cGMP evokes arteriolar vasorelaxation and is generated in smooth muscle by the soluble guanylyl cyclase (sGC) stimulated by endothelial NO. Phosphodiesterases (PDEs) degrade cGMP and may contribute in diameter regulation. We examined arteriolar cGMP levels in real-time in vivo upon NO and acetylcholine (ACh) and effects of PDE inhibition. Changes in cGMP were measured by FRET using the indicator protein cGi500. Arterioles in the cremaster muscle of anesthetized mice expressing cGi500 were exposed for imaging. They were stimulated with various NO donors (DEA/NO, SNP, SNAP) and ACh with or without PDE inhibition (non-specific: IBMX; PDE-5 specific: sildenafil). Furthermore, dilations induced by ACh and SNP were studied in sGC-deficient mice. DEA/NO and SNAP led to a fast, concentration-dependent rise of fluorescence ratio of cGi500 (by 4.0% at 10 mu mol/L), that declined on removal slowly. These ratio changes indicated cGMP increases as verified by inhibition of the sGC using ODQ. PDE inhibition increased cGMP levels slowly despite NO synthase inhibition. However, it did not amplify NO-induced ratio increases, but the decline after drug removal was decelerated. Interestingly, ACh did not modify cGMP levels and dilations were not impaired in sGC-deficient mice. FRET-based imaging allows reliable real-time assessment of arteriolar cGMP levels in vivo. PDE activity does not limit the amplitude, but the duration of cGMP signaling after stimulation. Furthermore, we conclude that ACh does not release endothelial NO in murine arterioles. Preliminary experiments demonstrate that simultaneous measurement of cGMP and diameter is feasible using mice with expression of cGi500 in smooth muscle cells.
Aim Hyperkalemia is prevalent in the elderly, and underlying molecular mechanisms remain incompletely understood. We hypothesized that Klotho deficiency, a feature of aging, impairs renal potassium homeostasis and contributes to hyperkalemia.Methods Klotho heterozygous knockout (KLhet) mice, characterized by reduced Klotho levels, were used to investigate determinants of potassium handling, including renal outer medullary potassium channel (ROMK)1, the epithelial sodium channel (ENaC), which provides the driving force for ROMK-mediated potassium secretion, and plasma aldosterone concentration (PAC).Results KLhet mice developed hyperkalemia with reduced urinary potassium excretion, accompanied by activation of renal mechanistic target of rapamycin complex 1 (mTORC1) signaling, indicated by increased phosphorylated S6, and decreased ROMK1 abundance in the plasma membrane fraction. Adrenal expression of aldosterone synthase Cyp11b2 and PAC were elevated, and ENaC activity was enhanced, as evidenced by increased cleavage of alpha- and gamma-ENaC subunits, partially compensating for impaired potassium excretion. Pharmacological ENaC inhibition with amiloride precipitated severe hyperkalemia in KLhet mice. In contrast, treatment with the mTORC1 inhibitor rapamycin or Klotho supplementation suppressed renal mTORC1 signaling, restored plasma membrane ROMK1 abundance, increased urinary potassium excretion, and normalized serum potassium levels, with reduced Cyp11b2 expression.Conclusion Klotho deficiency impairs renal potassium handling through mTORC1-dependent downregulation of ROMK, with compensatory hyperaldosteronism and ENaC activation. These findings highlight an important role of the Klotho-mTORC1 axis in distal potassium homeostasis and its potential as a therapeutic target for Klotho deficiency-associated hyperkalemia.
Diabetic cardiomyopathy (DCM), a diabetes-specific myocardial pathology, is a significant contributor to diabetes-related heart failure. Endoplasmic reticulum (ER) stress plays a crucial role in DCM, yet its upstream regulatory mechanisms remain incompletely elucidated. Cellular heterogeneity in DCM was investigated by analyzing single-cell transcriptomic data from the Gene Expression Omnibus. A mouse DCM model was established and validated using echocardiography, serum biochemical assays, and histological analysis. TNFAIP1 expression was detected by Western blot. A high glucose-induced DCM cellular model was constructed using HL-1 cardiomyocytes and neonatal cardiomyocytes (NMCMs). Cell injury and TNFAIP1 expression were assessed via TUNEL staining, Annexin V/PI staining, CellRox probe, RT-qPCR, and Western blot. Finally, a TNFAIP1-knockdown DCM mouse model was constructed, and the in vitro results were further validated by echocardiography, serum biochemical assays, histological analysis, and other methods. Single-cell analyses revealed the enrichment of TNFAIP1+ cardiomyocytes in DCM, with their differentially expressed genes enriched in p38 MAPK and apoptosis signaling pathways. TNFAIP1 expression was significantly upregulated in both DCM mouse and cellular models. TNFAIP1 overexpression activated p38 MAPK-mediated ER stress, increased ROS levels, and promoted apoptosis, whereas TNFAIP1 knockdown produced the opposite effects. These effects were significantly reversed by the p38 MAPK pathway inhibitor SB203580, the ER stress inhibitor TUDCA, and the ER stress inducer Thapsigargin. Finally, in vivo experiments further confirmed that TNFAIP1 knockdown ameliorated the progression of myocardial injury in DCM mice. TNFAIP1 promotes DCM progression via the p38 MAPK-mediated ER stress-apoptosis axis, identifying TNFAIP1 as a novel potential therapeutic target for DCM.
