Paricalcitol attenuates oxidative stress and inflammatory response in the liver of NAFLD rats by regulating FOXO3a and NFκB acetylation.

The lack of therapeutics along with complex pathophysiology made non-alcoholic fatty liver disease (NAFLD) a research hotspot. Studies showed that the deficiency of Vitamin D plays a vital role in NAFLD pathogenesis. While several research studies focused on vitamin D supplementation in NAFLD, there is still a need to understand the regulatory mechanism of direct vitamin D receptor activation in NAFLD. In the present study, we explored the role of direct Vitamin D receptor activation using paricalcitol in choline-deficient high-fat diet-induced NAFLD rat liver and its modulation on protein acetylation. Our results showed that paricalcitol administration significantly reduced the fat accumulation in HepG2 cells and the liver of NAFLD rats. Paricalcitol attenuated the elevated serum level of alanine transaminase, aspartate transaminase, insulin, low-density lipoprotein, triglyceride, and increased high-density lipoprotein in NAFLD rats. Paricalcitol significantly decreased the increased total protein acetylation by enhancing the SIRT1 and SIRT3 expression in NAFLD liver. Further, the study revealed that paricalcitol reduced the acetylation of NFκB and FOXO3a in NAFLD liver along with a decrease in the mRNA expression of IL1ß, NFκB, TNFα, and increased catalase and MnSOD. Moreover, total antioxidant activity, glutathione, and catalase were also elevated, whereas lipid peroxidation, myeloperoxidase, and reactive oxygen species levels were significantly decreased in the liver of NAFLD after paricalcitol administration. The study concludes that the downregulation of SIRT1 and SIRT3 in NAFLD liver was associated with an increased acetylated NFκB and FOXO3a. Paricalcitol effectively reversed hepatic inflammation and oxidative stress in NAFLD rats through transcriptional regulation of NFκB and FOXO3a, respectively, by inhibiting their acetylation.

Cholecalciferol Supplementation Impacts Behavior and Hippocampal Neuroglial Reorganization in Vitamin D-Deficient Rats.

Vitamin D deficiency (VDD) is widespread around the world and has been extensively documented to affect various health conditions, including the cognitive functioning of the brain. Serum 25-hydroxylated forms of vitamin D are traditionally used to determine vitamin D status. However, there is now evidence that cholecalciferol activation can occur and be controlled by locally expressed enzymes in the brain. This study aimed to investigate the effects of cholecalciferol supplementation on cognitive function in rats who underwent transient VDD in adulthood. Thirty-six adult Wistar rats were administered paricalcitol (seven doses of 32 ng injected every other day) along with a "vitamin D-free" diet to induce VDD, which was confirmed using a LC-MS/MS serum analysis of the cholecalciferol and 25-hydroxyvitamin D3 levels. Treatment was performed by including 1000 IU/kg and 10,000 IU/kg cholecalciferol in the diet. Cognitive performance was evaluated using the novel object recognition (NOR), Morris water maze (MWM), and radial arm maze (RAM) tests. An immunohistochemical analysis of the brain regions involved in learning and memory was performed by quantifying the neurons, astrocytes, and microglia labelled with anti-neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) antibodies, respectively. The vitamin D deficient group showed the lowest performance in both the MWM and RAM tests. In contrast, the cholecalciferol-treated groups exhibited a faster learning curve. However, no difference was detected between the groups in the NOR test. On the other hand, differences in the cellular organization of the hippocampus and amygdala were observed between the groups. Cholecalciferol supplementation decreased the density of the Iba-1- and GFAP-labeled cells in the hilus and cornu Ammonis 3 (CA3) regions of the hippocampus and in the amygdala. These results support vitamin D's substantial role in learning and memory. They also highlight that subtle changes of cognitive function induced by transient VDD could be reversed by cholecalciferol supplementation. Further studies are needed to better understand VDD and cholecalciferol's effects on the brain structure and function.

