Acute acalculous cholecystitis complicated by infectious mononucleosis caused by cytomegalovirus.

Key Clinical Message: When seeing patients who present with atypical lymphocytes and abdominal pain without accompanying symptoms of pharyngitis or lymphadenopathy, acalculous cholecystitis caused by CMV infection should be considered as a differential diagnosis. Abstract: A teenage man presented with a fever and epigastric pain. The patient tested positive for cytomegalovirus IgG and IgM. Abdominal ultrasonography and contrast-enhanced CT revealed hepatosplenomegaly and gallbladder wall thickening. MRI did not identify gallstones or tumorous lesions. He was diagnosed with infectious mononucleosis and acalculous cholecystitis caused by cytomegalovirus.

Comparison of water balance among healthy young and old adults and handicapped adults.

The body's water balance is changed by food and beverage intake, metabolism, and excretion. In this study, we performed a cross-sectional study that investigated the changes of water intake and water output in healthy Japanese young and elderly people and handicapped adults. Water balance was assessed by water intake from foods and beverages, metabolic water production, non-renal water losses (NRWL), and urine volume. Most of the parameters did not change with aging in healthy adults. Estimated total water intake (ml/kg/day) increased with aging. In the healthy men, healthy women, and handicapped adults, daily water intake (median [interquartile range]) accounted for 49.4 (41.4-59.9) ml/kg, 42.9 (38.7-51.8) ml/kg, and 50.9 (43.8-74.0) ml/kg, respectively. Water loss from the kidney accounted for 19.2 (16.2-29.2) ml/kg, 22.0 (16.2-26.6) ml/kg, and 27.5 (22.7-47.2) ml/kg, respectively. NRWL accounted for 26.6 (18.5-35.2) ml/kg, 22.4 (16.2-28.8) ml/kg, and 23.5 (19.8-28.5) ml/kg, respectively. Our findings suggest that a daily total water intake of more than 50-55 ml/kg is required to prevent dehydration in healthy and handicapped adults. J. Med. Invest. 70 : 195-199, February, 2023.

A Prospective Observational Study Conducted at a Single Center in Japan to Validate a Previously Developed Predictive Formula of In-Hospital Mortality for Patients Aged ≥65 Years with Endogenous Diseases Transported by Ambulance.

BACKGROUND In 2019, we developed a predictive formula of in-hospital mortality for inpatients aged ≥65 years transported by ambulance for endogenous diseases. In this study, we aimed to validate this previously developed predictive formula. MATERIAL AND METHODS In this single-center prospective observational study, we enrolled all patients aged ≥65 years who were transported by ambulance and admitted to an acute care hospital in Japan for endogenous diseases. We calculated the score according to our developed formula using age, disturbance of consciousness, the shock index, and amount of oxygen administered, with each item scoring 1 point and then totaling them. We subsequently evaluated the in-hospital mortality rate, stratum-specific likelihood ratio, and area under the receiver operating characteristic curve (AUC) of the formula, using in-hospital mortality as the primary outcome. RESULTS In total, 325 patients were included in this study. Forty-two patients died during hospitalization. Multivariable logistic regression analysis (forced-entry method) revealed that disturbance of consciousness and oxygen administration 5 L/min or more were significantly associated with mortality during hospitalization. In contrast, aged ≥90 years and shock index of 1 or higher were not significant. The mortality and stratum-specific likelihood ratios for scores in the predictive formula of 3 and 4 were 40.9% and 4.66, and 66.7% and 13.48, respectively. The AUC of the predictive formula for in-hospital mortality was 0.670 (95% confidence interval: 0.574-0.767). CONCLUSIONS This study showed that our predictive formula, consisting of factors available immediately after ambulance transport in older patients, is feasible with sufficient discrimination ability and reliability.

Necessity of daily 1000-IU vitamin D supplementation for maintaining a sufficient vitamin D status.

The changes in the serum 25-hydroxyvitamin D (25(OH)D) concentrations after daily 1000-IU vitamin D intake for 3 months (3-month-VD), 6 months (6-month-VD) and then 6-month cessation of vitamin D in-take (6-month-VD cessation) were examined. The serum 25(OH)D levels in 11 male and 16 female subjects were 12.1±3.5 ng/mL at baseline, increased to 27.1±4.7 ng/mL at 3-month-VD, 28.5±5.1 ng/mL at 6-month-VD and decreased to 16.4±4.0 ng/mL at 6-month-VD cessation. The present study suggested that a vitamin D intake of 1000 IU/day is required to maintain the 25(OH) D concentration at 30 ng/mL or higher without vitamin D intoxication. J. Med. Invest. 69 : 135-140, February, 2022.

