Mitigation of benzyl butyl phthalate toxicity in male germ cells with combined treatment of parthenolide, N-acetylcysteine, and 3-methyladenine.

Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5 µM. Exposure to 50 µM BBP, approximating the IC50 of 53.9 µM, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50 µM BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.

Vascular, valvular and kidney calcification manifested in mouse models of adenine-induced chronic kidney disease.

BACKGROUND: Ectopic calcification (EC) involves multiple organ systems in chronic kidney disease (CKD). Previous CKD-animal models primarily focused on a certain histological abnormality but did not show the correlation with calcified development among various tissues. This study compared calcified deposition in various tissues during CKD progression in mice. METHODS: Male 8-week-old C57BL/6J mice were randomly allocated to the seven groups: a basic, adenine, high-phosphorus, or adenine and high-phosphorus diet for 12-16 weeks (Ctl16, A12, P16, or AP16, respectively); an adenine diet for 4-6 weeks; and a high-phosphorus or adenine and high-phosphorus diet for 10-12 weeks (A6 + P10, A4 + P12, or A4 + AP12, respectively). RESULTS: Compared to the Ctl16 mice, the P16 mice only displayed a slight abnormality in serum calcium and phosphorus; the A12 mice had the most serious kidney impairment; the A4 + P12 and A6 + P10 mice had similar conditions of CKD, mineral abnormalities, and mild calcification in the kidney and aortic valves; the A4 + AP12 and AP16 groups had severe kidney impairment, mineral abnormalities and calcification in the kidneys, aortic valves and aortas. Furthermore, calcium-phosphate particles were deposited not only in the tubulointerstitial compartment but in the glomerular and tubular basement membrane. The elemental composition of EC in various tissues matched the calcification of human cardiovascular tissue as determined by energy dispersive spectroscopy. CONCLUSIONS: The severity of CKD was unparalleled with the progression of mineral metabolism disorder and EC. Calcification was closely related in different tissues and observed in the glomerular and tubular basement membranes.

Previous CKD-animal models primarily focused on a certain histological abnormality but lacked investigations of the interplay of EC in various tissues. This study compared calcified deposition in several tissues during CKD progression in mice, which was closely related. The severity of CKD was unparalleled with the development of ectopic calcification. Glomerular and tubular basement membrane calcification was detected in CKD mice, which has been considered extremely rare in clinical.

Lack of Endothelial Nitric Oxide Synthase Accelerates Ectopic Calcification in Uremic Mice Fed an Adenine and High Phosphorus Diet.

Ectopic calcification is a risk of cardiovascular disease in chronic kidney disease (CKD) patients, and impaired endothelial nitric oxide synthase (eNOS) is involved in the CKD complications. However, whether eNOS dysfunction is a cause of ectopic calcification in CKD remains to be elucidated. To address this issue, we investigated the role of eNOS in ectopic calcification in mice with renal injury caused by an adenine and high-phosphorus (Ade + HP) diet. DBA/2J mice, a calcification-sensitive strain, were fed Ade + HP for 3 weeks. Expression levels of eNOS-related genes were reduced significantly in their calcified aorta. C57BL/6J is a calcification-resistant strain, and wild-type mice showed mild calcified lesions in the aorta and kidney when given an Ade + HP diet for 4 weeks. In contrast, a lack of eNOS led to the development of severe aortic calcification accompanied by an increase in runt-related transcription factor 2, an osteochondrogenic marker. Increased renal calcium deposition and the tubular injury score were remarkable in mice lacking eNOS-fed Ade + HP. Exacerbation of ectopic calcification by a lack of eNOS is associated with increased oxidative stress markers such as nicotinamide adenine dinucleotide phosphate oxidases. In conclusion, eNOS is critically important in preventing ectopic calcification. Therefore, the maintenance of eNOS is useful to reduce cardiovascular disease events and to improve prognosis in CKD patients.

Integration of network pharmacology and intestinal flora to investigate the mechanism of action of Chinese herbal Cichorium intybus formula in attenuating adenine and ethambutol hydrochloride-induced hyperuricemic nephropathy in rats.