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

The effect of vitamin D2 supplementation on vitamin D levels in humans: A time and dose-response meta-analysis of randomized controlled trials.

BACKGROUND: Inconsistencies exist regarding the influence of vitamin D2 (ergocalciferol) supplementation on serum vitamin D levels. These inconsistencies could be attributed to numerous factors, such as dosage, baseline vitamin D levels, and duration of intervention. Hence, this dose-response meta-analysis of randomized controlled trials was conducted to assess the efficacy of vitamin D2 supplementation on vitamin D levels. METHODS: Relevant studies were searched in PubMed/Medline, Web of Science, Embase, and Scopus, from their inception to 3 January 2023. Variable alterations were considered to calculate the pooled weighted mean difference (WMD) with 95% confidence interval (CI) using the random effects model. RESULTS: Pooled results from 33 study arms demonstrated that Vitamin D2 treatment significantly increases total vitamin D concentrations (WMD: 11.47 ng/mL, 95 %CI: 9.29 to 13.64, p < 0.001), 25(OH)D2 concentrations (WMD: 11.40 ng/mL, 95 %CI: 4.72 to 18.09, p = 0.001), and 1,25(OH)D concentrations (WMD: 5.61 ng/mL, 95 %CI: 0.74 to 10.48, p = 0.024), but decreases 25(OH)D3 concentrations (WMD: -4.63 ng/mL, 95 %CI: -6.46 to -2.81, p < 0.001). In subgroup analyses, increase in total vitamin D concentrations was more significant in vitamin D2 doses >2000 IU/day (WMD: 13.82 ng/mL), studies with duration ≤12 weeks (WMD: 12.53 ng/mL), participants aged ≥60 years (WMD: 14.40 ng/mL), and trials with basal 25(OH)D concentrations <20 ng/mL (WMD: 11.47 ng/mL). CONCLUSIONS: This meta-analysis indicates that the supplementation of vitamin D2 significantly increases the serum concentrations of total vitamin D, 25(OH)D2, and 1,25(OH)D, but decreases 25(OH)D3 concentrations. Careful consideration of patient characteristics, dosage, and treatment duration is recommended for vitamin D2 supplementation.

Doxercalciferol alleviates UVB-induced HaCaT cell senescence and skin photoaging.

Prolonged or excessive ultraviolet (UV) exposure can lead to premature skin aging. Doxercalciferol (Dox), an analog of vitamin D2, is chiefly used to treat endocrine diseases, cardiovascular diseases, kidney diseases, etc. To date, research on Dox in alleviating photoaging and UV-induced inflammation is scarce. In this research, we evaluated the function of Dox in ultraviolet radiation B (UVB)-induced photoaging and explored the potential mechanism in human keratinocytes (Hacat) and BALB/c mice. First, we established a stable UVB-induced photoaging cell model. Then, we found that the senescence ß-galactosidase (SA-ß-Gal) positive rate, senescence-related protein (p16), aging-related genes (p21 and p53), senescence-associated secretory phenotype (SASP), inflammatory driving factors (IL-1ß and IL-6) and matrix metalloproteinases (MMPs) (MMP1 and MMP9) were upregulated in HaCaT cells after UVB irradiation. At the same time, the effect of UVB on the back skin of BALB/c mice showed a consistent trend. Dox effectively alleviated the aforementioned changes caused by UVB radiation. Mechanistically, we found that UVB activated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways, and Dox inhibited UVB-activated NF-κB and MAPK. Furthermore, Dox inhibited UVB-induced skin photoaging and damage in mice. In summary, Dox has been improved to inhibit photoaging, which may help to develop therapies to delay skin photoaging.

Paricalcitol and Extended-Release Calcifediol for Treatment of Secondary Hyperparathyroidism in Non-Dialysis Chronic Kidney Disease: Results From a Network Meta-Analysis.

