The Role of Organic Cation Transporters in the Pharmacokinetics, Pharmacodynamics and Drug-Drug Interactions of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) decisively contributed in revolutionizing the therapeutic approach to cancer, offering non-invasive, tolerable therapies for a better quality of life. Nonetheless, degree and duration of the response to TKI therapy vary depending on cancer molecular features, the ability of developing resistance to the drug, on pharmacokinetic alterations caused by germline variants and unwanted drug-drug interactions at the level of membrane transporters and metabolizing enzymes. A great deal of approved TKIs are inhibitors of the organic cation transporters (OCTs). A handful are also substrates of them. These transporters are polyspecific and highly expressed in normal epithelia, particularly the intestine, liver and kidney, and are, hence, arguably relevant sites of TKI interactions with other OCT substrates. Moreover, OCTs are often repressed in cancer cells and might contribute to the resistance of cancer cells to TKIs. This article reviews the OCT interactions with approved and in-development TKIs reported in vitro and in vivo and critically discusses the potential clinical ramifications thereof.

Prognostic Impact of the Angiogenic Gene POSTN and Its Related Genes on Lung Adenocarcinoma.

Background: The function of angiogenesis-related genes (ARGs) in lung adenocarcinoma (LUAD) remains poorly documented. This study was designed to reveal ARGs in LUAD and related networks. Methods: We worked with sequencing data and clinical information pertaining to LUAD from public databases. ARGs were retrieved from the HALLMARK_ANGIOGENESIS gene set. Differential analysis and Kaplan-Meier (K-M) analysis were performed to authenticate the ARGs associated with LUAD. Weighted gene correlation network analysis was performed on the mining hub genes linked to the abovementioned genes, and functional enrichment analysis was done. Subsequently, Cox regression analyses were used to construct the prognostic gene. POSTN and microvessel density were detected using immunohistochemistry. Results: POSTN, an ARG that was highly expressed in patients with LUAD and was closely associated with their weak overall survival was identified. Differentially expressed genes associated with POSTN were mainly enriched in entries related to the tubulointerstitial system, immune response, and epithelial cells. A positive correlation was demonstrated between POSTN expression and tumor microvessel density in LUAD. Subsequently, a prognostic gene signature was constructed and revealed that 4 genes may predict the survival of LUAD patients. Furthermore, the ESTIMATE and CIBERSORT analyses suggested that our risk scoring system may be implicated in altering the immune microenvironment of patients with LUAD. Finally, a ceRNA network was constructed based on the prognostic genes, and the regulatory networks were examined. Conclusion: POSTN, a novel prognostic gene signature associated with ARGs, was constructed for the prognosis of patients with LUAD. This signature may alter the immune microenvironment by modulating the activation of the tubulointerstitial system, epithelial cells, and immune cells, ultimately affecting patient survival.

SR-BI as a target of natural products and its significance in cancer.

Scavenger receptor class B type I (SR-BI) protein is an integral membrane glycoprotein. SR-BI is emerging as a multifunctional protein, which regulates autophagy, efferocytosis, cell survival and inflammation. It is well known that SR-BI plays a critical role in lipoprotein metabolism by mediating cholesteryl esters selective uptake and the bi-directional flux of free cholesterol. Recently, SR-BI has also been identified as a potential marker for cancer diagnosis, prognosis, or even a treatment target. Natural products are a promising source for the discovery of new drug leads. Multiple natural products were identified to regulate SR-BI protein expression. There are still a number of challenges in modulating SR-BI expression in cancer and in using natural products for modulation of such protein expression. In this review, our purpose is to discuss the relationship between SR-BI protein and cancer, and the molecular mechanisms regulating SR-BI expression, as well as to provide an overview of natural products that regulate SR-BI expression.

Antioxidant Effects of Protocatechuic Acid and Protocatechuic Aldehyde: Old Wine in a New Bottle.