CONTEXT: Cichorium intybus L. (Asteraceae) formula (CF) has been applied as a folk medicine to treat hyperuricemic nephropathy (HN). However, the exact mechanism remains unclear. OBJECTIVE: To explore the therapeutic effect and mechanism of CF on HN. MATERIALS AND METHODS: Through network pharmacological methods, the targets of the active component of CF against HN were obtained. Subsequently, Male Wistar rats were divided into control, HN, allopurinol (50 mg/kg), CF high-dose (8.64 g/kg) and CF low-dose (2.16 g/kg) groups. The HN model was induced via intragastric administration of adenine (100 mg/kg) and ethambutol hydrochloride (250 mg/kg) for 3 weeks. After CF treatment, biochemical indicators including UA, UREA and CREA were measured. Then, HE staining, qRT-PCR and gut microbiota analysis were conducted to further explore the mechanism. RESULTS: The network pharmacology identified 83 key targets, 6 core genes and 200 signalling pathways involved in the treatment of HN. Compared to the HN group, CF (8.64 g/kg) significantly reduced the levels of UA, UREA and CREA (from 2.4 to 1.57 µMol/L, from 15.87 to 11.05 mMol/L and from 64.83 to 54.83 µMol/L, respectively), and mitigated renal damage. Furthermore, CF inhibited the expression of IL-6, TP53, TNF and JUN. It also altered the composition of gut microbiota, and ameliorated HN by increasing the relative abundance of some probiotics. CONCLUSIONS: This work elucidated the therapeutic effect and underlying mechanism by which CF protects against HN from the view of the biodiversity of the intestinal flora, thus providing a scientific basis for the usage of CF.

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

ABCC4 single-nucleotide polymorphisms as markers of tenofovir disoproxil fumarate-induced kidney impairment.

Recently, the use of antiretroviral drug tenofovir disoproxil fumarate (TDF) is increased, thanks to the new co-formulation with doravirine, the availability of booster-free regimens, and its advantageous lipid-lowering effect. The aim of our study was to identify genetic markers that contribute to assess the risk of TDF-related renal toxicity. We have retrospectively investigated, in 179 HIV positive patients treated with TDF, the association between the main variants in ABCC2, ABCC4, and ABCC10 genes and four safety endpoints, three clinically relevant as renal outcomes and a higher tenofovir plasma concentration. In patients with an annual eGFR decline >5 mL/min/1.73 m2 a difference in genotype frequencies was observed for ABCC10 c.1875 + 526 G>A (3 subjects AA vs. 44 GG + GA, p = 0.045). In patients with an eGFR decrement >25%, plus a decline in GFR category and TDF discontinuation, a difference was observed for ABCC4 c.*38T>G (35 subjects TG + GG vs. 18 TT, p = 0.052). At univariate analysis OR was 1.39 [(95% CI 1.00-1.96) p = 0.054] and at multivariate analysis OR was 1.49 [(95% CI 1.00-2.22) p = 0.049]. The stronger associations were found between the tenofovir accumulation and ABCC4 c.*38T>G and c.3348G>A: the percentage of these patients was higher in the TG + GG (p = 0.011) and in the AA (p = 0.004) genotype, respectively. The logistic regression analysis confirmed these significant relationships. No significant association was observed in patients with eGFR < 60 mL/min/1.73m2 and with the studied ABCC2 polymorphisms. Our results show a major role for a combined determination of ABCC4/ABCC10 variants as an indicator of tenofovir toxicity in the clinical practice.

Evidence of an intestinal phosphate transporter alternative to type IIb sodium-dependent phosphate transporter in rats with chronic kidney disease.

BACKGROUND: Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. METHODS: CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). RESULTS: In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. CONCLUSIONS: This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.

Huangjinsan ameliorates adenine-induced chronic kidney disease by regulating metabolic profiling.

Chronic kidney disease is an increasingly serious public health problem worldwide. Our recent studies have shown that Huangjinsan has a renal protective effect on chronic kidney disease, but the specific mechanism by which this effect occurs is not clear. To study the therapeutic effect of Huangjinsan on chronic kidney disease and to explore its possible mechanism of action through nontargeted metabolomics methods, a chronic kidney disease rat model was induced by adenine, and the Huangjinsan extract was given by oral gavage. Body weight, the kidney index, pathological sections, and a series of biochemical indicators were measured. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used to analyze the changes in the plasma metabolome. Huangjinsan significantly reduced indicators of kidney damage, including total protein, albumin, the total protein to creatinine ratio, and the albumin to creatinine ratio in urine, as well as IL-2, MCP-1α, and blood urea levels in plasma. Based on nontargeted metabolomics, 13 metabolites related to chronic kidney disease were discovered. These metabolites are closely related to glycerophospholipid metabolism, arginine and proline metabolism, and sphingolipid metabolism. We found that Huangjinsan can restore the renal function of adenine-induced chronic kidney disease by regulating the metabolic profile.

Adropin and spexin hormones regulate the systemic inflammation in adenine-induced chronic kidney failure in rat.

