Vancomycin relieves tacrolimus-induced hyperglycemia by eliminating gut bacterial beta-glucuronidase enzyme activity.

Up to 40% of transplant recipients treated long-term with tacrolimus (TAC) develop post-transplant diabetes mellitus (PTDM). TAC is an important risk factor for PTDM, but is also essential for immunosuppression after transplantation. Long-term TAC treatment alters the gut microbiome, but the mechanisms of TAC-induced gut microbiota in the pathogenesis of PTDM are poorly characterized. Here, we showed that vancomycin, an inhibitor of bacterial beta-glucuronidase (GUS), prevents TAC-induced glucose disorder and insulin resistance in mice. Metagenomics shows that GUS-producing bacteria are predominant and flourish in the TAC-induced hyperglycemia mouse model, with upregulation of intestinal GUS activity. Targeted metabolomics analysis revealed that in the presence of high GUS activity, the hydrolysis of bile acid (BAs)-glucuronic conjugates is increased and most BAs are overproduced in the serum and liver, which, in turn, activates the ileal farnesoid X receptor (FXR) and suppresses GLP-1 secretion by L-cells. The GUS inhibitor vancomycin significantly eliminated GUS-producing bacteria and inhibited bacterial GUS activity and BAs levels, thereby enhancing L-cell GLP-1 secretion and preventing hyperglycemia. Our results propose a novel clinical strategy for inhibiting the bacterial GUS enzyme to prevent hyperglycemia without requiring withdrawal of TAC treatment. This strategy exerted its effect through the ileal bile acid-FXR-GLP-1 pathway.

Peripheral Klotho protects the kidney and brain by regulating M2a/M2c macrophage polarization in d-gal-treated aged mice.

In elderly individuals, aging can cause changes in the structure and function of one or more organs, increasing their susceptibility to various damage factors, especially the heart, kidney, brain and other important organs. Therefore, the incidence of cardiovascular disease, neurodegenerative diseases and chronic kidney disease in the elderly population is significantly higher than that in the general population. In our previous study, the hearts of aged mice did not express the antiaging protein Klotho (KL), but peripheral elevation of KL may significantly delay cardiac aging. The kidney and brain are the main organs that produce KL, but the effects and mechanism of peripheral KL supplementation on the kidney and hippocampus are still unclear. To study the effect and possible mechanism of KL against kidney and hippocampus aging, 60 male BALB/c mice were randomly divided into the Adult group, the KL group, the D-gal-induced Aged group, and the KL + Aged group. The results showed that KL increased anti-inflammatory M2a/M2c macrophages in the kidney and hippocampus of aging mice, significantly reduced tissue inflammation and oxidative stress, and improved organ function and aging status. More importantly, we demonstrate that despite the impermeable bloodbrain barrier in mice, peripherally administered KL surprisingly enhances M2-type microglia polarization, induces cognitive enhancement and reduces neuroinflammation. Cellular experimental results suggest that KL may play a role in delaying senescence by regulating the TLR4/Myd88/NF-κB signaling pathway to regulate macrophage polarization and reduce aging-related inflammation and oxidative stress.

Opioid-induced microbial dysbiosis disrupts irinotecan (CPT-11) metabolism and increases gastrointestinal toxicity in a murine model.

BACKGROUND AND PURPOSE: Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhoea. The chemotherapeutic irinotecan (CPT-11) causes severe gastrointestinal (GI) toxicity due to deconjugation of inactive metabolite SN-38 glucuronide (SN-38G) by bacterial ß-glucuronidases to the active 7-ethyl-10-hydroxycamptothecin (SN-38). Opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11 anti-tumour efficacy and GI toxicity are exacerbated by opioid co-administration. EXPERIMENTAL APPROACH: Eight-week-old C57BL/6 male mice were co-administration with CPT-11 ± opioid. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and quantitative real-time polymerase chain reaction were used to determine the severity of intestinal tissue damage. Human liver microsome In vitro assay was performed to confirm the effects of opioids on CPT-11 metabolism. KEY RESULTS: Gut microbiome analysis showed that morphine treatment induced enrichment of ß-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11 anti-tumour efficacy, this seemed to be microbiome independent. CONCLUSION AND IMPLICATIONS: Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.

Macrophages and derived-TNF-α promote lipopolysaccharide-induced upregulation of endogenous ß-glucuronidase in the epithelial cells of the bile duct: A possible facilitator of hepatolithiasis formation.