CONTEXT: Secondary hyperparathyroidism (SHPT) is a complication of chronic kidney disease (CKD) affecting mineral and bone metabolism and characterized by excessive parathyroid hormone (PTH) production and parathyroid hyperplasia. OBJECTIVE: The objective of this analysis was to compare the efficacy and adverse effects of extended-release calcifediol (ERC) and paricalcitol (PCT) by assessing their effect on the biomarkers PTH, calcium, and phosphate in patients with non-dialysis CKD (ND-CKD). METHODS: A systematic literature research was performed in PubMed to identify randomized control trials (RCTs). Quality assessment was done with the GRADE method. The effects of ERC vs PCT were compared using random effects in a frequentist setting. RESULTS: Nine RCTs comprising 1426 patients were included in the analyses. The analyses were performed on 2 overlapping networks, due to nonreporting of outcomes in some of the included studies. No head-to-head trials were identified. No statistically significant differences in PTH reduction were found between PCT and ERC. Treatment with PCT showed statistically significant increases in calcium compared with ERC (0.2 mg/dL increase; 95% CI, -0.37 to -0.05 mg/dL). No differences in effects on phosphate were observed. CONCLUSION: This network meta-analysis showed that ERC is comparable in lowering PTH levels vs PCT. ERC displayed avoidance of potentially clinically relevant increases in serum calcium, offering an effective and well-tolerated treatment option for the management of SHPT in patients with ND-CKD.

Paricalcitol protects against hydrogen peroxide-induced injury in endothelial cells through suppression of apoptosis.

The vascular endothelium is one of the main targets of oxidative stress which plays an important role in the pathophysiology of vascular damage. Recent studies show that vitamin D can positively regulate endothelial functions in various chronic diseases and in cases of increased oxidative stress. In our study, we investigated the restorative and protective potentials of paricalcitol which is frequently used in patients with chronic renal failure, a vitamin D analogue, in human umbilical vein endothelial cells (HUVEC) before and after H2O2-induced oxidative stress. Paricalcitol treatment after the oxidative stress induced by H2O2 increased cell viability in endothelial cells depending on the dose that was used. While paricalcitol (500 nM) decreased caspase-3 activity and mitochondrial membrane potential loss, it increased nitric oxide (NO) production and reduced glutathione (GSH) levels. Paricalcitol treatment before oxidative stress increased cell viability. It increased NO production and mitochondrial membrane potential while significantly reducing caspase-3 activity. While paricalcitol caused a significant inhibition of protein disulfide isomerase (PDI) reductase activity in healthy endothelial cells, it did not cause a significant change on the PDI reductase activity under oxidative stress conditions. Present study showed that paricalcitol has restorative and protective effects on endothelial cells against oxidative injury, but these effects occur at high concentrations of paricalcitol.

Can paricalcitol increase the effectiveness of n-acetylcysteine in contrast induced acute kidney prophylaxis in rats? A biochemical and histopathological study / ¿Puede el paricalcitol aumentar la eficacia de la n-acetilcisteína en la profilaxis renal aguda inducida por contraste en ratas? Un estudio bioquímico e histopatológico

SUMMARY: N-Acetylcysteine (NAC) is used for contrast induced acut kidney injury (CI-AKI) prophylaxis because of its antioxidant effects. Paricalcitol, which has reno-protective effects, is likely to provide a more effective prophylaxis when added to NAC treatment. The study was designed based on this hypothesis. The study was organised to include 4 groups each consisting of 7 rats. Group 1 was the control group, and Group 2 included rats with CI-AKI. Rats in Group 3 were administered NAC at a dose of 100 mg/kg via oral gavage once a day for 5 days. Rats in group 4 were administered paricalcitol at a dose of 0.4 mcg/kg once a day for 5 days in addition to NAC. CI-AKI was induced after the treatments in both groups. The study was terminated on the sixth day. Samples were collected from the rats' sera and kidney tissues to study oxidant and antioxidant parameters; kidney function tests were also studied. There were significant differences between the contrast nephropathy group (Group 2) and NAC and NAC+paricalcitol groups with respect to serum urea and creatinine levels. When the same groups were compared regarding oxidant (TOS-MDA) and antioxidant (TAC-Paraoxonase) parameters, we observed that the oxidant parameters increased in serum and kidney tissue samples with NAC use, and that effect was strengthened by the addition of paricalcitol to NAC treatment. However, despite increased antioxidant effectiveness, we observed no decrease in urea and creatinine levels when paricalcitol was added for CI-AKI in rats. There was no significant difference between Group 3 and Group 4. Paricalcitol provides a more potent antioxidant effect in both serum and kidney tissue samples when added to NAC treatment in rats with CI-AKI. Despite increased antioxidant parameters, however, paricalcitol does not provide a significant decrease in urea and creatinine levels.

