARB in mice with juxtaglomerular cell-specific deficiency of the AC-stimulatory Gsα. The ACEI captopril and quinaprilate and the ARB candesartan significantly increased plasma renin concentration (PRC) to 20 to 40 times basal PRC in wild-type mice but did not significantly alter PRC in Gsα-deficient mice. Captopril also completely abrogated renin stimulation in wild-type mice after co-administration of propranolol, indomethacin, and L-NAME. Treatment with enalapril and a low-NaCl diet for 7 days led to a 35-fold increase in PRC among wild-type mice but no significant change in PRC among Gsα-deficient mice. Three different pharmacologic inhibitors of AC reduced the stimulatory effect of captopril by 70% to 80%. In conclusion, blockade of angiotensin II stimulates renin synthesis and release indirectly through the action of ligands that activate the cAMP/PKA pathway in a Gsα-dependent fashion, including catecholamines, prostaglandins, and nitric oxide.Angiotensin II regulates renin secretion through a homeostatic mechanism that has been called the “short feedback loop,” with renin synthesis and secretion inhibited by increases and stimulated by reductions of angiotensin II concentration.¹ The cellular mechanisms underlying the feedback effects of angiotensin II are not entirely clear. There is good experimental support for the notion that the acute inhibition is mediated, at least in part, by a direct type 1 angiotensin II receptor (AT_1A)-dependent effect of the peptide on cell calcium.^1,2 Angiotensin II inhibits renin release in kidney slices³ and in isolated juxtaglomerular granular (JG) cells, where the effect is blocked by losartan.^4,5 In the isolated JG cell, angiotensin II increases intracellular calcium levels, with increases correlating with the reduction of renin release.⁵On the other hand, it is unclear whether the stimulatory arm of the short feedback loop (i.e., the increase of renin in response to a reduction of angiotensin II) can be explained by the reverse of the same mechanism. In the intact animal, acute administration of angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) causes a prompt and large increase in plasma renin concentration (PRC), reflecting stimulation of renin secretion. Chronic inhibition of ACE or AT_1A receptors leads to very marked and maintained increases in renal renin mRNA and renin content associated with recruitment of renin-generating cells in more proximal parts of the afferent arterioles.⁶ A similar potent upregulation of renin expression is found in ACE- or AT_1A-receptor-deficient mice.^7,8 There is no good in vitro model of the stimulatory effect of angiotensin II blockade on cell calcium or renin release. Furthermore, monitoring JG cell calcium in the intact animal is not practical, and therefore data that would speak directly to the question whether the application of ACEI or ARB causes measurable reductions of JG cell calcium are not available. Such an effect is by no means certain because cytosolic calcium in the JG cell is likely to be influenced by several other ligands that couple via G proteins to phospholipase C as well as by the cellular mechanisms that control calcium exchange across the plasma membrane and across boundaries of intracellular organelles.The experiments presented here were performed to further investigate the mechanisms by which angiotensin II blockade stimulates renin secretion. The growing body of data suggesting that renin may have direct, angiotensin-independent effects through the renin receptor⁹ gives added urgency to understanding the biologic mechanisms of this phenomenon, because the high circulating levels of renin produced by ACEI or ARB treatment may have pathophysiological effects. Thus, in the study presented here we asked the specific question whether the stimulation of renin release by ACEI and ARB requires the presence of Gsα as an adenylyl cyclase (AC) activator. We found that in mice in which AC-dependent generation of cAMP in JG cells is suppressed by a cell-specific knockout of Gsα, the acute and chronic stimulatory effects of ACEI or ARB were virtually abolished. These observations support the hypothesis that angiotensin blockade in vivo enhances renin release indirectly through influencing levels of one or more ligands that act through the Gsα-dependent pathway for activation of AC in JG cells. Because the effect of juxtaglomerular Gsα deletion to abrogate renin stimulation by ACEI is fully mimicked by combined administration of indomethacin, propranolol, and L-NAME, we conclude that prostaglandins, catecholamines, and nitric oxide (NO) are the main factors in stimulating the cAMP/protein kinase A pathway during angiotensin II blockade.     