BACKGROUND: Hepatolithiasis is prevalent in Southeast Asian regions, and the role of endogenous ß-glucuronidase (ß-GD) in the formation of hepatolithiasis is being gradually recognised. Revealing the regulation mechanism of the expression of endogenous ß-GD will provide new therapeutic strategies for intervening in the formation of hepatolithiasis. METHODS: Liver specimens from patients with hepatolithiasis were examined by immunohistochemistry to assess the expression of macrophage markers including CD68, CD80, and CD206, as well as that of TNF-α and endogenous ß-GD, compared with that in normal liver samples. HiBEpiC cells were co-cultured directly or indirectly with induced M2 macrophages or directly stimulated with TNF-α, and the expression of the endogenous ß-GD was examined. A PKC inhibitor, chelerythrine, and an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), were used to elucidate the possible regulation mechanism. RESULTS: The expression of macrophage markers including CD68 and CD206, as well as that of TNF-α and endogenous ß-GD significantly increased in liver specimens from patients with hepatolithiasis compared with that in normal liver samples. The expression of CD68, CD206 and TNF-α was positively correlated with that of endogenous ß-GD. When HiBEpiC cells were co-cultured directly or indirectly with M2 macrophages, following stimulation with lipopolysaccharide (LPS), the expression of endogenous ß-GD was significantly higher in the indirect co-culture group than that in the direct co-culture group, or in HiBEpiC cells or M2 macrophages cultured alone. Further experiments revealed that following stimulation with LPS, TNF-α secretion increased in both the indirect and direct co-culture groups compared with that in HiBEpiC cells cultured alone. TNF-α increased the expression of endogenous ß-GD in HiBEpiC cells, in a dose- and time-dependent manner. In addition, TNF-α significantly increased the expression levels of p-P65 and proliferating cell nuclear antigen (PCNA), and PDTC effectively inhibited the TNF-α-induced expression of PCNA and ß-GD. CONCLUSIONS: Infiltration of macrophages, especially M2 macrophages, may be involved in the hepatolithiasis formation. LPS activates the macrophages, inducing the secretion of TNF-α, which can further increase the expression of endogenous ß-GD in the epithelial cells of the bile duct, possibly via the NF-κB/PCNA signalling cascade.

Gut microbiota activity in chickens from two genetic lines and with outdoor-preferring, moderate-preferring, and indoor-preferring ranging profiles.

Despite the existing research into the gut microbiome of meat chickens, the associations between gut microbiome composition, its activity and chicken outdoor ranging frequency remain unexplored. The aim of this study was to determine the gut microbiota composition, activity and metabolic products in chickens of 2 different lines and 3 ranging profiles. Sixty non-beak trimmed birds, either Sasso or Green-legged Partridge were housed with access to outdoor ranges from wk. 5 to 10 of age. Outdoor ranges were video recorded to obtain frequencies of the birds' range use. The information about relative abundance of selected bacterial groups in the ceca including Lactobacillus spp., E. coli, Bifidobacterium spp., and Clostridium spp. was obtained with the PCR method. Gut microbiota activity was assessed based on the glycolytic activity of bacterial enzymes including, α-glucosidase, ß-glucosidase, α-galactosidase, ß-galactosidase, and ß-glucuronidase as well as based on the concentration of short-chain fatty acids (SCFA) in the caecal digesta. Statistical analysis was conducted by generalized linear mixed models, applying the breed and ranging profile as fixed effects and pen as a random factor. The lowest relative abundance of Bifidobacterium spp. was found in the cecal content of indoor-preferring Sasso birds (0.01 ± 0.001), as compared to all other birds in the experiment (ranging from 0.03 ± 0.01 to 0.11 ± 0.07; P = 0.0002). The lowest relative abundance of E. coli was identified for all outdoor-preferring birds and indoor- preferring Sasso birds (0.01 ± 0.001; P = 0.0087). Cecal activity of: α-glucosidase, ß-glucuronidase and ß-galactosidase was higher in Green-legged Partridges, as compared to Sasso (P = 0.013; P = 0.008; P = 0.004). Valeric acid concentrations were higher in moderate Green-legged Partridges than in Sasso of the same ranging profile (2.03 ± 0.16 vs. 1.5 ± 0.17; 0.016). The majority of the current results confirmed an effect of genotype and ranging profile on the various analyzed parameters. In outdoor-preferring birds, the consumption of pasture originating feed sources as a supplement to the indoor accessible cereal-based diet likely caused the positive effects on the birds' microbial profile.