RESUMEN: Debido a sus efectos atioxidantes la N- acetilcisteína (NAC) se usa para la profilaxis de la lesión renal aguda inducida por contraste (CI-AKI). Es probable que el paricalcitol, que tiene efectos renoprotectores, proporcione una profilaxis más eficaz cuando se agrega al tratamiento con NAC. En base a esta hipótesis el estudio fue diseñado para incluir cuatro grupos cada uno compuesto por siete ratas. El grupo 1 fue el grupo control y el grupo 2 incluyó ratas con CI-AKI. A las ratas del Grupo 3 se les administró NAC con una dosis de 100 mg/kg por sonda oral una vez al día, durante 5 días. A las ratas del grupo 4 se les administró paricalcitol a una dosis de 0,4 mcg/kg una vez al día durante 5 días, además de NAC. Se indujo CI-AKI después de los tratamientos en ambos grupos. El estudio finalizó el sexto día. Se recolectaron muestras de suero y tejidos renales de ratas para estudiar los parámetros oxidantes y antioxidantes; También se estudiaron las pruebas de función renal. Hubo diferencias significativas entre el grupo de nefropatía por contraste (Grupo 2) y los grupos NAC y NAC+paricalcitol con respecto a los niveles séricos de urea y creatinina. Cuando se compararon los mismos grupos con respecto a los parámetros oxidantes (TOS-MDA) y antioxidantes (TAC-Paraoxonase), observamos que los parámetros oxidantes aumentaron en muestras de suero y tejido renal con el uso de NAC, y ese efecto se vio reforzado por la adición de paricalcitol a tratamiento NAC. Sin embargo, a pesar de una mayor eficacia antioxidante, no observamos una disminución en los niveles de urea y creatinina cuando se agregó paricalcitol para CI-AKI en ratas. No hubo diferencias significativas entre el Grupo 3 y el Grupo 4. El paricalcitol proporciona un efecto antioxidante más potente tanto en muestras de suero como de tejido renal cuando se agrega al tratamiento con NAC en ratas con CI-AKI. Sin embargo, a pesar del aumento de los parámetros antioxidantes, el paricalcitol no proporciona una disminución sig- nificativa en los niveles de urea y creatinina.

Effects of Paricalcitol on Body Composition in Vitamin D-Deficient Rats.

Background: Paricalcitol has been proposed for the treatment of secondary hyperparathyroidism in patients with renal failure and vitamin D deficiency (VDD); however, VDD is related to a range of clinical complaints. We aimed to investigate the effects of paricalcitol on body composition in VDD rats. Methods: Thirty adult male rats aged 10 weeks were randomly divided into three groups of 10, comprising control, VDD, and VDD plus paricalcitol (32 ng/rat intraperitoneal injection) (VDD+P), at the Animal Lab of the Endocrinology and Metabolism Research Center, Shiraz, Iran, in 2020. Body composition was assessed after three weeks via serum biochemical tests and dual-energy X-ray absorptiometry. Finally, the data were analyzed by using the paired-sample t test, the one-way ANOVA, and the Tukey post hoc test. Results: Global lean mass and fat mass were lower in the VDD and VDD+P groups than in the controls (P<0.001). Global fat percentage was reduced significantly in the VDD+P group (P=0.029). Conclusion: Paricalcitol reduced global fat mass and fat percentage in a rat model with VDD. Evaluation of insulin and adiponectin levels is suggested to clarify the physiology of paricalcitol in VDD states.