Influence of FGF23 and Klotho on male reproduction: Systemic vs direct effects.

Currently, no treatment exists to improve semen quality in most infertile men. Here, we demonstrate systemic and direct effects of Fibroblast growth factor 23 (FGF23) and Klotho, which normally regulate vitamin D and mineral homeostasis, on testicular function. Direct effects are plausible because KLOTHO is expressed in both germ cells and spermatozoa and forms with FGFR1 a specific receptor for the bone-derived hormone FGF23. Treatment with FGF23 increased testicular weight in wild-type mice, while mice with global loss of either FGF23 or Klotho had low testicular weight, reduced sperm count, and sperm motility. Mice with germ cell-specific Klotho (gcKL) deficiency neither had a change in sperm count nor sperm motility. However, a tendency toward fewer pregnancies was detected, and significantly fewer Klotho heterozygous pups originated from gcKL knockdown mice than would be expected by mendelian inheritance. Moreover, gcKL mice had a molecular phenotype with higher testicular expression of Slc34a2 and Trpv5 than wild-type littermates, which suggests a regulatory role for testicular phosphate and calcium homeostasis. KLOTHO and FGFR1 were also expressed in human germ cells and spermatozoa, and FGF23 treatment augmented the calcium response to progesterone in human spermatozoa. Moreover, cross-sectional data revealed that infertile men with the highest serum Klotho levels had significantly higher serum Inhibin B and total sperm count than men with the lowest serum Klotho concentrations. In conclusion, this translational study suggests that FGF23 and Klotho influence gonadal function and testicular mineral ion homeostasis both directly and indirectly through systemic changes in vitamin D and mineral homeostasis.

Cathepsin K-deficient osteocytes prevent lactation-induced bone loss and parathyroid hormone suppression.

Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.

In vivo evidence for an interplay of FGF23/Klotho/PTH axis on the phosphate handling in renal proximal tubules.

Phosphate homeostasis is primarily maintained in the renal proximal tubules, where the expression of sodium/phosphate cotransporters (Npt2a and Npt2c) is modified by the endocrine actions of both fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). However, the specific contribution of each regulatory pathway in the proximal tubules has not been fully elucidated in vivo. We have previously demonstrated that proximal tubule-specific deletion of the FGF23 coreceptor Klotho results in mild hyperphosphatemia with little to no change in serum levels of FGF23, 1,25(OH)2D3, and PTH. In the present study, we characterized mice in which the PTH receptor PTH1R was specifically deleted from the proximal tubules, either alone or in combination with Klotho ( PT-PTH1R-/- and PT-PTH1R/KL-/-, respectively). PT-PTH1R-/- mice showed significant increases in serum FGF23 and PTH levels, whereas serum phosphate levels were maintained in the normal range, and Npt2a and Npt2c expression in brush border membrane (BBM) did not change compared with control mice. In contrast, PT-PTH1R/KL-/- mice displayed hyperphosphatemia and an increased abundance of Npt2a and Npt2c in the renal BBM, along with increased circulating FGF23 levels. While serum calcium was normal, 1,25(OH)2D3 levels were significantly decreased, leading to extremely high levels of PTH. Collectively, mice with a deletion of PTH1R alone in proximal tubules results in only minor changes in phosphate regulation, whereas deletion of both PTH1R and Klotho leads to a severe disturbance, including hyperphosphatemia with increased sodium/phosphate cotransporter expression in BBM. These results suggest an important interplay between the PTH/PTH1R and FGF23/Klotho pathways to affect renal phosphate handling in the proximal tubules.

Parathyroid hormone controls paracellular Ca2+ transport in the thick ascending limb by regulating the tight-junction protein Claudin14.