The role of gut microbial ß-glucuronidase in drug disposition and development.

Gut microbial ß-glucuronidase (gmGUS) is involved in the disposition of many endogenous and exogenous compounds. Preclinical studies have shown that inhibiting gmGUS activity affects drug disposition, resulting in reduced toxicity in the gastrointestinal tract (GIT) and enhanced systemic efficacy. Additionally, manipulating gmGUS activity is expected to be effective in preventing/treating local or systemic diseases. Although results from animal studies are promising, challenges remain in developing drugs by targeting gmGUS. Here, we review the role of gmGUS in host health under physiological and pathological conditions, the impact of gmGUS on the disposition of phenolic compounds, models used to study gmGUS activity, and the perspectives and challenges in developing drugs by targeting gmGUS.

Mechanism of the Fibroblast Growth Factor 23/α-Klotho Axis in Peripheral Blood Mononuclear Cell Inflammation in Alzheimer's Disease.

Alzheimer's disease (AD) is a prevalent type of dementia and threatens the health of most elderly people and poses a huge burden to families and society. The fibroblast growth factor 23 (FGF23)/α-Klotho axis is associated with multiple aging-related diseases. Hence, this study explored the mechanism of the FGF23/α-Klotho axis in AD. FGF23/α-Klotho protein contents and levels of inflammatory cytokines in AD patients were measured, and the correlation between FGF23/α-Klotho protein contents and inflammatory cytokines was analyzed. FGF23 and α-Klotho expressions were blocked in peripheral blood mononuclear cells (PBMCs) in AD patients (AD-PBMCs) to assess the effects on cell inflammation and the Wnt/ß-catenin pathway activation. The Wnt/ß-catenin pathway was inhibited to evaluate cell inflammation. Combined treatments of the cells were conducted to verify the role of the FGF23/α-Klotho axis and the Wnt/ß-catenin pathway in inflammation in AD-PBMCs. Increased FGF23 protein concentration and reduced α-Klotho protein concentration were observed in AD patients and correlated with inflammatory cytokine levels. FGF23 inhibition or α-Klotho overexpression reduced the production of inflammatory cytokines and activated the Wnt/ß-catenin pathway in AD-PBMCs. Blocking the Wnt/ß-catenin pathway increased inflammatory cytokine production in AD-PBMCs and annulled the effects of the FGF23/α-Klotho axis on AD-induced cell inflammation. We concluded that the FGF23/α-Klotho axis can regulate the AD-induced cell inflammation through the Wnt/ß-catenin pathway.

Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts.

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study, we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electric cell-substrate impedance sensing (ECIS), increased significantly during and after syncytialization, whereas the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared with nonsyncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with matrix metalloproteinase-9 (MMP-9), but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP-9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

Protective effects of klotho on palmitate-induced podocyte injury in diabetic nephropathy.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.

Klotho attenuated Doxorubicin-induced cardiomyopathy by alleviating Dynamin-related protein 1 - mediated mitochondrial dysfunction.

Doxorubicin (Dox)-induced cardiotoxicity could lead to dilated cardiomyopathy and heart failure. Our previous study reported the protective effects of Klotho against hyperglycemia-induced cardiomyopathy. We investigated whether Klotho alleviated Dox-induced cardiotoxicity. Neonatal rat ventricular cardiomyocytes and H9c2 cells were incubated with 5 µM Dox for 24 h with or without Klotho (0.1 µg/mL). Dox-induced cardiotoxicity model was approached in C57BL/6 mice. Cardiac function and serum enzyme activity, apoptosis and mitochondrial dysfunction were measured. We found that pretreatment with Klotho significantly reduced Dox-induced apoptosis in cardiomyocytes. In Dox-treated mice, Klotho also suppressed cardiac cell death and improved cardiac function. Moreover, the expression of Dynamin-related protein 1 (Drp1) was increased after Dox-treatment both in vitro and in vivo, which was related to apoptosis in cardiomyocytes. In vitro experiments, Drp1 ser 616 phosphorylation post-Dox stimulation could be significantly attenuated by Klotho or Drp1 specific inhibitor Mdivi-1. Overexpression of Drp1 in cardiomyocytes increased Dox-induced heart injury which could also be attenuated by Klotho. This study demonstrated that Klotho alleviated Dox-induced cardiotoxicity by reducing apoptosis and mitochondrial fission through down-regulating Drp1 expression. Our findings highlighted new targets for the therapy of Dox-induced cardiomyopathy.