Paricalcitol Improves Hypoxia-Induced and TGF-ß1-Induced Injury in Kidney Pericytes.

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-ß1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-ß1 (5 ng/mL) or hypoxia (1% O2 and 5% CO2). TGF-ß1 increased α-SMA and other fibrosis markers but reduced PDGFRß expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-ß1-induced cell migration of pericytes. Hypoxia increased TGF-ß1, α-SMA and other fibrosis markers but reduced PDGFRß expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-ß1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-ß1, α-SMA upregulation, and PDGFRß downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-ß1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.

Role of vitamin D/VDR nuclear translocation in down-regulation of NF-κB/NLRP3/caspase-1 axis in lupus nephritis.

BACKGROUND: Vitamin D receptor (VDR) and NLRP3 inflammasome play critical roles in lupus nephritis (LN) pathogenesis. AIM OF THE STUDY: This study explored the therapeutic effect of VDR agonist on LN and its molecular mechanism to inhibit NLRP3 signalling. METHODS: C57BL/6 mice, lupus-prone MRL/lpr mice, and VDR agonist paricacitol-treated MRL/lpr mice (300 ng/kg/mouse per dose, 5 times/week for 8 weeks from 8 weeks old) were used to assess kidney histopathology and measure proteinuria, serum anti-ds-DNA antibody and expression of NF-κB/NLRP3/caspase-1/IL-1ß/IL-18 axis. We used mouse renal tubular epithelial cells (mRTECs) to identify protein-protein interactions and examine the effects of paricalcitol. RESULTS AND CONCLUSION: LN pathogenesis decreased after paricalcitol treatment. We observed a marked improvement in renal pathology and a time-dependent decrease urine protein and serum anti-dsDNA antibody levels. In 16-week-old MRL/lpr LN mice, the upregulated expression of NLRP3/caspase-1/IL-1ß/IL-18 axis was significantly downregulated after paricalcitol treatment. Paricalcitol can reverse the apoptosis induced by anti-dsDNA antibody via the NF-κB/NLRP3/caspase-1/IL-1ß/IL-18 axis in mRTECs. Furthermore, paricalcitol suppressed NF-κB nuclear translocation by competitively binding to importin-4. In summary, the VDR agonist can alleviate LN by modulating the NF-κB/NLRP3/caspase-1/IL-1ß/IL-18 axis and suppressing the NF-κB nuclear translocation.

VDR/Atg3 Axis Regulates Slit Diaphragm to Tight Junction Transition via p62-Mediated Autophagy Pathway in Diabetic Nephropathy.

Foot process effacement is an important feature of early diabetic nephropathy (DN), which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62-mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase3 and caspase8. Interestingly, the expression of VDR in podocytes was decreased under diabetes conditions, which impaired autophagic flux through downregulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking the p62-mediated autophagy pathway in DN.

Vitamin D suppresses bleomycin-induced pulmonary fibrosis by targeting the local renin-angiotensin system in the lung.