Phenolic compounds are naturally present as secondary metabolites in plant-based sources such as fruits, vegetables, and spices. They have received considerable attention for their antioxidant, anti-inflammatory, and anti-carcinogenic properties for protection against many chronic disorders such as neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. They are categorized into various groups based on their chemical structure and include phenolic acids, flavonoids, curcumins, tannins, and quinolones. Their structural variations contribute to their specific beneficial effects on human health. The antioxidant property of phenolic compounds protects against oxidative stress by up-regulation of endogenous antioxidants, scavenging free radicals, and anti-apoptotic activity. Protocatechuic acid (PCA; 3,4-dihydroxy benzoic acid) and protocatechuic aldehyde (PAL; 3,4-dihydroxybenzaldehyde) are naturally occurring polyphenols found in vegetables, fruits, and herbs. PCA and PAL are the primary metabolites of anthocyanins and proanthocyanidins, which have been shown to possess pharmacological actions including antioxidant activity in vitro and in vivo. This review aims to explore the therapeutic potential of PCA and PAL by comprehensively summarizing their pharmacological properties reported to date, with an emphasis on their mechanisms of action and biological properties.

The Role of the Carnitine/Organic Cation Transporter Novel 2 in the Clinical Outcome of Patients With Locally Advanced Esophageal Carcinoma Treated With Oxaliplatin.

Esophageal cancer is the ninth most common malignancy worldwide, ranking sixth in mortality. Platinum-based chemotherapy is commonly used for treating locally advanced esophageal cancer, yet it is ineffective in a large portion of patients. There is a need for reliable molecular markers with direct clinical application for a prospective selection of patients who can benefit from chemotherapy and patients in whom toxicity is likely to outweigh the benefit. The cytotoxic activity of platinum derivatives largely depends on the uptake and accumulation into cells, primarily by organic cation transporters (OCTs). The aim of the study was to investigate the impact of OCT expression on the clinical outcome of patients with esophageal cancer treated with oxaliplatin. Twenty patients with esophageal squamous cell carcinoma (SCC) were prospectively enrolled and surgical specimens used for screening OCT expression level by western blotting and/or immunostaining, and for culture of cancer cells. Sixty-seven patients with SCC who received oxaliplatin and for whom follow-up was available were retrospectively assessed for organic cation/carnitine transporter 2 (OCTN2) expression by real time RT-PCR and immunostaining. OCTN2 staining was also performed in 22 esophageal adenocarcinomas. OCTN2 function in patient-derived cancer cells was evaluated by assessing L-carnitine uptake and sensitivity to oxaliplatin. The impact of OCTN2 on oxaliplatin activity was also assessed in HEK293 cells overexpressing OCTN2. OCTN2 expression was higher in tumor than in normal tissues. In patient-derived cancer cells and HEK293 cells, the expression of OCTN2 sensitized to oxaliplatin. Patients treated with oxaliplatin who had high OCTN2 level in the tumor tissue had a reduced risk of recurrence and a longer survival time than those with low expression of OCTN2 in tumor tissue. In conclusion, OCTN2 is expressed in esophageal cancer and it is likely to contribute to the accumulation and cytotoxic activity of oxaliplatin in patients with esophageal carcinoma treated with oxaliplatin.

The Role of Mitochondria in Drug-Induced Kidney Injury.

The kidneys utilize roughly 10% of the body's oxygen supply to produce the energy required for accomplishing their primary function: the regulation of body fluid composition through secreting, filtering, and reabsorbing metabolites and nutrients. To ensure an adequate ATP supply, the kidneys are particularly enriched in mitochondria, having the second highest mitochondrial content and thus oxygen consumption of our body. The bulk of the ATP generated in the kidneys is consumed to move solutes toward (reabsorption) or from (secretion) the peritubular capillaries through the concerted action of an array of ATP-binding cassette (ABC) pumps and transporters. ABC pumps function upon direct ATP hydrolysis. Transporters are driven by the ion electrochemical gradients and the membrane potential generated by the asymmetric transport of ions across the plasma membrane mediated by the ATPase pumps. Some of these transporters, namely the polyspecific organic anion transporters (OATs), the organic anion transporting polypeptides (OATPs), and the organic cation transporters (OCTs) are highly expressed on the proximal tubular cell membranes and happen to also transport drugs whose levels in the proximal tubular cells can rapidly rise, thereby damaging the mitochondria and resulting in cell death and kidney injury. Drug-induced kidney injury (DIKI) is a growing public health concern and a major cause of drug attrition in drug development and post-marketing approval. As part of the article collection "Mitochondria in Renal Health and Disease," here, we provide a critical overview of the main molecular mechanisms underlying the mitochondrial damage caused by drugs inducing nephrotoxicity.