Antiinflammatory Actions of Klotho: Implications for Therapy of Diabetic Nephropathy.

Klotho was initially introduced as an antiaging molecule. Klotho deficiency significantly reduces lifespan, and its overexpression extends it and protects against various pathological phenotypes, especially renal disease. It was shown to regulate phosphate and calcium metabolism, protect against oxidative stress, downregulate apoptosis, and have antiinflammatory and antifibrotic properties. The course of diabetes mellitus and diabetic nephropathy resembles premature cellular senescence and causes the activation of various proinflammatory and profibrotic processes. Klotho was shown to exert many beneficial effects in these disorders. The expression of Klotho protein is downregulated in early stages of inflammation and diabetic nephropathy by proinflammatory factors. Therefore, its therapeutic effects are diminished in this disorder. Significantly lower urine levels of Klotho may serve as an early biomarker of renal involvement in diabetes mellitus. Recombinant Klotho administration and Klotho overexpression may have immunotherapeutic potential for the treatment of both diabetes and diabetic nephropathy. Therefore, the current manuscript aims to characterize immunopathologies occurring in diabetes and diabetic nephropathy, and tries to match them with antiinflammatory actions of Klotho. It also gives reasons for Klotho to be used in diagnostics and immunotherapy of these disorders.

Anti-aging Klotho Protects SH-SY5Y Cells Against Amyloid ß1-42 Neurotoxicity: Involvement of Wnt1/pCREB/Nrf2/HO-1 Signaling.

Alzheimer's disease (AD) is considered a prevalent neurological disorder with a neurodegenerative nature in elderly people. Oxidative stress and neuroinflammation due to amyloid ß (Aß) peptides are strongly involved in AD pathogenesis. Klotho is an anti-aging protein with multiple protective effects that its deficiency is involved in development of age-related disorders. In this study, we investigated the beneficial effect of Klotho pretreatment at different concentrations of 0.5, 1, and 2 nM against Aß1-42 toxicity at a concentration of 20 µM in human SH-SY5Y neuroblastoma cells. Our findings showed that Klotho could significantly and partially restore cell viability and decrease reactive oxygen species (known as ROS) and improve superoxide dismutase activity (SOD) in addition to reduction of caspase 3 activity and DNA fragmentation following Aß1-42 challenge. In addition, exogenous Klotho also reduced inflammatory biomarkers consisting of nuclear factor-kB (NF-kB), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) in Aß-exposed cells. Besides, Klotho caused downregulation of Wnt1 level, upregulation of phosphorylated cyclic AMP response element binding (pCREB), and mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) with no significant alteration of epsilon isoform of protein kinase C (PKCε) after Aß toxicity. In summary, Klotho could alleviate apoptosis, oxidative stress, and inflammation in human neuroblastoma cells after Aß challenge and its beneficial effect is partially exerted through appropriate modulation of Wnt1/pCREB/Nrf2/HO-1 signaling.

Klotho attenuates angiotensin II­induced cardiotoxicity through suppression of necroptosis and oxidative stress.

Hyperglycemia is known to lead to cardiac injury and inflammation through the reactive oxygen species (ROS)­Toll­like receptor 4 (TLR4)­necroptosis pathway. Similarly, angiotensin II (Ang II) activates the TLR4­nuclear factor κB (NF­κB) p65 pathway, while the protein Klotho is known to inhibit this pathway, protecting cardiac cells from Ang II­induced injury. However, there is currently a lack of data on whether necroptosis participates in Ang II­induced cardiac injury and whether the Klotho protein has an effect on this process. The present study aimed to explore whether inhibition of the TLR4/NF­κB p65 necroptosis pathway is involved in the Klotho protein­mediated protection against the Ang II­induced cardiac injury and inflammation. H9c2 cardiac cells were incubated with 0.01 mM Ang II. Western blotting was used to assess the expression of receptor­interacting protein kinase 3 (RIP3), mixed­lineage kinase domain­like protein (MLKL), TLR4 and NF­κB p65. The present study also assessed injury indexes: Inflammatory cytokine expression, mitochondrial membrane potential (ΔΨm), apoptosis, ROS production and cell viability. The expression of TLR4, phosphorylated (p)­NF­κB p65, RIP3 and MLKL were increased by incubation with Ang II in H9c2 cells. The pretreatment of H9c2 cells with necrostatin­1 (Nec­1, an inhibitor of necroptosis) or TAK­242 (a small molecule inhibitor of TLR4) attenuated the upregulation of RIP3 and MLKL caused by Ang II. Klotho protein cotreatment also reversed the Ang II­induced upregulation of TLR4, p­NF­κB p65, RIP3 and MLKL. Furthermore, Ang II decreased cell viability and upregulated the secretion of inflammatory cytokines, ΔΨm loss and ROS generation blocked by pretreatment with Nec­1 or Klotho protein. Thus, it was determined that Klotho can attenuate the Ang II­induced necroptosis of cardiomyocytes through the TLR4/NF­κB p65 pathway, which suggests that Klotho could be a potential therapeutic drug against Ang II­induced cardiotoxicity.