Renal Ca2+ reabsorption is essential for maintaining systemic Ca2+ homeostasis and is tightly regulated through the parathyroid hormone (PTH)/PTHrP receptor (PTH1R) signaling pathway. We investigated the role of PTH1R in the kidney by generating a mouse model with targeted deletion of PTH1R in the thick ascending limb of Henle (TAL) and in distal convoluted tubules (DCTs): Ksp-cre;Pth1rfl/fl Mutant mice exhibited hypercalciuria and had lower serum calcium and markedly increased serum PTH levels. Unexpectedly, proteins involved in transcellular Ca2+ reabsorption in DCTs were not decreased. However, claudin14 (Cldn14), an inhibitory factor of the paracellular Ca2+ transport in the TAL, was significantly increased. Analyses by flow cytometry as well as the use of Cldn14-lacZ knock-in reporter mice confirmed increased Cldn14 expression and promoter activity in the TAL of Ksp-cre;Pth1rfl/fl mice. Moreover, PTH treatment of HEK293 cells stably transfected with CLDN14-GFP, together with PTH1R, induced cytosolic translocation of CLDN14 from the tight junction. Furthermore, mice with high serum PTH levels, regardless of high or low serum calcium, demonstrated that PTH/PTH1R signaling exerts a suppressive effect on Cldn14. We therefore conclude that PTH1R signaling directly and indirectly regulates the paracellular Ca2+ transport pathway by modulating Cldn14 expression in the TAL. Finally, systemic deletion of Cldn14 completely rescued the hypercalciuric and lower serum calcium phenotype in Ksp-cre;Pth1rfl/fl mice, emphasizing the importance of PTH in inhibiting Cldn14. Consequently, suppressing CLDN14 could provide a potential treatment to correct urinary Ca2+ loss, particularly in patients with hypoparathyroidism.

Klotho expression in osteocytes regulates bone metabolism and controls bone formation.

Osteocytes within the mineralized bone matrix control bone remodeling by regulating osteoblast and osteoclast activity. Osteocytes express the aging suppressor Klotho, but the functional role of this protein in skeletal homeostasis is unknown. Here we identify Klotho expression in osteocytes as a potent regulator of bone formation and bone mass. Targeted deletion of Klotho from osteocytes led to a striking increase in bone formation and bone volume coupled with enhanced osteoblast activity, in sharp contrast to what is observed in Klotho hypomorphic (kl/kl) mice. Conversely, overexpression of Klotho in cultured osteoblastic cells inhibited mineralization and osteogenic activity during osteocyte differentiation. Further, the induction of chronic kidney disease with high-turnover renal osteodystrophy led to downregulation of Klotho in bone cells. This appeared to offset the skeletal impact of osteocyte-targeted Klotho deletion. Thus, our findings establish a key role of osteocyte-expressed Klotho in regulating bone metabolism and indicate a new mechanism by which osteocytes control bone formation.

p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo.

p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.

In vivo evidence for a limited role of proximal tubular Klotho in renal phosphate handling.

Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3 synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo.

Fibroblast growth factor-23 and cardiac structure and function.

BACKGROUND: Fibroblast growth factor-23 (FGF-23) is a phosphaturic factor previously associated with left ventricular hypertrophy and systolic dysfunction among individuals with chronic kidney disease. Whether FGF-23 acts directly to induce left ventricular hypertrophy, potentially independent of its klotho coreceptor, remains uncertain. We investigated associations of FGF-23 with cardiac structural abnormalities among individuals with a broad range of kidney function and explored potential biological mechanisms using cardiac magnetic resonance imaging and histology in klotho-null mice, an established model of constitutively elevated FGF-23. METHODS AND RESULTS: Among 887 participants with coronary artery disease in the Heart and Soul Study, FGF-23 was modestly associated with worse left ventricular ejection fraction (-1.0% per standard deviation increase in lnFGF-23; standard error, 0.4%), but was not associated with the overall prevalence of concentric hypertrophy (odds ratio, 1.5; CI, 0.9 to 2.4) or eccentric hypertrophy (odds ratio, 1.1; CI, 0.9 to 1.3). FGF-23 was only associated with concentric hypertrophy among individuals with diminished kidney function (eGFR <60 mL/min per 1.73 m(2); odds ratio, 2.3; CI, 1.0 to 5.3; P-interaction=0.28). Comparing klotho-null with wild-type mice, null mice did not have greater left ventricular mass (P=0.37) or a lower ejection fraction (P=0.94). CONCLUSIONS: Together, our results suggest that FGF-23 is unlikely to have major effects on cardiovascular structure and function among patients free of substantial chronic kidney disease, and these effects may not be independent of the klotho coreceptor.

Interleukin 27 inhibits atherosclerosis via immunoregulation of macrophages in mice.