Untargeted Metabolomics Reveals Anaerobic Glycolysis as a Novel Target of the Hepatotoxic Antidepressant Nefazodone.

Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 µM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.

Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p.

Chronic inflammation plays a crucial role in vascular calcification. However, only a few studies have revealed the mechanisms underlying the development of inflammation under high-phosphate conditions in chronic kidney disease (CKD) patients. Here, we show that inflammation resulting from the activation of the TGFBR1/TAK1 pathway is involved in calcification in CKD rats or osteogenic medium-cultured human aortic smooth muscle cells (HASMCs). Moreover, miR-135a-5p is demonstrated to be a key regulator of the TGFBR1/TAK1 pathway, which has been reported to be decreased in CKD rats. We further reveal that farnesoid X receptor (FXR) activation increases miR-135a-5p expression, thereby inhibiting the activation of the TGFBR1/TAK1 pathway, ultimately resulting in the attenuation of vascular inflammation and calcification in CKD rats. Our findings provide advanced insights into the mechanisms underlying the development of inflammation in vascular calcification, and evidence that FXR activation could serve as a therapeutic strategy for retarding vascular calcification in CKD patients.

Obeticholic Acid Ameliorates Valproic Acid-Induced Hepatic Steatosis and Oxidative Stress.

Farnesoid X receptor (FXR), or NR1H4, protects the liver from insults of various etiologies. A role of FXR in drug-induced liver injury has also been hypothesized yet only marginally investigated. The aim of this study was to assess the effect of FXR activation on gene expression and phenotype of the liver of mice treated with valproic acid (VPA), or 2-propylpentanoic acid, a prototypical hepatotoxic drug. Obeticholic acid (OCA) was used to activate FXR both in mice and in human hepatocellular carcinoma (Huh-7) cells. Next-generation sequencing of mouse liver tissues was performed from control, VPA, and VPA + OCA-treated mice. Pathway analysis validation was performed using real-time reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry, and fluorometric assays. FXR activation induced antioxidative pathways, which was confirmed by a marked reduction in VPA-induced lipid peroxidation and endoplasmic reticulum stress. In vitro, VPA-induced oxidative stress was independent of lipid accumulation, stemmed from the cytoplasm, and was mitigated by OCA. In the liver of the mice treated with OCA, the levels of cytochrome P450 potentially involved in VPA metabolism were increased. The hepatic lipid-lowering effect observed in animals cotreated with VPA and OCA in comparison with that of animals treated with VPA was associated with regulation of the genes involved in the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) pathway. In conclusion, pronounced antioxidant activity, repression of the PPARγ pathway, and higher expression of P450 enzymes involved in VPA metabolism may underlie the hepatoprotective of FXR activation during VPA treatment. SIGNIFICANCE STATEMENT: Valproic acid-induced oxidative stress occurs in absence of lipid accumulation and is not of mitochondrial origin. Valproic acid exposure induces the expression of the steatogenic nuclear receptor peroxisome proliferator-activated γ (PPARγ) and its downstream target genes. Constitutive activation of the farnesoid X receptor (FXR) reduces PPARγ hepatic expression and induces hepatic antioxidant activity. The variability in FXR expression level/activity, for instance in individuals carrying loss-of-function genetic variants of the FXR gene, could contribute to valproic acid pharmacokinetic and toxicokinetic profile.

Effects of Farnesiferol B on Ischemia-Reperfusion-Induced Renal Damage, Inflammation, and NF-κB Signaling.

