Adenine-induced Rats Research Articles

Effects of Eucommia ulmoides leaf extract on xanthine oxidase and chronic kidney disease induced by adenine in rats

The aim of this study was to explore the uric acid-lowering effect and renal protective effect of Eucommia ulmoides leaf extract (EULE). The results of xanthine oxidase inhibition assay showed EULE exhibited a high inhibition rate similar to that of allopurinol, with an IC50 value of 1.53 mg/mL. A chronic kidney disease (CKD) model was established in adenine-induced rats to investigate the therapeutic effect of EULE on CKD. The results demonstrated EULE could reduce blood pressure and improve renal index. Additionally, EULE could regulate serum and urine indicators of renal function injury, and restore renal tissue morphology. Mechanistically, EULE was found to downregulate levels of malondialdehyde (MDA), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), while upregulating total antioxidant capacity (T-AOC), thereby alleviating inflammatory response in rats, leading to a reduction in renal damage. the Our findings provide potential applications of EULE as a natural product for the improvement of renal injury.

Discovery of DS-1093a: An oral hypoxia-inducible factor prolyl hydroxylase inhibitor for the treatment of renal anemia

Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We discovered DS44470011 in our previous study, which showed potent in vitro activity and in vivo efficacy based on HIF-PHD inhibition. However, DS44470011 was also found to exert genotoxic effects. By converting the biphenyl structure, which is suspected to be the cause of this genotoxicity, to a 1-phenylpiperidine structure, we were able to avoid genotoxicity and further improve the in vitro activity and in vivo efficacy. Furthermore, through the optimization of pyrimidine derivatives, we discovered DS-1093a, which has a wide safety margin with potent in vitro activity and an optimal pharmacokinetic profile. DS-1093a achieved an increase in hemoglobin levels in an adenine-induced rat model of chronic kidney disease after its continuous administration for 4 days.

Niaodukang mixture inhibits micro-inflammation in CKD rats by enhancing MiR-146a levels in enterogenous exosomes

Ethnopharmacological relevanceThe Niaodukang mixture (NDK) is a preparation known for its ability to lower serum creatine levels in individuals with chronic kidney disease (CKD) and is commonly administered at medical facilities like the Zhongshan Hospital of Traditional Chinese Medicine. The initial use of NDK was mainly to treat CKD. Our hospital frequently utilizes NDK, which consists of Rheum officinaleBaill., Salvia miltiorrhiza Bunge., Astragalus aaronii (Eig) Zohary., Carthamus tinctorius L., and Sanguisorba officinalis L., for treating patients with CKD-MBD. It has the effects of eliminating dampness and turbidity and dredging kidney collaterals. However, The impact and process of NDK in chronic kidney disease remain unknown. Aim of the studyTo determine whether microRNA-146a (miR-146a) is associated with CKD micro-inflammationand whether NDK protects against CKD micro-inflammation by modulating the miR-146a/nuclear factor kappa-B (NF-κB) signaling pathway. Materials and methods(1) An adenine-induced rat model of chronic kidney disease was created through the use of materials and methods. The levels of miR-146a in exosomes from plasma and ileum were determined by RT-PCR. (2) Human cloned colon adenocarcinoma (Caco-2)cellswere stimulated with lipopolysaccharide (LPS)and transfected with miR-146a mimic and inhibitor. Following that, the Western blot and RT-PCR techniques were used to measure the protein and mRNA quantities of Toll-like receptor 4 (TLR4), NF-κB, and TNF receptor-associated factor 6 (TRAF6). (3) Enzyme-linked immunosorbent assay (ELISA) was used to identify serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). (4) Plasma exosomes were extracted, and the exosomes in intestinal tissues were extracted via ultrahigh-speed centrifugation.Negative staining electron microscopy was used to analyze the morphology of exosomes and the ultrastructure of intestinal tissue and exosomes. The particle size of the exosomes was measured using nanoparticle tracking analysis. ResultsThe pathological characteristics of CKD rats included those associated with systemic micro-inflammation, which may be associated with the release of exosomes in intestinal tissue. NDK suppressed the inflammatory response in Caco-2 cells and decreased the levels of IL-1β, IL-6, and TNF-α in rats with CKD. The expression of miR-146a, which regulates inflammation, differed between plasma-derived and enterogenous exosomes in CKD rats, which may be due to stimulation of ileal exosome release into the blood. NDK effectively reduced the levels of TRAF6, NF-κB, and TLR4 in the ileum tissue of CKD rats. ConclusionNDK can effectively improve micro-inflammation in CKD ratsby enhancing the release of enterogenous exosomes, thereby enhancing the release of exosome-associated miR-146a and inhibiting micro-inflammation.

