Uremic Rats Research Articles

The use of LP533401 as a therapeutic option for renal osteodystrophy affects, renal calcium handling, vitamin D metabolism, and bone health in uremic rats

ABSTRACTBackground: Klotho is a key regulator of phosphate and Ca2+-transport in the kidney. Recently, we showed that treatment with LP533401 improved bone health in rats with chronic kidney disease (CKD) via the normalization of serum phosphate resulting from the reduced renal expression of phosphate cotransporters, including Klotho.Methods: We evaluated the effect of LP533401 therapy on Klotho-expression-dependent Ca2+-transporters, renal calcium handling, and the potential consequences for the bone of uremic rats.Results: Treatment with LP533401 and its vehicle resulted in the inhibition of transient receptor potential vanilloid receptor subtypes 5 and 6 (TRPV5, TRPV6) and calbindin (CaBP-28k, CaBP-9k) expression. The compensatory acceleration in renal expression of Na+/Ca2+-exchanger, 25-hydroxyvitamin d-1α-hydroxylase (CYP27B1), the intensification of vitamin D metabolism, and disruption of sophisticated balance between 1,25-dihydroxyvitamin D–serotonin was observed, especially in rats treated with LP533401. The imbalance between 1,25-dihydroxyvitamin D–serotonin levels led to intensified bone remodeling and improvement in bone geometry, mineral status, and strength in animals treated with LP533401.Conclusion: The modulation of circulating serotonin and its relation to other regulators of calcium handling can play an important role in calcium homeostasis and bone integrity in CKD rats treated with LP533401.

WISP1 silencing confers protection against epithelial-mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/β-catenin signaling pathway in uremia.

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial-mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/β-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following theestablishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/β-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/β-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/β-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/β-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/β-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Influence of colonic dialysis using Gubenxiezhuo on the distribution of gut microflora in uremia rats.

This study aimed to explore the underlying function of Gubenxiezhuo dialysis on the distribution of gut microflora uremia. A uremia rat model was constructed, and the morphology of renal tissue was determined using the hematoxylin-eosin (H&E) staining. Moreover, the blood samples were collected and the expression of IL-1β, IL-6, and CRP was determined using enzyme-linked immunosorbent assay. Following these experiments, the gut tissues of rats were collected and the distribution of gut microbiota was explored using real-time PCR. Compared with the control group, inflammatory infiltration, apoptosis, and bleeding were significantly upregulated in kidney of uremia rats, and Gubenxiezhuo dialysis could obviously ameliorate these changes. Expression of IL-1β, IL-6, and CRP were significantly elevated in uremic rats and Gubenxiezhuo could significantly attenuate these elevations (p < 0.01). In addition, Gubenxiezhuo dialysis also could attenuate the upregulations of Acinetobacter, Bacillus cereus, Proteus vulgaris, Shigella flexneri, and Escherichia coli , and the downregulation of Bifidobacterium, Lactobacillus, and Helicobacter in the uremia rats ( p < 0.05). Gubenxiezhuo dialysis could significantly ameliorate the inflammatory to modulate the distribution of gut microbiota in uremia.

EFFECTS OF RENAL DYSFUNCTION ON HEALING OF COLONIC ANASTOMOSIS: EXPERIMENTAL STUDY IN WISTAR RATS.

ABSTRACTBackground: Chronic kidney disease affects more than 500 million people worldwide. In this context, the uremic toxins present are related to worsening in tissue healing. Aim: Evaluate on healing of colonic anastomosis in uremic rats, serum and anatomopathological indicators, which may be related to the change tissue repair process. Methods: Twenty Wistar rats, were randomly separated into two groups. In the sham group they were submitted to 5/6 nephrectomy simulation in left kidney, simulation right nephrectomy, median laparotomy, colotomy and colorraphy. In the uremia group, they were submitted to 5/6 nephrectomy of the left kidney, total nephrectomy of the right kidney and median laparotomy, colotomy and colorraphy. Were collected for serum urea, creatinine and CRP dosages and the colonic segments were studied for evaluation of granulation tissue, collagen maturation, microvascular and myofibroblasts density, and cell viability. Through histochemical processing, microvascular density was evaluated by anti-CD34 monoclonal antibody marking, cell viability by cell proliferation nuclear antigen screening and myofibroblasts density with monoclonal anti-α-actin antibody. Computerized histometry was used for evaluations of collagens type I and III by the coloration of picrosirius. Results: The group submitted to nephrectomy 5/6, compared to the sham group, show urea increase (p<0.0000) and higher C reactive protein (p=0.0142). Decrease of granulation tissue formation (border reepithelialization p=0,0196, angiofibroblast proliferation p=0.0379), mean collagen I (p=0,0009) and collagen III (p=0,016), microvascular density (p=0,0074), cell proliferation nuclear antigen (p<0,0000) and myofibroblasts (p<0,0001). Conclusion: The uremia induced by nephrectomy 5/6 model establishes negative impact in the colonic wound healing.