BACKGROUND: G-protein-coupled bile acid receptor (TGR5), a membrane bile acid receptor, regulates macrophage reactivity, and attenuates inflammation in different disease models. However, the regulatory effects of TGR5 in ischemia/reperfusion (I/R)-induced kidney injury and inflammation have not yet been extensively studied. Therefore, we hypothesize that Farnesiferol B, a natural TGR5 agonist, could alleviate renal I/R injury by reducing inflammation and macrophage migration through activating TGR5. METHODS: Mice were treated with Farnesiferol B before I/R or sham procedures. Renal function, pathological analysis, and inflammatory mediators were examined. In vitro, the regulatory effects of Farnesiferol B on the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in macrophages were investigated. RESULTS: After I/R, Farnesiferol B-treated mice displayed better renal function and less tubular damage. Farnesiferol B reduced renal oxidative stress and inflammation significantly. In vitro, Farnesiferol B treatment alleviated lipopolysaccharide (LPS)-induced macrophage migration and activation, as well as LPS-induced NF-κB activation through TGR5. CONCLUSIONS: Farnesiferol B could protect kidney function from I/R-induced damage by attenuating inflammation though activating TGR5 in macrophages. Farnesiferol B might be a potent TGR5 ligand for the treatment of I/R-induced renal inflammation.

Renal Reabsorption of Folates: Pharmacological and Toxicological Snapshots.

Folates are water-soluble B9 vitamins that serve as one-carbon donors in the de novo synthesis of thymidylate and purines, and in the conversion of homocysteine to methionine. Due to their key roles in nucleic acid synthesis and in DNA methylation, inhibiting the folate pathway is still one of the most efficient approaches for the treatment of several tumors. Methotrexate and pemetrexed are the most prescribed antifolates and are mainly used in the treatment of acute myeloid leukemia, osteosarcoma, and lung cancers. Normal levels of folates in the blood are maintained not only by proper dietary intake and intestinal absorption, but also by an efficient renal reabsorption that seems to be primarily mediated by the glycosylphosphatidylinositol- (GPI) anchored protein folate receptor α (FRα), which is highly expressed at the brush-border membrane of proximal tubule cells. Folate deficiency due to malnutrition, impaired intestinal absorption or increased urinary elimination is associated with severe hematological and neurological deficits. This review describes the role of the kidneys in folate homeostasis, the molecular basis of folate handling by the kidneys, and the use of high dose folic acid as a model of acute kidney injury. Finally, we provide an overview on the development of folate-based compounds and their possible therapeutic potential and toxicological ramifications.

Renal glycosuria as a novel early sign of colistin-induced kidney damage in mice.

The polymixin colistin represents a last resort antibiotic for multidrug resistant infections, but its use is limited by the frequent onset of acute drug-induced kidney injury (DIKI). It is essential to closely monitor kidney function prior to and during colistin treatment in order to pinpoint early signs of injury and minimise long-term renal dysfunction. To facilitate this, a mouse model of colistin-induced nephrotoxicity was used to uncover novel early markers of colistin-induced DIKI. Increased urinary levels of kidney injury molecule 1 (Kim-1) as well as glycosuria were observed in colistin-treated mice, where alterations of established clinical markers of acute kidney injury (serum creatinine and albuminuria) and emerging markers such as cystatin C were inaccurate in flagging renal damage as confirmed by histology. A direct interaction of colistin with renal glucose reabsorption was ruled out by a cis-inhibition assay in mouse brush border membrane vesicles (BBMV). Immunohistochemical examination and protein quantification by western blotting showed a marked reduction in the protein amount of sodium-glucose transporter 2 (Sglt2), the main kidney glucose transporter, in renal tissue from colistin-treated mice in comparison to control animals. Consistently, BBMV isolated from treated mouse kidneys also showed a reduction in ex vivo glucose uptake when compared to BBMV isolated from control kidneys. These findings support pathology observations of colistin-induced proximal tubule damage at the site of the brush border membrane, where Sglt2 is expressed, and open avenues for the study of glycosuria as a sensitive, specific, and accessible marker of DIKI during colistin therapy.

microRNA-206 modulates the hepatic expression of the organic anion-transporting polypeptide 1B1.