Pioglitazone treatment mitigates cardiovascular bioprosthetic degeneration in a chronic kidney disease model.

Chronic kidney disease (CKD) is a risk factor for the development of cardiovascular diseases, e.g., atherosclerosis and calcific aortic valve disease, leading inevitably to valve replacement surgery. CKD patients with bioprosthetic cardiovascular grafts, in turn, have a higher risk of premature graft degeneration. Peroxisome proliferator-activated receptor gamma (PPARγ) activation by pioglitazone has cardio-renal protective properties, and research using a heterotopic valve implantation model has shown anti-degenerative effects of PPARγ activation on bioprosthetic valved grafts (BVG) in rats. The present work aims to analyze a potential protective effect of pioglitazone treatment on BVG in an adenine-induced rat model of CKD. BVG of Sprague Dawley rats were heterotopically implanted in Wistar rats in an infrarenal position for 4 and 8weeks. Animals were distributed into three groups for each time point: 1) control group receiving standard chow, 2) CKD group receiving 0.25% adenine and 3) CKD + pioglitazone group (300mg per kg of 0.25% adenine chow). BVG function was analyzed by echocardiography. Plasma analytes were determined and explanted grafts were analyzed by semi-quantitative real-time PCR, Western blot analysis, histology and immunohistology.PPARγ activation significantly reduced CKD-induced calcification of aortic and valvular segments of BVG by 44% and 53%, respectively. Pioglitazone treatment significantly also reduced CKD-induced intima hyperplasia by 60%. Plasma analysis revealed significantly attenuated potassium and phosphate levels after pioglitazone treatment. Moreover, PPARγ activation led to significantly decreased interleukin-6 gene expression (by 57%) in BVG compared to CKD animals. Pioglitazone treatment leads to functional improvement of BVG. This study broadens the understanding of the potential value of PPARγ activation in cardio-renal diseases and delineates pioglitazone treatment as a valuable option to prevent bioprosthetic graft failure in CKD. Further mechanistic studies, e.g., using small molecules activating PPARγ signaling pathways, are necessary for the evaluation of involved mechanisms. Additionally, the translation into pre-clinical studies using large animals is intended as the next research project.

Impaired renal ischemia reperfusion recovery after bilateral renal artery ligation in rats treated with adenine: role of renal mitochondria.

Vascular calcification (VC) and ischemia reperfusion (IR) injury is characterised to have mitochondrial dysfunction. However, the impact of dysfunctional mitochondria associated with vascular calcified rat kidney challenged to IR is not explored and is addressed in the present study. Male Wistar rats were treated with adenine for 20 days to induce chronic kidney dysfunction and VC. After 63 days, renal IR protocol was performed with subsequent recovery for 24h and 7 days. Various mitochondrial parameters and biochemical assays were performed to assess kidney function, IR injury and its recovery. Adenine-induced rats with VC, decreased creatinine clearance (CrCl), and severe tissue injury demonstrated an increase in renal tissue damage and decreased CrCl after 24h of IR (CrCl in ml: IR-0.220.02, VC-IR-0.050.01). Incidentally, the 24h IR pathology in kidney was similar in both VC-IR and normal rat IR. But, the magnitude of dysfunction was higher with VC-IR due to pre-existing basal tissue alterations. We found severed deterioration in mitochondrial quantity and quality supported by low bioenergetic function in both VC basal tissue and IR challenged sample. However, post 7 days of IR, unlike normal rat IR, VC rat IR did not improve CrCl and corresponding mitochondrial damage in terms of quantity and its function were observed. Based on the above findings, we conclude that IR in VC rat adversely affect the post-surgical recovery, mainly due to the ineffective renal mitochondrial functional restoration from the surgery.