Chronic kidney disease is one of the major global health problems. Chronic renal failure is stimulated by many cytokines and chemokines. Adropin and spexin (SPX) are peptides hormones. These peptides could affect inflammatory conditions, but this is unclear. Due to the limited information, we planned to investigate the impact of adropin and SPX hormones on systemic inflammation in adenine induced chronic kidney failure rat model. Chronic kidney failure was induced by administering adenine hemisulfate. Renal functions were measured by an autoanalyzer. Granulocyte colony-stimulating factor (G-CSF), interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, tumor necrosis factor-alpha, Eotaxin, growth-regulated oncogene-alpha, IP-10, monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1α, MIP-2, and RANTES levels were determined by Luminex. We observed an increase in 24-h urine volume and serum creatinine. Blood urea nitrogen (BUN) and urine protein levels were also significantly higher in the chronic kidney failure (CKF) group. Urine protein and 24-h urine volume were reduced with adropin and SPX treatments. Furthermore, G-CSF, IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-17A, and GRO-α significantly increased by CKF induction; however, these cytokines and chemokines significantly decreased by adropin treatment in the CKF group. Furthermore, adropin increased IP-10, MCP-1, MIP-1α, and MIP-2 levels. In addition, SPX treatment had a more limited effect, decreasing only G-CSF, IFN-γ, and IL-5 levels. The combined adropin + SPX treatment significantly reduced G-CSF, IFN-γ, IL-4, IL-5, IL-12, and IL-17A. Furthermore, IP-10, MCP-1, MCP-3, and MIP-2 were significantly increased by these combined treatments. Our findings indicate that renal functions and inflammatory response were modulated by adropin and SPX peptides. These peptides may have protective effects on systemic inflammation and renal failure progression.

[Five year experience in ibrutinib therapy for relapsed and refractory mantle cell lymphoma in real world Russian clinical practice].

BACKGROUND: Mantle cell lymphoma (MCL) is a rare and clinically aggressive lymphoma subtype. Current approaches have greatly improved patients outcomes, but relapse is inevitable. In phase IIIII clinical trials, ibrutinib has shown significant activity in patients with relapsed or refractory (R/R) MCL. AIM: To assess efficacy and toxicity of ibrutinib monotherapy in patients with R/R MCL in routine practice outside of clinical trials. MATERIALS AND METHODS: The study enrolled patients with confirmed R/R MCL who had received at least one line of previous chemotherapy. ECOG 24, cytopenia, infectious complications, hemorrhagic syndrome were not exclusion criteria. Patients received daily oral ibrutinib 560 mg until progression or unacceptable toxicity. RESULTS: From May 2015 to September 2020 ibrutinib therapy was started in 106 patients with R/R MCL in 16 regions of Russia. The median age was 66 years; ECOG2 18%, blastoid variant (or Ki6740% or WBC50109/l) 43%. The median number of previous treatment lines was 2 (111). The ORR was 78.4% (CRR 27.4%). The median PFS was 13.6 months and OS 23.2 months. In the blastoid group the median PFS was 4.4 months vs 36.5 months in the alternative group (p0.001), the median OS 9.0 vs 41.0 (p=0.001). The median OS of patients after progression on ibrutinib was 3.2 months. The common complications are hemorrhages (63%), diarrhea (62%), myalgia and muscle cramps (60%), infections (31%), skin and nail toxicity 15%, arrhythmia 8%. None of recipients had to completely discontinue ibrutinib therapy due to complications. CONCLUSION: Ibrutinib is effective and well tolerated in routine practice of R/R MCL treatment and our results are consistent with international clinical trials. The favorable toxicity profile and the high response rate made it possible to prescribe ibrutinib in severe somatic status, cytopenia, and even in the presence of infectious complications.

The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

Effect of Chronic Kidney Disease on Hepatic Clearance of Drugs in Rats.

The pharmacokinetics of some hepatically cleared drugs have been reported to fluctuate in patients with renal impairment, but the definitive factors have not been clarified. We compared the pharmacokinetics of some drugs with different hepatic elimination processes in a chronic kidney disease (CKD) rat model, to optimize their administration during kidney injury. We chose indocyanine green (ICG), midazolam (MDZ), and acetaminophen (APAP) as reference drugs to determine changes in hepatic clearance pathways in presence of CKD. Drugs were intravenously administered via the jugular vein to the CKD model rats, previously established by adenine administration, and then, blood, bile, and urine samples were collected. The plasma concentration of ICG, which is eliminated into the bile without biotransformation, increased; and its total body clearance (CLtot) significantly decreased in the CKD group compared to the control group. Moreover, the plasma concentrations of MDZ and APAP, metabolized in the liver by CYP3A and Ugt1a6 enzymes, respectively, were higher in the CKD group than in the control group. The biliary clearances of APAP and its derivative APAP-glucuronide increased in the CKD group, whereas their renal clearances were markedly decreased with respect to those in the control group. Altogether, plasma concentrations of some hepatically eliminated drugs increased in the CKD rat model, but depending on their pharmacokinetic characteristics. This study provides useful information for optimizing the administration of some hepatically cleared drugs in CKD patients.