BACKGROUND & AIMS: The organic anion-transporting polypeptide 1B1 (OATP1B1) is an anion exchanger expressed at the hepatocyte sinusoidal membrane, which mediates the uptake of several endogenous metabolites and drugs. OATP1B1 expression level and activity are major sources of inter-patient variability of pharmacokinetics and pharmacodynamics of several drugs. Besides the genotype, factors that contribute to the inter-individual variability in OATP1B1 expression level are practically unknown. The aim of this work was to uncover novel epigenetic mechanisms of OATP1B1 regulation. METHODS: A functional screening strategy to assess the effect of microRNAs on the uptake of estrone-3-sulphate, an OATP1B1 substrate, into human hepatocellular carcinoma (Huh-7) cells was used. microRNA-206 (miR-206) expression in human liver tissues was measured by real-time RT-PCR. OATP1B1 expression in Huh-7 and in human liver tissues was assessed by real-time RT-PCR, Western blotting and immunostaining. The mRNA-miRNA interaction was assessed by reporter assay. RESULTS: miR-206 mimic repressed mRNA and protein expression of OATP1B1 in Huh-7 cells. The intracellular accumulation of estrone-3-sulphate was reduced by 30% in cells overexpressing miR-206. The repressive effect of miR-206 on the activity of the firefly luciferase gene 2 under the control of the OATP1B1 3' untranslated region was lost upon deletion of the predicted miR-206 binding site. Hepatic miR-206 level negatively correlated with OATP1B1 mRNA and protein levels extracted from normal human liver tissues. CONCLUSIONS: miR-206 exerts a suppressive effect on OATP1B1 expression by an epigenetic mechanism. Individuals with high hepatic levels of miR-206 appear to display lower level of OATP1B1.

Low expression level of ASK1-interacting protein-1 correlated with tumor angiogenesis and poor survival in patients with esophageal squamous cell cancer.

OBJECTIVE: To investigate the expression of tumor suppressor protein ASK1-interacting protein-1 (AIP1) in human esophageal squamous cell carcinoma (ESCC) and its role in tumor progression, angiogenesis, and prognosis. METHODS: A total of 117 biopsy samples were obtained from ESCC patients. None of the patients had distant metastasis before surgery, and did not receive preoperative chemotherapy or radiotherapy. Immunohistochemistry was used to detect the expression of AIP1 protein and vascular endothelial growth factor receptor 2 (VEGFR2) in ESCC specimens collected from 117 patients who underwent esophageal cancer radical surgery. Microvessel density (MVD) was evaluated by immunohistochemical staining of vascular endothelial CD34. The correlation between AIP1 protein and clinicopathological characteristics, tumor angiogenesis, and prognosis was analyzed. RESULTS: The downregulation of AIP1 protein in esophageal carcinoma tissues was detected in 63 cases. This downregulation significantly correlated with lymph node metastasis, clinicopathological staging, and tumor MVD (P<0.05). Survival analysis showed that ESCC patients with a low expression of AIP1, a high expression of VEGFR2, and a high level of MVD had a lower 5-year survival rate (P<0.05). Multivariate analysis confirmed that the downregulation of AIP1 significantly affected patient survival. CONCLUSION: The downregulation of AIP1 correlated with ESCC progression, tumor angiogenesis, and poor prognosis. AIP1 could be a promising biomarker for predicting ESCC prognosis and a potential target for anti-angiogenic therapy.

Fluorocholine Transport Mediated by the Organic Cation Transporter 2 (OCT2, SLC22A2): Implication for Imaging of Kidney Tumors.

[18F]fluorocholine is the fluorinated analog of [11C]choline and is used in positron emission tomography to monitor tumor metabolic activity. Although important to optimize its use and expand the clinical indications, the molecular determinants of fluorocholine cellular uptake are poorly characterized. In this work, we described the influx kinetics of fluorocholine mediated by the organic cation transporter 2 (OCT2, SLC22A2) and compared with that of choline. Then we characterized the expression pattern of OCT2 in renal cell carcinoma (RCC). In HEK293 cells stably transfected with OCT2 fluorocholine influx, kinetics was biphasic, suggesting two independent binding sites: a high-affinity (Km = 14 ± 8 µM, Vmax = 1.3 ± 0.5 nmol mg-1 min-1) and a low-affinity component (Km = 1.8 ± 0.3 mM, Vmax = 104 ± 4.5 nmol mg-1 min-1). Notably, choline was found to be transported with sigmoidal kinetics typical of homotropic positive cooperativity (h = 1.2, 95% confidence interval 1.1-1.3). OCT2 mRNA expression level was found significantly decreased in primary but not in metastatic RCC. Tissue microarray immunostaining of 216 RCC biopsies confirmed that the OCT2 protein level was consistent with that of the mRNA. The kinetic properties described in this work suggest that OCT2 is likely to play a dominant role in [18F]fluorocholine uptake in vivo. OCT2-altered expression in primary and metastatic cancer cells, as compared with the surrounding tissues, could be exploited in RCC imaging, especially to increase the detection sensitivity for small metastatic lesions, a major clinical challenge during the initial staging of RCC.