Recombination Signal Binding Protein for Ig-κJ Region Regulates Juxtaglomerular Cell Phenotype by Activating the Myo-Endocrine Program and Suppressing Ectopic Gene Expression

Recombination signal binding protein for Ig-κJ region (RBP-J), the major downstream effector of Notch signaling, is necessary to maintain the number of renin-positive juxtaglomerular cells and the plasticity of arteriolar smooth muscle cells to re-express renin when homeostasis is threatened. We hypothesized that RBP-J controls a repertoire of genes that defines the phenotype of the renin cell. Mice bearing a bacterial artificial chromosome reporter with a mutated RBP-J binding site in the renin promoter had markedly reduced reporter expression at the basal state and in response to a homeostatic challenge. Mice with conditional deletion of RBP-J in renin cells had decreased expression of endocrine (renin and Akr1b7) and smooth muscle (Acta2, Myh11, Cnn1, and Smtn) genes and regulators of smooth muscle expression (miR-145, SRF, Nfatc4, and Crip1). To determine whether RBP-J deletion decreased the endowment of renin cells, we traced the fate of these cells in RBP-J conditional deletion mice. Notably, the lineage staining patterns in mutant and control kidneys were identical, although mutant kidneys had fewer or no renin-expressing cells in the juxtaglomerular apparatus. Microarray analysis of mutant arterioles revealed upregulation of genes usually expressed in hematopoietic cells. Thus, these results suggest that RBP-J maintains the identity of the renin cell by not only activating genes characteristic of the myo-endocrine phenotype but also, preventing ectopic gene expression and adoption of an aberrant phenotype, which could have severe consequences for the control of homeostasis.     

Surveillance of γδ T Cells Predicts Cytomegalovirus Infection Resolution in Kidney Transplants

Cytomegalovirus (CMV) infection in solid-organ transplantation is associated with increased morbidity and mortality, particularly if a CMV mutant strain with antiviral resistance emerges. Monitoring CMV–specific T cell response could provide relevant information for patient care. We and others have shown the involvement of Vδ2^neg γδ T cells in controlling CMV infection. Here, we assessed if Vδ2^neg γδ T cell kinetics in peripheral blood predict CMV infection resolution and emergence of a mutant strain in high–risk recipients of kidney transplants, including 168 seronegative recipients receiving organs from seropositive donors (D+R−) and 104 seropositive recipients receiving antithymocyte globulins (R+/ATG). Vδ2^neg γδ T cell percentages were serially determined in patients grafted between 2003 and 2011. The growing phase of Vδ2^neg γδ T cells was monitored in each infected patient, and the expansion rate during this phase was estimated individually by a linear mixed model. A Vδ2^neg γδ T cell expansion rate of ˃0.06% per day predicted the growing phase. The time after infection at which an expansion rate of 0.06% per day occurred was correlated with the resolution of CMV DNAemia (r=0.91; P<0.001). At 49 days of antiviral treatment, Vδ2^neg γδ T cell expansion onset was associated with recovery, whereas absence of expansion was associated with recurrent disease and DNAemia. The appearance of antiviral–resistant mutant CMV strains was associated with delayed Vδ2^neg γδ T cell expansion (P<0.001). In conclusion, longitudinal surveillance of Vδ2^neg γδ T cells in recipients of kidney transplants may predict CMV infection resolution and antiviral drug resistance.     

Multiple Genes of the Renin-Angiotensin System Are Novel Targets of Wnt/β-Catenin Signaling

Activation of the renin-angiotensin system (RAS) plays an essential role in the pathogenesis of CKD and cardiovascular disease. However, current anti-RAS therapy only has limited efficacy, partly because of compensatory upregulation of renin expression. Therefore, a treatment strategy to simultaneously target multiple RAS genes is necessary to achieve greater efficacy. By bioinformatics analyses, we discovered that the promoter regions of all RAS genes contained putative T-cell factor (TCF)/lymphoid enhancer factor (LEF)-binding sites, and β-catenin induced the binding of LEF-1 to these sites in kidney tubular cells. Overexpression of either β-catenin or different Wnt ligands induced the expression of all RAS genes. Conversely, a small-molecule β-catenin inhibitor ICG-001 abolished RAS induction. In a mouse model of nephropathy induced by adriamycin, either transient therapy or late administration of ICG-001 abolished established proteinuria and kidney lesions. ICG-001 inhibited renal expression of multiple RAS genes in vivo and abolished the expression of other Wnt/β-catenin target genes. Moreover, ICG-001 therapy restored expression of nephrin, podocin, and Wilms’ tumor 1, attenuated interstitial myofibroblast activation, repressed matrix expression, and inhibited renal inflammation and fibrosis. Collectively, these studies identify all RAS genes as novel downstream targets of Wnt/β-catenin. Our results indicate that blockade of Wnt/β-catenin signaling can simultaneously repress multiple RAS genes, thereby leading to the reversal of established proteinuria and kidney injury.     