Biomarker identification and pathway analysis of Astragalus membranaceus and Curcuma zedoaria couplet medicines on adenine-induced chronic kidney disease in rats based on metabolomics.

Background: Chronic kidney disease (CKD) is usually insidious, and most affected individuals are asymptomatic until the disease becomes advanced. The effective treatment of CKD would rely on the incorporation of multidisciplinary approaches. Astragalus membranaceus (AM) and Curcuma zedoaria (CZ) have been widely used in the treatment of CKD. However, the mechanism of AM and CZ in the treatment of CKD is still unclear. Methods: This study was designed to evaluate the effects of AM and CZ on adenine-induced rats and to investigate the underlying mechanism by using metabolomic analysis. Addition of 0.75% adenine to the diet of rats for 3weeks induced the animal model of CKD. The rats in the treatment group were treated with AM and CZ (2.1g/kg/day) for 4weeks. Blood and kidney samples were collected for biochemical and histological examination. Ultra-high-performance liquid chromatography/Q Exactive HFX mass spectrometer (UHPLC-QE-MS) was applied to analyze metabolic profiling variations in the kidney. Results: The results showed that AM and CZ could significantly reduce serum creatinine (Scr) and blood urea nitrogen (BUN) levels in CKD rats and alleviate renal pathological injury. By comparing the endogenous components of the normal group and the model group in positive ion mode and negative ion mode, a total of 365 and 155 different metabolites were screened, respectively. A total of 117 and 73 metabolites with significantly different expressions were identified between model group and AM and CZ group in positive ion mode and negative ion mode, respectively. The pivotal pathways affected by AM and CZ included nicotinate and nicotinamide metabolism, and glycine, serine and threonine metabolism. Furthermore, significant changes in metabolites in CKD rats after AM and CZ therapies were observed, including L-Threonine, D-pantothenic acid, and nicotinamide. Moreover, we found that AM and CZ significantly reduced renal fibrosis and inflammation in CKD rats, which may be related to the regulation of SIRT1/JNK signaling pathway. Conclusion: In conclusion, AM and CZ significantly reduced renal fibrosis and inflammation in CKD rats, which may be related to the regulation of SIRT1/JNK signaling pathway. Furthermore, L-Threonine, D-pantothenic acid, and nicotinamide may be potential biomarkers for the progression and treatment of CKD.

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Shenkang injection improves chronic kidney disease by inhibiting multiple renin-angiotensin system genes by blocking the Wnt/β-catenin signalling pathway.