Effect of Klotho protein during porcine oocyte maturation via Wnt signaling.

Klotho protein is well-known as an anti-aging agent, however, several studies have suggested that Klotho protein also increases antioxidant activity and the reproductive system, as Klotho protein is closely associated with Wnt signaling. The objective of our study was to investigate the enhancement of porcine oocyte in vitro maturation via the Klotho protein-Wnt signaling pathway. Following immunohistochemistry and ELISA, we treated cells with Klotho protein during in vitro maturation. Lithium Chloride, a specific activator of Wnt signaling, was subsequently co-administered with Klotho protein. Mature oocytes subjected to treatments were used for the analysis of embryonic development, qRT-PCR, and immunocytochemistry. Treatment with 5pg/ml Klotho protein significantly increased cumulus cell expansion, blastocyst formation rates, and the total cell number of blastocysts. During cotreatment with 5mM Lithium Chloride and 5pg/ml Klotho protein, blastocyst formation rates were the highest in Klotho protein-treated oocytes and the lowest in Lithium Chloride-treated oocytes. Expression levels of Wnt signaling-related transcripts and proteins were significantly impacted by Klotho protein and Lithium Chloride. Moreover, cellular ATP levels and antioxidant activities were enhanced by Klotho protein treatment. These findings suggest a significant involvement of the Klotho protein-Wnt signaling mechanism in porcine oocyte maturation.

Ameliorating Effect of Klotho Protein on Rat Heart during I/R Injury.

An essential procedure for the treatment of myocardial infarction is restoration of blood flow in the obstructed infarct artery, which may cause ischaemia/reperfusion (I/R) injury. Heart I/R injury manifests in oxidative stress, metabolic and morphological disorders, or cardiac contractile dysfunction. Klotho protein was found to be produced in the heart tissue and participate in antioxidation or ion homeostasis. The aim of this study was to examine an influence of Klotho protein on the heart subjected to I/R injury. Wistar rats served as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischaemia, and isolated rat cardiomyocytes underwent chemical I/R in vitro, with or without recombinant Klotho protein administration. Haemodynamic parameters of heart function, cell contractility, markers of I/R injury and oxidative stress, and the level of contractile proteins such as myosin light chain 1 (MLC1) and troponin I (TnI) were measured. The treatment of hearts subjected to I/R injury with Klotho protein resulted in a recovery of heart mechanical function and ameliorated myocyte contractility. This improvement was associated with decreased tissue injury, enhanced antioxidant capacity, and reduced release of MLC1 and TnI. The present research showed the contribution of Klotho to cardioprevention during I/R. Thus, Klotho protein may support the protection from I/R injury and prevention of contractile dysfunction in the rat heart.

Recombinant Klotho protein enhances cholesterol efflux of THP-1 macrophage-derived foam cells via suppressing Wnt/ß-catenin signaling pathway.