Murine research on atherosclerosis has been conducted largely on the C57BL/6 (B6) strain background, and most commonly, the B6.129P2-Apoe tm1Unc/J (B6-ApoEKO) strain. The use of the B6 background strain limits translatability for immunotherapy development due to inherent species differences between human and mouse immune systems. A proposed solution to this limitation is a humanized mouse model of atherosclerosis that would support the engraftment of human lymphoid and myeloid cell populations. The development of such a model has been hindered by the lack of a mouse strain that both develops atherosclerosis and supports human immune cell engraftment. In this paper, we report on a NOD.Cg-Prkdc scid Il2rg tm1Wjl/Sz (NSG) immunodeficient mouse strain that has a 383 bp deletion in the Apoe gene which prevents ApoE protein expression. When maintained on a high fat diet, NSG-ApoEKO mice develop hypercholesteremia and atherosclerosis at levels similar to B6-ApoEKO mice. While atherosclerosis in B6-ApoEKO mice has been widely studied in many laboratories under multiple conditions, our initial study of disease development in NSG-ApoEKO mice was limited and future comparative studies with B6-ApoEKO mice are necessary. NSG-ApoEKO mice concomitantly support human cell engraftment while developing atherosclerosis, thereby making them useful to study human-specific immune aspects.
Obesity and its associated metabolic disorders have become major global public health challenges. Increasing evidence indicates that mitophagy plays a pivotal role in the pathogenesis of obesity and related metabolic diseases. This study aimed to investigate the role and underlying mechanisms of E3 ubiquitin ligase RNF126 in obesity and non-alcoholic fatty liver disease (NAFLD). Using Rnf126 +/- mice fed a high-fat diet (HFD), we found RNF126 deficiency markedly exacerbated body weight gain, elevated serum ALT and AST levels, inflammatory responses, apoptosis, and insulin resistance. Histological analysis with H&E and Oil Red O staining revealed that RNF126 deficiency aggravated hepatic inflammatory cell infiltration, lipid droplet accumulation, and adipocyte hypertrophy in HFD-fed mice. HFD feeding significantly inhibited RNF126 expression in liver and muscle. Moreover, Rnf126 +/- mice exhibited reduced oxygen consumption, carbon dioxide production and heat generation without changes in food intake. Mechanistically, qPCR and Western blot analyses demonstrated that RNF126 deficiency suppressed the mRNA and protein expression of UCP-1, SIRT1, and PGC-1 alpha in the liver and white adipose tissue, indicating impaired mitochondrial biogenesis. Moreover, the protein levels of cleaved PINK1, Parkin, and LC3 were reduced, while p62 expression was increased in the liver of Rnf126 +/- mice, indicating inhibited autophagic activity. Consistently, oxidative stress was significantly elevated, as evidenced by increased MDA levels and decreased SOD and GSH contents. Rnf126 +/- mice livers displayed obvious mitochondrial swelling and partial cristae disorganization with an increase of mtDNA expression. Mitochondrial complex-III enzymatic activity, hepatic ATP, TOM20 protein expression and complex-III subunit UQCRFS1 expression were substantially decreased in HFD-fed Rnf126 +/- mice. At the cellular level, RNF126 overexpression in palmitic acid-treated HepG2 cells restored cleaved PINK1, Parkin, and LC3 protein levels, while reducing p62 expression, indicating that RNF126 promotes mitophagy. Furthermore, ROS fluorescence assays showed that RNF126 overexpression attenuated intracellular ROS accumulation. Rescue experiments demonstrated that the protective effects of RNF126 on mitochondrial membrane potential, lipid accumulation, and mitophagy marker expression are dependent on its ubiquitin ligase activity. Overall, RNF126 alleviates HFD-induced lipid accumulation and hepatic injury by promoting mitophagy and maintaining redox homeostasis, suggesting its potential as a novel therapeutic target for NAFLD.