MicroRNA-122-5p ameliorates tubular injury in diabetic nephropathy via FIH-1/HIF-1α pathway

Diabetes kidney disease (DKD) affects approximately one-third of diabetes patients, however, the specific molecular mechanism of DKD remains unclear, and there is still a lack of effective therapies. Here, we demonstrated a significant increase of microRNA-122-5p (miR-122-5p) in renal tubular cells in STZ induced diabetic nephropathy (DN) mice. Moreover, inhibition of miR-122-5p led to increased cell death and serve tubular injury and promoted DN progression following STZ treatment in mice, whereas supplementation of miR-122-5p mimic had kidney protective effects in this model. In addition, miR-122-5p suppressed the expression of factor inhibiting hypoxia-inducible factor-1 (FIH-1) in vitro models of DN. microRNA target reporter assay further verified FIH-1 as a direct target of miR-122-5p. Generally, FIH-1 inhibits the activity of HIF-1α. Our in vitro study further indicated that overexpression of HIF-1α by transfection of HIF-1α plasmid reduced tubular cell death, suggesting a protective role of HIF-1α in DN. Collectively, these findings may unveil a novel miR-122-5p/FIH-1/HIF-1α pathway which can attenuate the DN progression.     

Grg3/TLE3 and Grg1/TLE1 Induce Monohormonal Pancreatic β-Cells While Repressing α-Cell Functions

In the pancreas, α- and β-cells possess a degree of plasticity. In vitro differentiation of pluripotent cells yields mostly α- and polyhormonal β-like cells, indicating a gap in understanding of how functional monohormonal β-cells are formed and of the endogenous repressive mechanisms used to maintain β-cell identity. We show that the corepressor Grg3 is expressed in almost all β-cells throughout embryogenesis to adulthood. However, Grg3 is expressed in fewer nascent α-cells and is progressively lost from α-cells as endocrine cells mature into adulthood. We show that mouse Grg3^+/− β-cells have increased α-specific gene expression, and Grg3^+/− pancreata have more α-cells and more polyhormonal cells, indicating that Grg3 is required for the physiologic maintenance of monohormonal β-cell identity. Ectopic expression of Grg3 in α-cells represses glucagon and Arx, and the addition of Pdx1 induces Glut2 expression and glucose-responsive insulin secretion. Furthermore, we found that Grg1 is the predominant Groucho expressed in human β-cells but acts functionally similarly to Grg3. Overall, we find that Grg3 and Grg1 establish a monohormonal β-cell identity, and Groucho family members may be useful tools or markers for making functional β-cells.     

Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII^endo+/−) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII^endo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII^endo+/− mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII^endo+/+ counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β–induced canonical Smad2 signaling was reduced in TβRII^+/− ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TβRII^+/− ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII^+/− ECs compared with TβRII^+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.     

WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling

With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca²⁺-activated K⁺ (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the α subunit of BK (HEK-BKα cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKα channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKα protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKα expression and decreased ERK1/2 phosphorylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKα expression. Similarly, pretreatment of HEK-BKα cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKα expression in a dose-dependent manner. Knockdown of WNK1 expression also increased the ubiquitination of BKα channels. Notably, mice fed a high-K⁺ diet for 10 days had significantly higher renal protein expression levels of BKα and WNK1 and lower levels of ERK1/2 phosphorylation compared with mice fed a normal-K⁺ diet. These data suggest that WNK1 enhances BK channel function by reducing ERK1/2 signaling-mediated lysosomal degradation of the channel.     

Mesenchymal Stem Cells From Infants Born to Obese Mothers Exhibit Greater Potential for Adipogenesis: The Healthy Start BabyBUMP Project

Maternal obesity increases the risk for pediatric obesity; however, the molecular mechanisms in human infants remain poorly understood. We hypothesized that mesenchymal stem cells (MSCs) from infants born to obese mothers would demonstrate greater potential for adipogenesis and less potential for myogenesis, driven by differences in β-catenin, a regulator of MSC commitment. MSCs were cultured from the umbilical cords of infants born to normal-weight (prepregnancy [pp] BMI 21.1 ± 0.3 kg/m²; n = 15; NW-MSCs) and obese mothers (ppBMI 34.6 ± 1.0 kg/m²; n = 14; Ob-MSCs). Upon differentiation, Ob-MSCs exhibit evidence of greater adipogenesis (+30% Oil Red O stain [ORO], +50% peroxisome proliferator–activated receptor (PPAR)-γ protein; P < 0.05) compared with NW-MSCs. In undifferentiated cells, total β-catenin protein content was 10% lower and phosphorylated Thr41Ser45/total β-catenin was 25% higher (P < 0.05) in Ob-MSCs versus NW-MSCs (P < 0.05). Coupled with 25% lower inhibitory phosphorylation of GSK-3β in Ob-MSCs (P < 0.05), these data suggest greater β-catenin degradation in Ob-MSCs. Lithium chloride inhibition of GSK-3β increased nuclear β-catenin content and normalized nuclear PPAR-γ in Ob-MSCs. Last, ORO in adipogenic differentiating cells was positively correlated with the percent fat mass in infants (r = 0.475; P < 0.05). These results suggest that altered GSK-3β/β-catenin signaling in MSCs of infants exposed to maternal obesity may have important consequences for MSC lineage commitment, fetal fat accrual, and offspring obesity risk.