Idiopathic pulmonary fibrosis (IPF) is a severe disorder leading to progressive and irreversible loss of pulmonary function. In this study we investigated the anti-fibrotic effect of vitamin D using a mouse model of IPF. Lung fibrosis was induced with bleomycin in vitamin D-sufficient and vitamin D-deficient C57BL/6 mice. We found that treatment with active vitamin D analog paricalcitol prevented mouse body weight loss and alleviated lung fibrosis, whereas vitamin D deficiency severely aggravated lung injury. At the molecular level, paricalcitol treatment suppressed the induction of fibrotic inducer TGF-ß and extracellular matrix proteins α-SMA, collagen type I and fibronectin in the lung, whereas vitamin D deficiency exacerbated the induction of these proteins. Interestingly, bleomycin treatment activated the local renin-angiotensin system (RAS) in the lung, manifested by the induction of renin, angiotensinogen, angiotensin II and angiotensin receptor type 1 (AT1R). Paricalcitol treatment suppressed the induction of these RAS components, whereas vitamin D deficiency enhanced the activation of the lung RAS. We also showed that treatment of bleomycin-induced vitamin D-deficient mice with AT1R antagonist losartan relieved weight loss, substantially ameliorated lung fibrosis and markedly blocked TGF-ß induction in the lung. Moreover, we demonstrated that in lung fibroblast cultures, TGF-ß and angiotensin II synergistically induced TGF-ß, AT1R, α-SMA, collagen type I and fibronectin, whereas 1,25-dihydroxyvitamin D markedly suppressed the induction of these fibrotic markers. Collectively, these observations strongly suggest that vitamin D mitigates lung fibrosis by blocking the activation of the lung RAS in this mouse model of IPF.

Yes-associated protein regulates the hepatoprotective effect of vitamin D receptor activation through promoting adaptive bile duct remodeling in cholestatic mice.

Mounting clinical evidence has revealed that the vitamin D receptor (VDR) is associated with cholestatic liver injury, although the functions of VDR in this condition remain largely unexplored. Here, we investigated the effects of VDR activation on bile duct ligation (BDL) mice, and the underlying mechanisms were further investigated. A low-calcemic VDR agonist, paricalcitol (PAL, 200 ng/kg), was intraperitoneally injected into BDL mice every other day for 5 days or 28 days. Liver histology, liver function indicators, cholangiocyte proliferation, fibrosis scores, and inflammation were evaluated. Mice treated with PAL were rescued from the decreased survival rate induced by BDL and liver damage was reduced. Mechanistically, PAL promoted cholangiocyte proliferation, which was likely conducive to proliferating bile duct maturation and increased branching of bile ducts. PAL treatment also increased the expression of Yes-associated protein (YAP) and its target protein epithelial cell adhesion molecule (EpCam) and decreased the level of inactive cytoplasmic phosphorylated YAP. YAP knockdown abrogated PAL-induced primary bile duct epithelial cell proliferation, confirmed with YAP inhibitor administration. In addition, BDL-induced liver fibrosis and inflammatory cell infiltration were reduced by PAL treatment at both day 5 and day 28 post-BDL. In conclusion, VDR activation mitigates cholestatic liver injury by promoting adaptive bile duct remodeling through cholangiocytic YAP upregulation. Because PAL is an approved clinical drug, it may be useful for treatment of cholestatic liver disease. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Preclinical Therapy with Vitamin D3 in Experimental Encephalomyelitis: Efficacy and Comparison with Paricalcitol.

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). MS and its animal model called experimental autoimmune encephalomyelitis (EAE) immunopathogenesis involve a plethora of immune cells whose activation releases a variety of proinflammatory mediators and free radicals. Vitamin D3 (VitD) is endowed with immunomodulatory and antioxidant properties that we demonstrated to control EAE development. However, this protective effect triggered hypercalcemia. As such, we compared the therapeutic potential of VitD and paricalcitol (Pari), which is a non-hypercalcemic vitamin D analog, to control EAE. From the seventh day on after EAE induction, mice were injected with VitD or Pari every other day. VitD, but not Pari, displayed downmodulatory ability being able to reduce the recruitment of inflammatory cells, the mRNA expression of inflammatory parameters, and demyelination at the CNS. Lower production of proinflammatory cytokines by lymph node-derived cells and IL-17 by gut explants, and reduced intestinal inflammation were detected in the EAE/VitD group compared to the EAE untreated or Pari groups. Dendritic cells (DCs) differentiated in the presence of VitD developed a more tolerogenic phenotype than in the presence of Pari. These findings suggest that VitD, but not Pari, has the potential to be used as a preventive therapy to control MS severity.

Adjunctive host-directed therapies for pulmonary tuberculosis: a prospective, open-label, phase 2, randomised controlled trial.