Chronic kidney disease (CKD) is a major worldwide public health problem. The increase in the number of patients with CKD and end-stage kidney disease requesting renal dialysis or transplantation will progress to epidemic proportions in the next several decades. Although blocking the renin-angiotensin system (RAS) has been used as a first-line standard therapy in patients with hypertension and CKD, patients still progress towards end-stage kidney disease, which might be closely associated with compensatory renin expression subsequent to RAS blockade through a homeostatic mechanism. The Wnt/β-catenin signalling pathway is the master upstream regulator that controls multiple intrarenal RAS genes. As Wnt/β-catenin regulates multiple RAS genes, we inferred that this pathway might also be implicated in blood pressure control. Therefore, discovering new medications to synchronously target multiple RAS genes is necessary and essential for the effective treatment of patients with CKD. We hypothesized that Shenkang injection (SKI), which is widely used to treat CKD patients, might ameliorate CKD by inhibiting the activation of multiple RAS genes via the Wnt/β-catenin signalling pathway. To test this hypothesis, we used adenine-induced CKD rats and angiotensin II (AngII)-induced HK-2 and NRK-49F cells. Treatment with SKI inhibited renal function decline, hypertension and renal fibrosis. Mechanistically, SKI abrogated the increased protein expression of multiple RAS elements, including angiotensin-converting enzyme and angiotensin II type 1 receptor, as well as Wnt1, β-catenin and downstream target genes, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in adenine-induced rats, which was verified in AngII-induced HK-2 and NRK-49F cells. Similarly, our results further indicated that treatment with rhein isolated from SKI attenuated renal function decline and epithelial-to-mesenchymal transition and repressed RAS activation and the hyperactive Wnt/β-catenin signalling pathway in both adenine-induced rats and AngII-induced HK-2 and NRK-49F cells. This study first revealed that SKI repressed epithelial-to-mesenchymal transition by synchronously targeting multiple RAS elements by blocking the hyperactive Wnt/β-catenin signalling pathway.

The Dysregulation of Eicosanoids and Bile Acids Correlates with Impaired Kidney Function and Renal Fibrosis in Chronic Renal Failure

Chronic renal failure (CRF) is an irreversible deterioration of the renal functions that characterized by fluid electrolyte unbalance and metabolic-endocrine dysfunctions. Increasing evidence demonstrated that metabolic disturbances, especially dyslipidemia and profound changes in lipid and lipoprotein metabolism were involved in CRF. Identification of lipids associated with impaired kidney functions may play important roles in the understanding of biochemical mechanism and CRF treatment. Ultra-performance liquid chromatography coupled with high-definition mass spectrometry-based lipidomics was performed to identify important differential lipids in adenine-induced CRF rats and investigate the undergoing anti-fibrotic mechanism of Polyporus umbellatus (PPU) and ergone (ERG). Linear correlation analysis was performed between lipid species intensities and creatinine levels in serum. Adenine-induced rats exhibited declining kidney function and renal fibrosis. Compared with control rats, a panel of lipid species was identified in the serum of CRF rats. Our further study demonstrated that eight lipids, including leukotrienes and bile acids, presented a strong linear correlation with serum creatinine levels. In addition, receiver operating characteristics analysis showed that eight lipids exhibited excellent area under the curve for differentiating CRF from control rats, with high sensitivity and specificity. The aberrant changes of clinical biochemistry data and dysregulation of eight lipids could be significantly improved by the administration of PPU and ergone. In conclusion, CRF might be associated with the disturbance of leukotriene metabolism, bile acid metabolism and lysophospholipid metabolism. The levels of eicosanoids and bile acids could be used for indicating kidney function impairment in CRF. PPU could improve renal functions and either fully or partially reversed the levels of eicosanoids and bile acids.

Neointimal hyperplasia in the inferior vena cava of adenine-induced chronic kidney disease rats with aortocaval fistulas

The failure of autologous arteriovenous fistulas (AVFs) occurs primarily due to stenosis in the anastomotic site, which is mainly related to the development of neointimal hyperplasia (NIH). Therefore, we conducted a study to establish a novel approach to create aortocaval fistulas (ACFs) in adenine-induced (AD) chronic kidney disease (CKD) rats to study the NIH in the inferior vena cava. Ten adult female rats received a 0.75% adenine-rich diet for 4weeks to induce CKD and underwent ACF surgery. Ten healthy rats served as controls. A 5-10-mm segment of a vein immediately adjacent to that the portion of the vein used for creating the fistula was surgically removed at the time of creating the fistula, and reconstruction of the failed fistula from the same patient was used as controls. ACF was assessed using duplex scans and histopathological analyses. At the end of the experiment, AD rats showed higher serum creatinine and urea nitrogen than those of vehicle-treated rats. Remarkable histological changes in kidney tissues demonstrated successful CKD models. Sections of the ACF in AD rats and veins removed at the time of the reconstruction of the failed fistula of the patient demonstrated that the eccentric neointima formation is irregularly thickened, with several small vessels within a more cellular region of the neointima. Immunohistochemistry demonstrated the presence of myofibroblasts, contractile smooth muscle cells and macrophages within the neointima. Our rat models with ACFs showed typical features of NIH in the formation of fistula stenosis, which can resemble clinical findings in uremic patients.