Cardioprotective Effects of Paricalcitol Alone and in Combination With FGF23 Receptor Inhibition in Chronic Renal Failure: Experimental and Clinical Studies

In uremic animals, vitamin D receptor (VDR) agonists like paricalcitol (Pc) attenuate cardiac hypertrophy, but this effect has not been replicated consistently in humans with chronic kidney disease. Elevated fibroblast growth factor 23 (FGF23) levels cause cardiac hypertrophy with activation of the myocardial calcineurin/nuclear factor of activated T cell (NFAT) axis and may antagonize the cardioprotective effects of VDR agonist therapy. We hypothesized that the effectiveness of Pc may depend on the prevailing circulating levels of FGF23 and could be potentiated by the combined administration of a pan-FGF23 receptor (FGFR) blocker agent (PD173074). In rats with 5/6 nephrectomy treated with Pc or PD173074 or both agents concurrently, myocardial mRNA expression of renin-angiotensin system, VDR, FGFR4, and calcineurin/NFAT target genes was determined. In adolescents on hemodialysis, we analyzed sequential echocardiograms, blood pressures and serial FGF23 measurements, and their relations to the cumulative administered dose of parenteral Pc. The ratio of Pc dose/plasma levels of FGF23 correlated inversely (P < 0.005) with the cardiac mass in uremic rats and in hemodialysis patients, independently of hypertension. Despite persistently elevated FGF23 levels and myocardial FGFR4 activation, Pc suppressed upregulated myocardial calcineurin/NFAT target genes, and the effects were amplified by coadministration of PD173074. The beneficial effects of Pc on uremic cardiac hypertrophy are counterbalanced by the increased FGF23 levels. Blockade of FGF23-mediated signaling increased the Pc-induced suppression of the myocardial calcineurin/NFAT system. Higher doses of Pc should be considered in the treatment of patients with uremic cardiomyopathy.

Efficacy of linalool to ameliorate uremia induced vascular calcification in wistar rats

BackgroundUremia is the condition generally associated with the last stage of chronic kidney disease (CKD) due to highly reduced glomerular filtration rate. Mortality of the patients diagnosed with Uremia generally occurs due to cardiovascular involvement. This occurs due to the transdifferentiation of vascular smooth muscle cells (VSMCs) into osteogenic cells in hyperphosphatemic condition that is associated with kidney failure promoting extra-osseous calcification. PurposeLinalool is an essential oil that has been recently studied for its procardiovascular effects, thus the aim of the study involved to identify its potential role on vascular calcification (VC). MethodsUremia was induced in male wistar rats, weighing 250–350 gm by giving adenine diet for 4 weeks followed by phosphate diet for next 4 weeks. Linalool was given orally at two different doses (100 mg/kg bodyweight and 150 mg/kg bodyweight) daily for 4 weeks with phosphate diet. ResultsLinalool being a moderate antioxidant probably scavenged superoxide radicals (in vitro analysis). Deposition of calcium was observed by alizarin and von-kossa stains in aorta of uremic rats whereas linalool co-administration prevents calcium deposition in aorta of uremic rats. Elevated mRNA expression of calcification markers, increased lipid peroxidation levels and increased levels of catalase and superoxide dismutase (SOD) was found in aorta of uremic animals. However, with supplementation of linalool reduction in the mRNA expression of calcification markers, lipid peroxidation and antioxidant enzymes were observed. ConclusionTherefore it can be concluded that linalool could be a promising therapeutic candidate for exploring its clinical application in VC.

Review: update on the management of calciphylaxis.

Calciphylaxis is a disease of significant morbidity and mortality, predominantly affecting dialysis patients. The term 'calciphylaxis' was coined by Seyle etal. in 1961 to describe calcium deposition in the skin and subcutaneous soft tissue of uremic rats in response to 'triggers' (e.g. trauma, metallic salts) after exposure to 'sensitizing agents' (e.g. vitamin D and parathyroid hormone). In humans, calciphylaxis, however, is not a disorder of induced hypersensitivity. Instead, it is a disorder of cutaneous microvascular occlusion caused by thrombosis and calcification. Progressive, excruciatingly painful, non-healing wounds develop in these patients, pre-disposing them to high risk of sepsis and death. Calciphylaxis has no approved therapies. Increased awareness and research in this field have facilitated identification of risk factors and causation pathways. Development of therapeutic options and wound care management, however, are still at a nascent stage. Certain therapies have shown a promise that needs evaluation in prospective clinical trials. It is hoped that ongoing research will help us better understand the pathogenesis of this complex disease and develop efficacious treatment options. In this review, we outline the components involved in calciphylaxis diagnosis and treatment.