BACKGROUND: Current tuberculosis treatments leave patients with clinically significant lung injury and increased all-cause mortality post-cure. Adjunctive host-directed therapies could protect the lungs, improve long-term survival, and shorten treatment duration; however, few have been tested clinically. Therefore, we aimed to assess the safety and preliminary efficacy of four host-directed therapies for tuberculosis. METHODS: In this prospective, open-label, phase 2, randomised controlled trial, patients with pulmonary tuberculosis were recruited at three clinical sites in South Africa. Eligible patients were aged 18-65 years, HIV-1-negative, and had rifampicin-susceptible Mycobacterium tuberculosis, a sputum Xpert cycle threshold of less than 20, and moderately advanced or far advanced disease on chest radiography. By use of numbers generated in blocks of ten and stratification by site, eligible patients were randomly assigned (1:1:1:1:1) to receive one of the four oral host-directed treatments plus standard tuberculosis treatment or standard treatment alone (the control group). Host-directed treatments were: CC-11050 (200 mg twice daily, taken with food; day 1-112); everolimus (0·5 mg/day; day 1-112); auranofin (3 mg/day for seven doses, then 6 mg/day; day 1-112); and ergocalciferol (5 mg on day 1, then 2·5 mg on day 28 and day 56). All study participants received oral rifabutin-substituted standard tuberculosis treatment for 180 days. Patients and clinicians were not masked to treatment assignment. Spirometry and sputum culture with solid and liquid media were done at baseline and up to 180 days at specified intervals throughout treatment. The primary endpoint was safety and tolerability up to day 210. Secondary preliminary efficacy endpoints were treatment effects on sputum microbiology (culture status at day 56 and the hazard ratio for stable culture conversion up to day 180) and lung function (FEV1 and forced vital capacity [FVC]) measured by spirometry at day 56, day 180, and day 540. Safety was analysed in the intention-to-treat population and preliminary efficacy primarily in the per-protocol population. The trial is registered at ClinicalTrials.gov, NCT02968927. Post-treatment follow-up was completed in 2020. FINDINGS: Between Nov 18, 2016, and Sept 27, 2018, 200 patients were screened and randomly assigned to different treatment groups (n=40 per group, apart from n=39 in the everolimus group after one patient withdrew consent). 11 treatment-emergent serious adverse events occurred either during treatment or within 30 days after treatment discontinuation, of which three were attributable to a host-directed treatment. Life-threatening thrombocytopenia occurred in an auranofin recipient; apparent intra-abdominal sepsis leading to death occurred in another auranofin recipient and was classified as a suspected unexpected serious adverse reaction. Tuberculous spondylitis occurred as an apparent paradoxical reaction in a patient receiving ergocalciferol. Two patients in the control group had life-threatening, treatment-attributable liver injury. No treatment-emergent, treatment-attributable serious adverse events occurred in patients receiving CC-11050 or everolimus. Mean FEV1 in the control group was 61·7% of predicted (95% CI 56·3-67·1) at baseline and 69·1% (62·3-75·8) at day 180. Patients treated with CC-11050 and everolimus had increased recovery of FEV1 at day 180 relative to the control group (mean difference from control group 6·30%, 95% CI 0·06-12·54; p=0·048; and 6·56%, 0·18-12·95; p=0·044, respectively), whereas auranofin and ergocalciferol recipients did not. None of the treatments had an effect on FVC during 180 days of follow-up or on measures of sputum culture status over the course of the study. INTERPRETATION: CC-11050 and everolimus were safe and reasonably well tolerated as adjunctive therapies for tuberculosis, and analysis of preliminary efficacy suggests they might also enhance the recovery of FEV1, a key measure of lung function and predictor of all-cause mortality. Further studies of these candidates are warranted. FUNDING: The Bill & Melinda Gates Foundation and the South African Medical Research Council.

Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-ß1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-ß1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-ß1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-ß1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Evaluation of the protective effect of paricalcitol and vitamin D3 at doxorubicin nephrotoxicity in rats with 99mTechnetium-dimercaptosuccinic acid renal scintigraphy and biochemical methods.

AIM: The present study aimed to examine the effect of paricalcitol (PRC) and vitamin D3 (vit D3) on doxorubicin (DOX)-induced nephrotoxicity in rats. MATERIALS AND METHODS: Forty-two Wistar rats were randomly categorized into six groups: control; 2) PRC(0.5 µg/kg) and 3) vit D3(5.000 IU/kg) administered for 14 days; 4) DOX, 18 mg/kg administered on the 12th, 13th and 14th days of the study; 5) PRC (0.5 µg/kg, +DOX(18 mg/kg); vit D3(5.000 IU)+DOX(18 mg/kg). On the 15th day of the experiment, 99mTc-DMSA uptake level and biochemical parameter in serum and tissue were assay. RESULTS: Activities of 99mTechnetium-Dimercaptosuccinic Acid (99mTc-DMSA) were lower in groups receiving DOX and/or PRC+DOX, vit D3+DOX than in control groups. The 99mTc-DMSA level in the group PRC+DOX and vit D3+DOX were importantly higher than DOX group. DOX caused an important increase in blood urea nitrogen (BUN), creatinine, Tumor Necrosis Factor-α(TNF- α), interleukin-6(IL-6) and nitric oxide(NO) levels compared to control groups. However, PRC and vit D3 pretreatments lowered them. Uptake of 99mTc-DMSA level was higher in groups PRC+DOX than in vit D3+DOX group. Administration of PRC and vit D3 alone did not change alterations all of parameters. CONCLUSION: The results indicated that PRC administration protects kidney in DOX-induced nephrotoxic rats. In addition, PRC has a stronger nephroprotective effect than vit D3.

Vitamin D/VDR attenuate cisplatin-induced AKI by down-regulating NLRP3/Caspase-1/GSDMD pyroptosis pathway.

Vitamin D/Vitamin D receptor (VDR) has been shown to inhibit the NF-κB-mediated inflammatory effects. Up-regulation of the NLRP3(Recombinant NLR Family, Pyrin Domain Containing Protein 3)/Caspase-1/GSDMD (Gasdermin D) pathway through NF-κb is one of the key mechanisms leading to pyroptosis. This study aims to explore the effects of vitamin D/VDR on the pyroptosis pathway in cisplatin induced acute kidney injury (AKI) models. Our results showed that in wide type mice, renal function loss, tissue injury and cell death induced by cisplatin were alleviated by pretreatment of high-dose paricalcitol(a VDR agonist) accompanied with up-regulated VDR and decreased expression of NLRP3, GSDMD-N, Cleaved-Caspase-1 and mature Interleukin- 1ß (features of pyroptosis). While, in VDR knock out mice, cisplatin induced more severer renal injury and further increased pyroptosis related protein than the wild type mice and the effect of paricalcitol were also eliminated. In tubular cell specific VDR-over expressing mice, those renal injury index as well as pyroptosis phenotype were significantly reduced by low-dose paricalcitol pretreatment with upregulated VDR expression compared with WT mice. In vitro data using gain and lose function experiments in Human tubular epithelial cell (HK-2) were consistent with the observation as in vivo work. Our further experiments in both animal and cell culture work has found that the level of IκBα(Inhibitor of NF-κB) were decreased and the nuclear level of NF-κB p65 of renal tubular cells were increased after cisplatin injury while VDR activation by paricalcitol could reverse up-regulation of nuclear NF-κB p65 with reduced cell pyroptosis. These data suggested that vitamin D/VDR could alleviate cisplatin-induced acute renal injury partly by inhibiting NF-κB-mediated NLRP3/Caspase-1/GSDMD pyroptosis.

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells.

BACKGROUND: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. METHODS: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). RESULTS: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. CONCLUSIONS: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs' structure might lead to new treatment options against ovarian cancer.