Severe chronic kidney disease environment reduced calcium-sensing receptor expression in parathyroid glands of adenine-induced rats even without high phosphorus diet

BackgroundChronic kidney disease (CKD) disrupts mineral homeostasis and its main underlying cause is secondary hyperparathyroidism (SHPT). We previously reported that calcium-sensing receptor (CaSR) mRNA and protein expression in parathyroid glands (PTGs) significantly decreased in a CKD rat model induced by a 5/6 nephrectomy that were fed a high phosphorus diet. However, there was a significant difference in the severity of CKD between high phosphorus and adequate phosphorus diet groups. Thus, it was unclear whether CKD environment or the high phosphorus diet influenced CaSR expression, and the underlying mechanism remains largely unknown.MethodsCKD was induced in rats with 0.75% adenine-containing diet. CKD and control rats were maintained for 5 days and 2 weeks on diets with 0.7% or 1.3% phosphorus. For gene expression analysis, quantitative real-time polymerase chain reaction was performed with TaqMan probes. Protein expression was analyzed by immunohistochemistry.ResultsPTG CaSR expression significantly decreased in the presence of a severe CKD environment, even without the high phosphate load. Ki67 expressing cells in PTGs were significantly higher only in the CKD rats fed a high phosphorus diet. Furthermore, among the many genes that could affect CaSR expression, only vitamin D receptor (VDR) and glial cells missing 2 (Gcm2) showed significant changes. Moreover, Gcm2 was significantly reduced at an early stage without significant changes in serum calcium, phosphorus and 1,25(OH)2 vitamin D, and there was no significant reduction in CaSR and VDR expressions. Then, significantly elevated Ki67-positive cell numbers were also only observed in the early CKD PTGs with high-phosphorus diets.ConclusionsOur data suggest that the cause of the decreased PTG CaSR expression is the reduction in VDR and Gcm2 expression; Gcm2 may play a role in the onset and progression of SHPT.

Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α Inhibits Vascular Calcification Through Sirtuin 3-Mediated Reduction of Mitochondrial Oxidative Stress.

Aims: Vascular calcification is associated with cardiovascular death in patients with chronic kidney disease (CKD). Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) plays an important role in various cardiovascular diseases. However, its role in vascular calcification remains unknown. Results: Adenine-induced rat CKD model was used to induce arterial medial calcification. The level of PGC-1α decreased in abdominal aorta of CKD rats. Overexpression of PGC-1α significantly ameliorated calcium deposition in rat abdominal aorta, isolated carotid rings, and cultured vascular smooth muscle cells (VSMCs). Mitochondrial reactive oxygen species (mtROS) increased in calcifying aorta and VSMCs. Upregulation of PGC-1α inhibited, whereas PGC-1α depletion promoted β-glycerophosphate-induced mtROS production and calcium deposition. Moreover, PGC-1α increased superoxide dismutase 1 (SOD1) and SOD2 contents in vivo and in vitro, whereas SOD2 deletion eliminated PGC-1α-mediated mtROS change and promoted calcium deposition. Mechanistically, sirtuin 3 (SIRT3) expression declined in calcifying aorta and VSMCs, while PGC-1α overexpression restored SIRT3 expression. Inhibition of SIRT3 by 3-TYP or siRNA (small interfering RNA) reduced PGC-1α-induced upregulation of SOD1 and SOD2, and abolished the protective effect of PGC-1α on calcification of VSMCs. Importantly, PGC-1α was reduced in calcified femoral arteries in CKD patients. In phosphate-induced human umbilical arterial calcification, upregulation of PGC-1α attenuated calcium nodule formation, while this protective effect was abolished by SIRT3 inhibitor. Innovation: We showed for the first time that PGC-1α is an important endogenous regulator against vascular calcification. Induction of PGC-1α could be a potential strategy to treat vascular calcification in CKD patients. Conclusions: PGC-1α protected against vascular calcification by SIRT3-mediated mtROS reduction.