Knockdown of CK2α reduces P-cresol-induced fibrosis in human renal proximal tubule epithelial cells via the downregulation of profilin-1.

Renal fibrosis is one of the main causes of chronic kidney disease. Many studies have focused on fibroblasts and myofibroblasts involved in renal fibrogenesis. Recently, several studies have reported that renal proximal tubule epithelial cells are possible initiators of renal fibrosis. However, the mechanism through which cells induce renal fibrosis is poorly understood. In this study, we found that CK2α induces fibrosis in renal proximal tubule epithelial cells (TH1) by regulating the expression of profilin-1 (Pfn1). CKD mouse model and TH1 cells treated with P-cresol also showed an increased level of Pfn1. The knockdown of CK2α suppressed fibrosis in TH1 cells via the downregulation of Pfn1. In particular, CK2α knockdown inhibited the expression of stress fibers and fibrosis-related proteins in P-cresol-treated TH1 cells. Furthermore, the knockdown of CK2α inhibited mitochondrial dysfunction and restored cellular senescence and cell cycle in P-cresol-treated TH1 cells. These results indicate that CK2α induces renal fibrosis through Pfn1, which makes CK2α a key target molecule in the treatment of fibrosis related to chronic kidney disease.

The Time-Feature of Uric Acid Excretion in Hyperuricemia Mice Induced by Potassium Oxonate and Adenine.

Hyperuricemia is an important risk factor of chronic kidney disease, metabolic syndrome and cardiovascular disease. We aimed to assess the time-feature relationship of hyperuricemia mouse model on uric acid excretion and renal function. A hyperuricemia mouse model was established by potassium oxonate (PO) and adenine for 21 days. Ultra Performance Liquid Chromatography was used to determine plasma uric acid level. Hematoxylin-eosin staining was applied to observe kidney pathological changes, and Western blot was used to detect renal urate transporters' expression. In hyperuricemia mice, plasma uric acid level increased significantly from the 3rd day, and tended to be stable from the 7th day, and the clearance rate of uric acid decreased greatly from the 3rd day. Further study found that the renal organ of hyperuricemia mice showed slight damage from the 3rd day, and significantly deteriorated renal function from the 10th day. In addition, the expression levels of GLUT9 and URAT1 were upregulated from the 3rd day, while ABCG2 and OAT1 were downregulated from the 3rd day, and NPT1 were downregulated from the 7th day in hyperuricemia mice kidney. This paper presents a method suitable for experimental hyperuricemia mouse model, and shows the time-feature of each index in a hyperuricemia mice model.

Reduction of Secreted Frizzled-Related Protein 5 Drives Vascular Calcification through Wnt3a-Mediated Rho/ROCK/JNK Signaling in Chronic Kidney Disease.

Vascular calcification (VC) is commonly associated with bone loss in patients with chronic kidney disease (CKD). The Wingless-related integration site (Wnt) regulates osteoblast activation through canonical signaling pathways, but the common pathophysiology of these pathways during VC and bone loss has not been identified. A rat model of adenine-induced CKD with VC was used in this study. The rats were fed 0.75% adenine (2.5% protein, 0.92% phosphate) with or without intraperitoneal injection of calcitriol (0.08 µg/kg/day) for 4 weeks. Angiotensin II (3 µM)-induced VC was achieved in high phosphate medium (3 mM) through its effect on vascular smooth muscle cells (VSMCs). In an mRNA profiler polymerase chain reaction assay of the Wnt signaling pathway, secreted frizzled-related protein 5 (sFRP5) levels were significantly decreased in the CKD rat model compared with the control group. The repression of sFRP5 on VSMC trans-differentiation was mediated through Rho/Rho-associated coiled coil containing protein kinase (ROCK) and c-Jun N-terminal kinase (JNK) pathways activated by Wnt3a. In a proof of concept study conducted with patients with CKD, serum sFRP5 concentrations were significantly lower in subjects with VC than in those without VC. Our findings suggest that repression of sFRP5 is associated with VC in the CKD environment via activation of the noncanonical Wnt pathway, and thus that sFRP5 might be a novel therapeutic target for VC in CKD.

Obesity causes renal mitochondrial dysfunction and energy imbalance and accelerates chronic kidney disease in mice.