Drug-induced bile duct injury.

Drug-induced liver injury includes a spectrum of pathologies, some related to the mode of injury, some to the cell type primarily damaged. Among these, drug-induced bile duct injury is characterized by the destruction of the biliary epithelium following exposure to a drug. Most of the drugs associated with bile duct injury cause immune-mediated lesions to the epithelium of interlobular ducts. These share common histopathological features with primary biliary cholangitis, such as inflammation and necrosis at the expense of cholangiocytes and, if the insult persists, bile duct loss and biliary cirrhosis. Some drugs selectively target larger ducts. Such injury is often dose-dependent and thought to be the result of intrinsic drug toxicity. The histological changes resemble those seen in primary sclerosing cholangitis. This overview focuses on the clinical and pathological features of bile duct injury associated with drug treatment and on the immunological and biochemical effects that drugs exert on the biliary epithelium. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

TNF-α Deficiency Prevents Renal Inflammation and Oxidative Stress in Obese Mice.

BACKGROUND/AIMS: Obese patients and experimental animals exhibit high levels of inflammatory cytokines, such as tumor necrosis factor (TNF)-α. However, the role of TNF-α in the pathophysiologic process in obesity induced kidney damage is still unknown. METHODS: We used TNF-α deficient mice and wild-type (WT) C57/BJ6 mice controls to study the effect of TNF-α on inflammation and oxidative stress in kidney by the model of high-fat diet (HFD) and primary isolated mouse renal proximal tubule cells treated with a mixture of free fatty acids (FFA). RESULTS: Compared with the chow diet group, HFD-fed WT mice had higher urinary albumin and increased levels of renal fibrosis, glomerulosclerosis, inflammation, oxidative stress and apoptosis in the kidney. These changes were co-related with increased expression of TNF-α in the kidney and were attenuated by TNF-α deficiency. In vitro, accumulation of intracellular lipids induced TNF-α expression and oxidative stress in FFA treated primary proximal tubule cells. However, TNF-α inhibition with siRNA or TNF-α deficiency decreased the lipid induced oxidative stress in these cells. CONCLUSION: These findings suggest that TNF-α plays an important role in the HFD induced kidney damage, and targeting TNF-α and/or its receptors could be a promising therapeutic regimen for progressive nephropathy.

Vitamin D3 transactivates the zinc and manganese transporter SLC30A10 via the Vitamin D receptor.

Vitamin D3 regulates genes critical for human health and its deficiency is associated with an increased risk for osteoporosis, cancer, diabetes, multiple sclerosis, hypertension, inflammatory and immunological diseases. To study the impact of vitamin D3 on genes relevant for the transport and metabolism of nutrients and drugs, we employed next-generation sequencing (NGS) and analyzed global gene expression of the human-derived Caco-2 cell line treated with 500nM vitamin D3. Genes involved in neuropeptide signaling, inflammation, cell adhesion and morphogenesis were differentially expressed. Notably, genes implicated in zinc, manganese and iron homeostasis were largely increased by vitamin D3 treatment. An ∼10-fold increase in ceruloplasmin and ∼4-fold increase in haptoglobin gene expression suggested a possible association between vitamin D and iron homeostasis. SLC30A10, the gene encoding the zinc and manganese transporter ZnT10, was the chiefly affected transporter, with ∼15-fold increase in expression. SLC30A10 is critical for zinc and manganese homeostasis and mutations in this gene, resulting in impaired ZnT10 function or expression, cause manganese intoxication, with Parkinson-like symptoms. Our NGS results were validated by real-time PCR in Caco-2 cells, as well as in duodenal biopsies taken from healthy human subjects treated with 0.5µg vitamin D3 daily for 10 days. In addition to increasing gene expression of SLC30A10 and the positive control TRPV6, vitamin D3 also increased ZnT10 protein expression, as indicated by Western blot and cytofluorescence. In silico identification of potential vitamin D responsive elements (VDREs) in the 5'-flanking region of the SLC30A10 promoter and dual-luciferase reporter assay showed enhanced promoter activity in the presence of vitamin D receptor (VDR) and retinoid X receptor (RXR) constructs, as well as vitamin D3, but not when one of these factors was absent. Electrophoretic mobility shift assay (EMSA) and competition EMSA revealed binding of select sequences, namely, nt -1623/-1588 and nt -1758/-1723 relative to the transcription start site, to VDR-containing nuclear extracts. In conclusion, we have shown that vitamin D3 transactivates the SLC30A10 gene in a VDR-dependent manner, resulting in increased ZnT10 protein expression. Because SLC30A10 is highly expressed in the small intestine, it is possible that the control of zinc and manganese systemic levels is regulated by vitamin D3 in the intestine. Zinc, manganese and vitamin D are important for bone metabolism and brain health. Future examination of a possible role for supplementation or chelation of zinc and manganese, alongside vitamin D3 administration, will further our understanding of its potential benefit in the treatment of specific illnesses, such as osteoporosis and Parkinson's disease.