Omega-3 polyunsaturated fatty acids alleviate adenine-induced chronic renal failure via regulating ROS production and TGF-β/SMAD pathway.

To explore the role of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in adenine-induced rat chronic renal failure and its underlying mechanism. 30 Sprague Dawley (SD) rats were randomly assigned into three groups, namely sham group, adenine induction group (adenine group) and adenine induction + ω-3 PUFAs treatment group (ω-3 PUFAs group), with 10 rats in each group. Serum and kidney samples were collected after rats were sacrificed. Serum levels of Cr (creatinine) and BUN (urea nitrogen) were detected using commercial kits. HE (hematoxylin and eosin) staining was performed to evaluate the pathological changes of kidneys. Levels of oxidative stress indicators in rat kidney homogenate were detected by relative commercial kits, including SOD (superoxide dismutase), GSH (reduced glutathione), CAT (catalase), and T-AOC (total antioxidant capacity). Reactive oxygen species (ROS) production was also detected by immunofluorescence. Protein expressions of nuclear factor E2 related factor 2 (Nrf2) and transforming growth factor-beta (TGF-β)/SMAD pathway-related genes were detected by Western blot. Serum levels of Cr and BUN in ω-3 PUFAs group were remarkably decreased compared with those of adenine group. Higher contents of SOD, GSH, CAT and T-AOC were observed in ω-3 PUFAs group compared with those of adenine group. Besides, MAD content and ROS production were lower in ω-3 PUFAs group than those of adenine group. Pathological changes of kidneys were alleviated after ω-3 PUFAs treatment. Western blot results demonstrated that ω-3 PUFAs treatment remarkably upregulates Nrf2, HO-1, NQO1, but downregulates relative genes in TGF-β/SMAD pathway. ω-3 PUFAs alleviated adenine-induced chronic renal failure through enhancing antioxidant stress and inhibiting inflammatory response via regulating Nrf2 and TGF-β/SMAD pathway.

Calcitriol Accelerates Vascular Calcification Irrespective of Vitamin K Status in a Rat Model of Chronic Kidney Disease with Hyperphosphatemia and Secondary Hyperparathyroidism.

Patients with chronic kidney disease (CKD) have a markedly increased risk for developing cardiovascular disease. Nontraditional risk factors, such as increased phosphate retention, increased serum fibroblast growth factor 23 (FGF-23), and deficiencies in vitamins D and K metabolism, likely play key roles in the development of vascular calcification during CKD progression. Calcitriol [1,25-(OH)2-D3] is a key transcriptional regulator of matrix Gla protein, a vitamin K-dependent protein that inhibits vascular calcification. We hypothesized that calcitriol treatment would inhibit the development of vascular calcification and this inhibition would be dependent on vitamin K status in a rat model of CKD. Rats were treated with dietary adenine (0.25%) to induce CKD, with either 0, 20, or 80 ng/kg of calcitriol with low or high dietary vitamin K1 (0.2 or 100 mg/kg) for 7 weeks. Calcitriol at both lower (20 ng/kg) and moderate (80 ng/kg) doses increased the severity of vascular calcification, and contrary to our hypothesis this was not significantly improved by high dietary vitamin K1. Calcitriol had a dose-dependent effect on: 1) lowering serum parathyroid hormone, 2) increasing serum calcium, and 3) increasing serum FGF-23. Calcitriol treatment significantly increased aortic expression of the calcification genes Runx2 and Pit-1 These data also implicate impaired vitamin D catabolism in CKD, which may contribute to the development of calcitriol toxicity and increased vascular calcification. The present findings demonstrate that in an adenine-induced rat model of CKD calcitriol treatment at doses as low as 20 ng/kg can increase the severity of vascular calcification regardless of vitamin K status.