Chronic inflammation in arterial wall that is driven by immune cells and cytokines plays pivotal roles in the development of atherosclerosis. Interleukin 27 (IL-27) is a member of the IL-12 family of cytokines that consists of IL-27p28 and Epstein-Barr virus induced gene 3 (EBI3) and has anti-inflammatory properties that regulate T cell polarization and cytokine production. IL-27-deficient (Ldlr-/-Ebi3-/-) and IL-27 receptor-deficient (Ldlr-/-WSX-1-/-) Ldlr-/- mice were generated and fed with a high-cholesterol diet to induce atherosclerosis. Roles of bone marrow-derived cells in vivo and macrophages in vitro were studied using bone marrow reconstitution by transplantation and cultured peritoneal macrophages, respectively. We demonstrate that mice lacking IL-27 or IL-27 receptor are more susceptible to atherosclerosis compared with wild type due to enhanced accumulation and activation of macrophages in arterial walls. The number of circulating proinflammatory Ly6C(hi) monocytes showed no significant difference between wild-type mice and mice lacking IL-27 or IL-27 receptor. Administration of IL-27 suppressed the development of atherosclerosis in vivo and macrophage activation in vitro that was indicated by increased uptake of modified low-density lipoprotein and augmented production of proinflammatory cytokines. These findings define a novel inhibitory role for IL-27 in atherosclerosis that regulates macrophage activation in mice.

[Vascular endothelial dysfunction].

The endothelium modulates vascular tone, blood coagulation, cell growth, and inflammation throughout the circulatory system. Endothelial dysfunction has been proposed to be one of the initiating events of atherosclerosis and is characterized by unbalanced concentrations of vasodilating and vasoconstricting factors, the most important being nitric oxide (NO) and angiotensin II (AII), respectively. In endothelial cells, AII, the key effector of the renin-angiotensin system (RAS), stimulates the production of reactive oxygen species (ROS) and negatively regulates the NO signaling pathway and thereby induces endothelial dysfunction. RAS blockade with an angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin receptor blockers(ARBs) provides a rational approach to reverse endothelial dysfunction. In this review, attention has been specifically focused on recent findings and putative mechanisms of the beneficial effects of RAS blockade on endothelial function.

Angiotensin II increases expression of IP-10 and the renin-angiotensin system in endothelial cells.

Angiotensin II promotes vascular inflammation, which plays important roles in vascular injury. In this study, we found that angiotensin II-stimulated human endothelial cells increased the release of a CXC chemokine, IP-10, according to an antibody array. IP-10 expression was higher in the endothelium of coronary blood vessels in mice infused with angiotensin II than in control. Quantitative real-time PCR analysis revealed that angiotensin II significantly increased IP-10 mRNA expression compared to control. Pretreatment with valsartan, but not with PD123319, blocked angiotensin II-induced IP-10 mRNA expression. IP-10 levels in conditioned media detected by ELISA increased in response to angiotensin II compared to control, which was blocked by the pretreatment with valsartan. These data indicate that angiotensin II stimulates IP-10 production from endothelial cells via angiotensin II type 1 receptors. In endothelial cells, IP-10 significantly increased mRNA expression of renin, angiotensin-converting enzyme, and angiotensinogen. IP-10 also increased angiotensin II levels in conditioned media compared to control. Angiotensin II significantly increased mRNA expression of renin, angiotensin converting enzyme and angiotensinogen, which was blocked by neutralization of IP-10 with antibody in endothelial cells. IP-10 neutralization with antibody blocked angiotensin II-induced apoptosis and cell senescence in endothelial cells. These data indicate that IP-10 is involved not only in leukocyte-endothelial interaction but also in the circuit of endothelial renin-angiotensin system activation that potentially promotes atherosclerosis.

Simvastatin stimulates vascular endothelial growth factor production by hypoxia-inducible factor-1alpha upregulation in endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and plays an important pathophysiological role in the maintenance of tissue structure as well as regeneration after ischemic injury. Three-hydroxy-3methylglutaryl-CoA reductase inhibitors reduce vascular inflammation and induce angiogenesis. This study examined whether simvastatin stimulates VEGF expression in endothelial cells as well as the nature of its underlying mechanism. METHODS AND RESULTS: Simvastatin induced mRNA expression and protein secretion of VEGF in endothelial cells that were reversed by pretreatment with mevalonate and geranylgeranylpyrophosphate but not by farnesylpyrophosphate. Adenovirus-mediated expression of the dominant-negative mutant of RhoA induced VEGF mRNA and protein. Simvastatin increased hypoxia-inducible factor-1alpha (HIF-1alpha) protein level without changing its mRNA expression. Inhibition of RhoA had similar effects to simvastatin on VEGF expression. Inhibition of RhoA caused the translocation of HIF-1alpha to the nuclear fraction. Depletion of HIF-1alpha by RNA interference blocked simvastatin-induced VEGF mRNA expression. CONCLUSIONS: Simvastatin stimulates VEGF expression by RhoA downregulation and HIF-1alpha upregulation in endothelial cells. These data indicate a novel role for RhoA as a negative regulator of HIF-1alpha.

Male gametic cell-specific histone gH2A gene of Lilium longiflorum: genomic structure and promoter activity in the generative cell.

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5' upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5' upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.