BACKGROUND: Atherosclerosis (AS) is the basis of cardiovascular diseases, characterized by chronic inflammatory and lipid metabolism disorders. Although the anti-inflammatory effect of Klotho in AS has been clearly shown, its lipid-lowering effect is unclear. In this study, we examined the effects of recombinant Klotho (Re-KL) protein on lipid accumulation in foam cells. METHODS: THP-1 cells were exposed to 100 nM phorbol myristate acetate for 24 h and then to oxidized low-density lipoprotein (ox-LDL; 80 mg/mL) to induce foam cell formation. Subsequently, the foam cells were incubated with Re-KL and/or DKK1, an inhibitor of the Wnt/ß-catenin pathway. RESULTS: Oil red O staining and cholesterol intake assay revealed that the foam cell model was constructed successfully. Pre-treatment of the foam cells with Re-KL decreased total cholesterol level, up-regulated the expression of ATP binding cassette transporter A1 (ABCA1) and G1 (ABCG1), and down-regulated the expression of acyl coenzyme a-cholesterol acyltransferase 1 (ACAT1) and members of the scavenger family (SR-A1 and CD36). In addition, the expression of Wnt/ß-catenin pathway-related proteins in foam cells was significantly decreased by the stimulus of Re-KL. Interestingly, the effect of Re-KL was similar to that of DKK1 on foam cells. CONCLUSIONS: The Re-KL-induced up-regulation of reverse cholesterol transport capacity promotes cholesterol efflux and reduces lipid accumulation by suppressing the Wnt/ß-catenin pathway in foam cells.

Rostral ventrolateral medulla neuron activity is suppressed by Klotho and stimulated by FGF23 in newborn Wistar rats.

Hypertension often occurs in patients with chronic kidney disease (CKD). Considering the decrease in serum Klotho and increase in serum FGF23 levels in such patients, decreased Klotho and increased FGF23 levels were thought to be associated with hypertension. Presympathetic neurons at the rostral ventrolateral medulla (RVLM) contribute to sympathetic activity and regulation of blood pressure. Therefore, we hypothesized that Klotho would reduce the activities of RVLM neurons and FGF23 would stimulate them. Accordingly, this study examined the effects of Klotho and FGF23 on bulbospinal neurons in the RVLM. We used a brainstem-spinal cord preparation to record from RVLM presympathetic neurons and to evaluate the effects of Klotho and FGF23 on firing rate and membrane potentials of these neurons. Our results showed that Klotho-induced RVLM neuron hyperpolarization, while ouabain, a Na+/K+-ATPase inhibitor, suppressed the effects of Klotho on such neurons. Moreover, FGF23 induced RVLM neuron depolarization, while SU5402, an FGF23 receptor (FGFR1) antagonist, induced RVLM neuron hyperpolarization. Histological examinations revealed that Klotho, Na+/K+-ATPase, FGF23, and FGFR1 were present in RVLM neurons and that Klotho was localized in the same neurons as FGFR1. These results suggest that Klotho and FG23 regulate the activity of RVLM neurons. Klotho may reduce the activity of RVLM neurons via stimulating Na+/K+-ATPase on those neurons while FGF23 may activate those neurons via FGFR1.

Recombinant α-Klotho Protein Alleviated Acute Cardiorenal Injury in a Mouse Model of Lipopolysaccharide-Induced Septic Cardiorenal Syndrome Type 5.

BACKGROUND AND AIMS: Klotho is an aging-suppressor gene mainly expressed in the renal tubules. The klotho gene encodes the α-klotho protein, which has many functions. Previous studies have found that α-klotho protein has a cardiorenal protective function. α-Klotho deficiency renders the kidney more susceptible to injury and results in cardiovascular calcification and left ventricular hypertrophy in chronic kidney disease. However, the role of α-klotho in acute heart injury and acute kidney injury with sepsis remains unknown. This study aimed to investigate the effects and mechanisms of α-klotho in septic cardiorenal injury. METHODS: Male 8-week-old C57BL/6 mice were randomly assigned to the control group, lipopolysaccharide (LPS; 10 mg/kg) group, LPS (10 mg/kg)+α-klotho (0.01 mg/kg) group, and LPS (10 mg/kg)+α-klotho (0.02 mg/kg) group. Recombinant α-klotho was intraperitoneally injected an hour before LPS injection. Mice were euthanized at 24 h after LPS injection. The serum troponin, brain natriuretic peptide (BNP), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine levels were measured in all groups at 24 h. Biomarkers of mice heart apoptosis, inflammation, oxidative stress, and endoplasmic reticulum stress, such as caspase-3, interleukin 1 (IL-1), reactive oxygen species (ROS), and glucose-regulated protein 78 (GRP78), were also measured. RESULTS: α-Klotho was mainly expressed in mice kidneys and was undetectable in the control mice hearts. α-Klotho substantially decreased after LPS injection. In the LPS group, the serum troponin levels significantly increased as early as 6 h (p < 0.05) after LPS injection, while the BNP, NGAL, and creatinine levels significantly increased at 24 h (p < 0.05). Pretreatment with α-klotho significantly ameliorated acute cardiorenal injury. In the LPS+α-klotho (0.01 mg/kg) group, the levels of apoptosis, inflammation, and oxidative stress were decreased, while the level of endoplasmic reticulum stress was elevated. CONCLUSIONS: α-Klotho significantly alleviates acute cardiorenal injury in LPS-induced septic cardiorenal injury due to the inhibition of apoptosis, inflammation, and oxidation, as well as the regulation of endoplasmic reticulum stress levels.