The flavonoid rutin improves kidney and heart structure and function in an adenine-induced rat model of chronic kidney disease

Many foods contain rutin, a flavonoid with many health benefits. Here we tested rutin to reverse chronic kidney disease (CKD) and associated cardiovascular disease, using an established rodent model with adenine diet (0.25%) over 16weeks. Rutin was added to the diet for the final 8weeks. We measured kidney function (blood urea nitrogen/BUN, plasma creatinine/PCr, plasma uric acid, proteinuria); kidney structure (tubular damage, fibrosis, inflammation); cardiovascular function and structure (blood pressure, left ventricular stiffness, vascular responses, echocardiography, histology); and biomarkers for oxidative stress (HO-1), inflammation (PLA-2, TNF-α) and fibrosis (TGF-β). Adenine diet increased CKD characteristics (tubulointerstitial atrophy and fibrosis, inflammation, proteinuria, BUN, PCr, uric acid) and CKD-associated cardiovascular disease. Rutin improved kidney and heart structure and function, decreased HO-1 and PLA-2 expression, but did not reduce TGF-β or TNF-α. Decreased expression of HO-1 and PLA-2 appears to be a mechanistic pathway for reno- and cardioprotection.

Combination therapy with renin-angiotensin-aldosterone system inhibitor telmisartan and serine protease inhibitor camostat mesilate provides further renoprotection in a rat chronic kidney disease model

We previously reported that camostat mesilate (CM) had renoprotective and antihypertensive effects in rat CKD models. In this study, we examined if CM has a distinct renoprotective effect from telmisartan (TE), a renin-angiotensin-aldosterone system (RAS) inhibitor, on the progression of CKD. We evaluated the effect of CM (400 mg/kg/day) and/or TE (10 mg/kg/day) on renal function, oxidative stress, renal fibrosis, and RAS components in the adenine-induced rat CKD model following 5-weeks treatment period. The combination therapy with CM and TE significantly decreased the adenine-induced increase in serum creatinine levels compared with each monotherapy, although all treatment groups showed similar reduction in blood pressure. Similarly, adenine-induced elevation in oxidative stress markers and renal fibrosis markers were significantly reduced by the combination therapy relative to each monotherapy. Furthermore, the effect of the combination therapy on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was similar to that of TE monotherapy, and CM had no effect on both PRA and PAC, suggesting that CM has a distinct pharmacological property from RAS inhibition. Our findings indicate that CM could be a candidate drug for an add-on therapy for CKD patients who had been treated with RAS inhibitors.

1D.08

Objective: Cardiovascular disease is a major cause of mortality in patients with chronic kidney disease (CKD). Elevated serum phosphate and FGF23 are associated with cardiovascular disease in patients with CKD. Current therapy focuses on decreasing serum phosphorus using phosphate binders. PA21 is a new iron-based phosphate binder. Few studies have analysed how to suppress FGF23 up-regulation using phosphate binders. To evaluate the effects of PA21 compared with other phosphate binders as lanthanum carbonate (La) and sevelamer carbonate (Se) on serum FGF23, phosphorus, calcium, iPTH concentrations and to investigate a potential effect on the development of vascular calcifications in an adenine-induced rat model of CRF. Design and method: After induction of chronic renal failure through a 4 week adenine-diet, renal function was significantly impaired in all groups. All uremic rats developed severe hyperphosphatemia and serum PTH increased significantly. Phosphate binders were then given for 4 weeks to all uremic rats, except for the uremic control rats. The concentration of each binder (% of binder added to the diet) was chosen to deliver approximately the same amount of active pharmaceutical moiety to each rat: PA21 5% (corresponding to 1% iron), La 2% (1% lanthanum), Se 1.5% (1% sevelamer). A computer-assisted automated quantitative measurement was used to assess the degree of calcification from von Kossa stained vessel sections. Results: Hyperphosphatemia and increased serum PTH levels were controlled in the phosphate binder treated groups to the same extent. PA21 was the only phosphate binder that was associated with a decrease of FGF23. In uremic control rats, vascular calcifications were more prominently present in the thoracic aorta compared to the carotids and the abdominal aorta. Vascular calcifications of thoracic aorta were significantly decreased by the three phosphate binders to a similar extent. PA21 was more efficient than lanthanum carbonate to prevent calcifications in the upper part of the thoracic aorta. Conclusions: PA21 was as effective in the control of hyperphosphatemia, secondary hyperparathyroidism and vascular calcifications as La and Se. The role of FGF23 as a potential factor of calcification needs to be confirmed.

Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6–7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg−1 d−1.

The Acute Encephalopathy Induced by Intake of Sugihiratake Mushroom in the Patients with Renal Damage Might Be Associated with the Intoxication of Cyanide and Thiocyanate

A novel type of encephalopathy associated with the ingestion of Sugihiratake mushroom (Pleurocybella porrigens) occurred in patients with chronic renal failure treated on hemodialysis in fall, 2004 in Japan. To clarify the mechanism of encephalopathy onset, we, for the first time, purified the cyanogen glycoside fraction (CG) from Sugihiratake mushroom using reversed phase high-performance liquid chromatography and hydrophilic interaction chromatography. Furthermore, we investigated single dose toxicity of the CG in an adenine-induced rat model of chronic renal damage (CRD). Pathological examination of kidneys indicates the development of CRD. Oral administration of the CG induces the accumulation of thiocyanate in the hemolyzed blood and brain in CRD rats, although no morphological changes were found in the brain. No further enhancement of kidney damage is observed after the oral administration of the CG in CRD rats. This is the first experimental report to suggest that acute encephalopathy, induced by Sugihiratake mushroom intake in the patients with chronic renal failure, is associated with intoxication of cyanide and thiocyanate, presumably produced metabolically produced after the ingestion of Sugihiratake mushroom.

Advanced glycation end products suppress lysyl oxidase and induce bone collagen degradation in a rat model of renal osteodystrophy

Renal osteodystrophy (ROD) is a major problem in patients with renal insufficiency. The present study was designed to elucidate the role of bone collagen changes and osteoblast differentiation in a rat model of ROD pathogenesis induced by adenine. Typical characteristics of renal failure, including increased serum urea nitrogen, creatinine, inorganic phosphorus, and intact parathyroid hormone levels, and decreased serum calcium and 1,25(OH)2D3 levels, were observed in adenine-induced rats. Micro-computed tomography analysis of the femur in adenine-induced rats showed decreased bone mineral density and osteoporotic changes, confirmed by the three-point bending test. The cancellous bone histomorphometric parameters of the tibia showed increased osteoblast number, decreased osteoclast surface with peritrabecular fibrosis, and increased osteoid tissue, indicating a severe mineralization disorder similar to clinical ROD. Scanning and transmission electron microscopy revealed irregular alignment and increased diameter of bone collagen fibrils in adenine-induced rats. Protein expression analysis showed greater accumulation of advanced glycation end products (AGEs) in peritrabecular osteoblasts of adenine-induced rats than in the controls. In contrast, suppressed expression of runt-related transcription factor 2, alkaline phosphatase, secreted phosphoprotein 1 (Spp1), and lysyl oxidase (Lox) mRNA levels, particularly the amount of active LOX protein, were observed. In in-vitro experiments, mineralizing MC3T3-E1 osteoblastic cells stimulated with AGE-modified bovine serum albumin had attenuated the expression of Spp1 mRNA levels and active LOX protein, with a decrease in extracellular nodules of mineralization. These observations provide clues to ROD pathogenesis, as they indicate that the suppression of osteoblast differentiation and decreased active LOX protein associated with accumulation of AGEs in osteoblasts caused structural abnormalities of bone collagen fibrils and a severe mineralization disorder, leading to bone fragility.