High-serum phosphate and parathyroid hormone distinctly regulate bone loss and vascular calcification in experimental chronic kidney disease.

In chronic kidney disease (CKD), increases in serum phosphate and parathyroid hormone (PTH) aggravate vascular calcification (VC) and bone loss. This study was designed to discriminate high phosphorus (HP) and PTH contribution to VC and bone loss. Nephrectomized rats fed a HP diet underwent either sham operation or parathyroidectomy and PTH 1-34 supplementation to normalize serum PTH. In uraemic rats fed a HP diet, parathyroidectomy with serum PTH 1-34 supplementation resulted in (i) reduced aortic calcium (80%) by attenuating osteogenic differentiation (higher α-actin; reduced Runx2 and BMP2) and increasing the Wnt inhibitor Sclerostin, despite a similar degree of hyperphosphataemia, renal damage and serum Klotho; (ii) prevention of bone loss mostly by attenuating bone resorption and increases in Wnt inhibitors; and (iii) a 70% decrease in serum calcitriol levels despite significantly reduced serum Fgf23, calcium and renal 24-hydroxylase, which questions that Fgf23 is the main regulator of renal calcitriol production. Significantly, when vascular smooth muscle cells (VSMCs) were exposed exclusively to high phosphate and calcium, high PTH enhanced while low PTH attenuated calcium deposition through parathyroid hormone 1 receptor (PTH1R) signalling. In hyperphosphataemic CKD, a defective suppression of high PTH exacerbates HP-mediated osteogenic VSMC differentiation and reduces vascular levels of anti-calcifying sclerostin.

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

BackgroundDysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease (CKD). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD, not only by direct effects on the endothelium but also by an increment of α‐Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia‐induced endothelial damage and the extent to which this was dependent on increased α‐Klotho concentrations.Methods and ResultsIn a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial‐gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α‐Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real‐time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro‐permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial‐cadherin–based cell‐cell junctions and diminished F‐actin stress fiber organization, preventing the formation of endothelial intracellular gaps.ConclusionsOur results demonstrate that paricalcitol attenuates the CKD‐induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell‐cell interactions.

Effect of inhibition of CBP-coactivated β-catenin-mediated Wnt signalling in uremic rats with vascular calcifications.

Uremic vascular calcification is a regulated cell-mediated process wherein cells in the arterial wall transdifferentiate to actively calcifying cells resulting in a process resembling bone formation. Wnt signalling is established as a major driver for vessel formation and maturation and for embryonic bone formation, and disturbed Wnt signalling might play a role in vascular calcification. ICG-001 is a small molecule Wnt inhibitor that specifically targets the coactivator CREB binding protein (CBP)/β-catenin-mediated signalling. In the present investigation we examined the effect of ICG-001 on vascular calcification in uremic rats. Uremic vascular calcification was induced in adult male rats by 5/6-nephrectomy, high phosphate diet and alfacalcidol. The presence of uremic vascular calcification in the aorta was associated with induction of gene expression of the Wnt target gene and marker of proliferation, cyclinD1; the mediator of canonical Wnt signalling, β-catenin and the matricellular proteins, fibronectin and periostin. Furthermore, genes from fibrosis-related pathways, TGF-β and activin A, as well as factors related to epithelial-mesenchymal transition, snail1 and vimentin were induced. ICG-001 treatment had significant effects on gene expression in kidney and aorta from healthy rats. These effects were however limited in uremic rats, and treatment with ICG-001 did not reduce the Ca-content of the uremic vasculature.

Effect of Continuous Intravenous Calcium Loading on Fibroblast Growth Factor 23 in Normal and Uremic Rats.

Fibroblast growth factor 23 (FGF23) is associated with mortality in patients with CKD. However, the mechanisms underlying stimulation of FGF23 remain to be investigated. We examined whether hypercalcemia induced by continuous intravenous (CIV) calcium (Ca) infusion regulates FGF23 levels in normal rats (Normal) and 5/6 nephrectomized uremic rats (Nx). Microinfusion pumps were implanted in the Normal and Nx rats for CIV Ca infusion, and blood, urine, kidney, and tibia were collected. The results showed an increase in serum Ca-stimulated FGF23 independently of serum phosphate (P) and creatinine levels in Normal and Nx rats. FGF23 mRNA from the tibia was also increased by the Ca infusion. Despite high FGF23 levels after Ca infusion, urinary P excretion was decreased. Renal α-Klotho expression was significantly reduced by Ca infusion. These results suggest that intravenous Ca loading might stimulate FGF23 production from bone in normal and uremic rats. Reduction of renal P excretion suggests that the bioactivity of FGF23 is inhibited, and the decrease in renal α-Klotho expression might have a role in this pathological process. In conclusion, CIV Ca loading increased FGF23 in normal and uremic rats, and renal α-Klotho is necessary to maintain the bioactivity of FGF23 as a phosphaturic factor.