Farnesoid X Receptor Protects against Kidney Injury in Uninephrectomized Obese Mice.

Activation of the farnesoid X receptor (FXR) has indicated a therapeutic potential for this nuclear bile acid receptor in the prevention of diabetic nephropathy and obesity-induced renal damage. Here, we investigated the protective role of FXR against kidney damage induced by obesity in mice that had undergone uninephrectomy, a model resembling the clinical situation of kidney donation by obese individuals. Mice fed a high-fat diet developed the core features of metabolic syndrome, with subsequent renal lipid accumulation and renal injury, including glomerulosclerosis, interstitial fibrosis, and albuminuria. The effects were accentuated by uninephrectomy. In human renal biopsies, staining of 4-hydroxynonenal (4-HNE), glucose-regulated protein 78 (Grp78), and C/EBP-homologous protein, markers of endoplasmic reticulum stress, was more prominent in the proximal tubules of 15 obese patients compared with 16 non-obese patients. In mice treated with the FXR agonist obeticholic acid, renal injury, renal lipid accumulation, apoptosis, and changes in lipid peroxidation were attenuated. Moreover, disturbed mitochondrial function was ameliorated and the mitochondrial respiratory chain recovered following obeticholic acid treatment. Culturing renal proximal tubular cells with free fatty acid and FXR agonists showed that FXR activation protected cells from free fatty acid-induced oxidative stress and endoplasmic reticulum stress, as denoted by a reduction in the level of reactive oxygen species staining and Grp78 immunostaining, respectively. Several genes involved in glutathione metabolism were induced by FXR activation in the remnant kidney, which was consistent with a decreased glutathione disulfide/glutathione ratio. In summary, FXR activation maintains endogenous glutathione homeostasis and protects the kidney in uninephrectomized mice from obesity-induced injury.

Bile acids but not acidic acids induce Barrett's esophagus.

Barrett's esophagus (BE) is associated with the development of esophageal adenocarcinoma (EAC). Bile acids (BAs) refluxing into the esophagus contribute to esophageal injury, which results in BE and subsequent EAC. We developed two animal models to test the role of BAs in the pathogenesis of BE. We surgically generated BA reflux, with or without gastric acid, in rats. In a second experiment, we fed animals separately with BAs and gastric acid. Pathologic changes were examined and the expression of Muc2 and Cdx2 in BE tissue was tested by immunostaining. Inflammatory factors in the plasma, as well as differentiation genes in BE were examined through highly sensitive ELISA and semi-quantitative RT-PCR techniques. We found that BAs are sufficient for the induction of esophagitis and Barrett's-like metaplasia in the esophagus. Overexpression of inflammatory cells, IL-6, and TNF-α was observed both in animals fed with BAs and surgically generated BA reflux. Furthermore, elevated levels of Cdx2, Muc2, Bmp4, Kit19, and Tff2 (differentiation genes in BE) were found in BA-treated rats. In conclusion, BAs, but not gastric acid, are a major causative factor for BE. We confirmed that BAs contribute to the development of BE by inducing the inflammatory response in the esophagus. Inhibiting BAs may be a promising therapy for BE.