Klotho inhibits angiotensin II-induced cardiac hypertrophy, fibrosis, and dysfunction in mice through suppression of transforming growth factor-ß1 signaling pathway.

Recent studies have revealed critical roles of transforming growth factor-ß1 (TGF-ß1) and microRNA-132 (miR-132), a downstream mediator of TGF-ß1, in the pathogenesis of cardiac remodeling. In this study, we tested whether the antiaging protein klotho modifies angiotensin II (Ang II)-induced cardiac remodeling through regulating TGF-ß1-miR-132 axis. We found that both klotho and the TGF-ß1 inhibitor LY364947 significantly inhibited cardiac hypertrophy, fibrosis, and dysfunction in Ang II-infused mice, as evidenced by the ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL), cardiomyocyte cross-sectional area, fibrotic area, and expression of prohypertrophic genes (ANP, ß-MHC) and fibrotic marker genes (α-SMA, collagen I), echocardiographic parameters. Meanwhile, klotho also significantly inhibited Ang II-induced protein expression of TGF-ß1 and phosphorylated Smad2/3 in the heart tissues and cultured cardiomyocytes and cardiac fibroblasts. In vitro experiments demonstrated that Ang II-induced cardiomyocyte hypertrophy and proliferation and activation of cardiac fibroblasts were markedly inhibited by klotho, LY364947 or the miR-132 inhibitor anti-miR-132. Both klotho and the TGF-ß1 inhibitor LY364947 downregulated the miR-132 expression. Additionally, klotho decreased Ang II-induced protein expressions of cardiac fibroblast growth factor (FGF)23 in vivo and in vitro. The decreased protein levels of klotho in serum and renal tissues of Ang II-infused mice were elevated by klotho. Klotho downregulated the protein levels of TGF-ß1 in renal tissues of Ang II-infused mice. In conclusion, our results suggest that klotho prevents Ang II-induced cardiac remodeling and dysfunction through modifying the TGF-ß1-miR-132 axis, providing an experimental basis for clinical treatment on cardiac remodeling.

Klotho restrain RIG-1/NF-κB signaling activation and monocyte inflammatory factor release under uremic condition.

AIMS: Systemic inflammation is a main hallmark of chronic kidney disease (CKD), but the underlying mechanisms of pathogenesis of CKD-associated systemic inflammation is unclear. Current study was designed to investigate the relationship between indoxyl sulphate (IS) and CKD-associated systemic inflammation along with the protective effects of Klotho in CKD. METHODS: IS serum levels from patients were detected by high-performance liquid chromatography (HPLC), and Serum Klotho, IL-6 and TNF-α were measured separately by ELISA and Real-Time PCR analysis. Monocytes were incubated with or without Klotho, while the expressions of retinoic acid-inducible gene I (RIG-I) and NF-κB were analyzed through Western blot assay. Heterozygous kl/kl (kl/+) mice or WT mice were treated with 5/6 renal damage. Thereafter, the CKD mice were intraperitoneally injected with recombinant Klotho protein or PBS. KEY FINDINGS: It shows that in 286 CKD patients, the serum levels of inflammatory factors were positively related with IS, but negatively related with Klotho. Klotho significantly inhibited IS-induced RIG-I/NF-κB activation and productions of both IL-6 and TNF-α in cultured monocytes. In vivo, along with the increase of IS and decrease of Klotho in the serum, the activation of RIG-I/NF-κB signaling was observed in peripheral blood monocytes in both CKD mice and patients. Notably, higher levels of IL-6 and TNF-α were detected in kl+/- mice given CKD. Klotho administration has evidently attenuated RIG-I/NF-κB activation in monocytes and systemic inflammation in CKD mice. SIGNIFICANCE: The findings suggest that Klotho can suppress CKD-associated systemic inflammation through inhibiting IS-induced RIG-1/NF-κB activation and monocyte inflammatory factor release.