Obesity and metabolic syndrome are well-known risk factors for chronic kidney disease (CKD); however, less is known about the mechanism(s) by which metabolic syndrome might accelerate kidney disease. We hypothesized that metabolic syndrome should accelerate the development of kidney disease and that it might be associated with alterations in energy metabolism. We studied the pound mouse (which develops early metabolic syndrome due to a leptin receptor deletion) and wild-type littermates and compared the level of renal injury and muscle wasting after equivalent injury with oral adenine. Renal function, histology, and biochemical analyses were performed. The presence of metabolic syndrome was associated with earlier development of renal disease (12 mo) and earlier mortality in pound mice compared with controls. After administration of adenine, kidney disease was worse in pound mice, and this was associated with greater tubular injury with a decrease in kidney mitochondria, lower tissue ATP levels, and worse oxidative stress. Pound mice with similar levels of renal function as adenine-treated wild-type mice also showed worse sarcopenia, with lower tissue ATP and intracellular phosphate levels. In summary, our data demonstrate that obesity and metabolic syndrome accelerate the progression of CKD and worsen CKD-dependent sarcopenia. Both conditions are associated with renal alterations in energy metabolism and lower tissue ATP levels secondary to mitochondrial dysfunction and reduced mitochondrial number.

Renal oxygenation during the early stages of adenine-induced chronic kidney disease.

To assess whether renal hypoxia is an early event in adenine-induced chronic kidney disease, adenine (100 mg) or its vehicle was administered to male Sprague-Dawley rats by daily oral gavage for 7 days. Kidney oxygenation was assessed by 1) blood oximetry and Clark electrode in thiobutabarbital-anesthetized rats, 2) radiotelemetry in unanesthetized rats, and 3) expression of hypoxia-inducible factor (HIF)-1α and HIF-2α protein. After 7 days of treatment, under anesthesia, renal O2 delivery was 51% less, whereas renal O2 consumption was 65% less, in adenine-treated rats than in vehicle-treated rats. Tissue Po2 measured by Clark electrode was similar in the renal cortex but 44% less in the medulla of adenine-treated rats than in that of vehicle-treated rats. In contrast, in unanesthetized rats, both cortical and medullary tissue Po2 measured by radiotelemetry remained stable across 7 days of adenine treatment. Notably, anesthesia and laparotomy led to greater reductions in medullary tissue Po2 measured by radiotelemetry in rats treated with adenine (37%) than in vehicle-treated rats (16%), possibly explaining differences between our observations with Clark electrodes and radiotelemetry. Renal expression of HIF-1α was less after 7 days of adenine treatment than after vehicle treatment, whereas expression of HIF-2α did not differ significantly between the two groups. Renal dysfunction was evident after 7 days of adenine treatment, with glomerular filtration rate 65% less and serum creatinine concentration 183% greater in adenine-treated rats than in vehicle-treated rats. Renal cortical tissue hypoxia may not precede renal dysfunction in adenine-induced chronic kidney disease and so may not be an early pathological feature in this model.

Adenine acts in the kidney as a signaling factor and causes salt- and water-losing nephropathy: early mechanism of adenine-induced renal injury.

Chronic adenine feeding is extensively used to develop animal models of chronic renal failure with metabolic features resembling those observed in humans. However, the mechanism by which adenine induces renal failure is poorly understood. In this study, we examined the early effects of adenine on water metabolism and salt balance in rats placed in metabolic cages and fed control or adenine-containing diets for 7 days. Molecular and functional studies demonstrated that adenine-fed rats exhibited a significant reduction in food intake, polyuria, polydipsia, decreased urine osmolality, and increased salt wasting. These effects are independent of changes in food intake and result from a coordinated downregulation of water channel aquaporin-2 (AQP2) and salt transporter (Na+-K+-Cl- cotransporter 2; NKCC2) in the collecting duct and medullary thick ascending limb, respectively. As a result, adenine-fed rats exhibited massive volume depletion, as indicated by a significant body weight loss, increased blood urea nitrogen, and increased hematocrit and hemoglobin levels, all of which were significantly corrected with NaCl replacement. Adenine-induced urinary concentrating defect was not corrected by exogenous arginine vasopressin (AVP), and it correlated with reduced cAMP production in vivo and in vitro. In conclusion, adenine acts on renal tubules as a signaling molecule and causes nephrogenic diabetes insipidus with salt wasting, at least, by directly interfering with AVP V2 receptor signaling with subsequent downregulation of NKCC2 and AQP2 in the kidney. The combination of renal fluid loss and decreased food intake with subsequent massive volume depletion likely plays an important role in the development of early prerenal failure that progresses to chronic kidney disease in long-term adenine feeding.