Characterization of SNF472 pharmacokinetics and efficacy in uremic and non-uremic rats models of cardiovascular calcification

End-stage renal disease is strongly associated with progressive cardiovascular calcification (CVC) and there is currently no therapy targeted to treat CVC. SNF472 is an experimental formulation under development for treatment of soft tissue calcification. We have investigated the pharmacokinetics of SNF472 administration in rats and its inhibitory effects on CVC. SNF472 was studied in three rat models: (1) prevention of vitamin D3-induced CVC with an intravenous SNF472 bolus of 1 mg/kg SNF472, (2) inhibition of progression of vitamin D3-induced CVC with a subcutaneous SNF472 bolus of 10 or 60 mg/kg SNF472, starting after calcification induction, (3) CVC in adenine-induced uremic rats treated with 50 mg/kg SNF472 via i.v. 4h -infusion. Uremic rats presented lower plasma levels of SNF472 than control animals after i.v. infusion. CVC in non-uremic rats was inhibited by 60–70% after treatment with SNF472 and progression of cardiac calcification completely blocked. Development of CVC in uremic rats was inhibited by up to 80% following i.v. infusion of SNF472. SNF472 inhibits the development and progression of CVC in uremic and non-uremic rats in the same range of SNF472 plasma levels but using in each case the required dose to obtain those levels. These results collectively support the development of SNF472 as a novel therapeutic option for treatment of CVC in humans.

Effect of cross-linked chitosan iron (III) on vascular calcification in uremic rats.

Cross-linked chitosan iron (III) is a chitin-derived polymer with a chelating effect on phosphorus, but it is untested in vascular calcification. We evaluated this compound's ability to reduce hyperphosphatemia and its effect on vascular calcification in uremic rats using an adenine-based, phosphorus-rich diet for seven weeks. We used a control group to characterize the uremia. Uremic rats were divided according the treatment into chronic kidney disease, CKD-Ch-Fe(III)CL (CKD-Ch), CKD-calcium carbonate, or CKD-sevelamer groups. We measured creatinine, phosphorus, calcium, alkaline phosphatase, phosphorus excretion fraction, parathyroid hormone, and fibroblast growth factor 23. Vascular calcification was assessed using the aortic calcium content, and a semi-quantitative analysis was performed using Von Kossa and hematoxylin-eosin staining. At week seven, rats in the chronic kidney disease group had higher creatinine, phosphorus, phosphorus excretion fraction, calcium, alkaline phosphatase, fibroblast growth factor 23, and aortic calcium content than those in the Control group. Treatments with cross-linked chitosan iron (III) and calcium carbonate prevented phosphorus increase (20%-30% reduction). The aortic calcium content was lowered by 88% and 85% in the CKD-Ch and CKD-sevelamer groups, respectively. The prevalence of vascular changes was higher in the chronic kidney disease and CKD-calcium carbonate (62.5%) groups than in the CKD-Ch group (37.5%). In conclusion, cross-linked chitosan iron (III) had a phosphorus chelating effect similar to calcium carbonate already available for clinical use, and prevented calcium accumulation in the aorta. Impact statement Vascular calcification (VC) is a common complication due to CKD-related bone and mineral disorder (BMD) and is characterized by deposition of calcium in vessels. Effective therapies are not yet available but new phosphorus chelators can prevent complications from CV. We tested the effect of chitosan, a new phosphorus chelator, on the VC of uremic animals. It has recently been proposed that chitosan treatment may be effective in the treatment of hyperphosphataemia. However, its action on vascular calcification has not been investigated yet. In this study, we demonstrated that chitosan reduced the calcium content in the aorta, suggesting that this may be a therapeutic approach in the treatment of hyperphosphatemia by preventing CV.

High calcium, phosphate and calcitriol supplementation leads to an osteocyte-like phenotype in calcified vessels and bone mineralisation defect in uremic rats.

A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.

Therapeutic potential of probiotic Lactobacillus plantarum AD3 on acetaminophen induced uremia in experimental rats

Summary Background & aims Chronic kidney disease (CKD) is rapidly heading towards epidemic proportion globally, requires dialysis or kidney transplantation. No generally applicable therapies to slow progression of renal disease are available. The aim of this study was to evaluate Lactobacillus plantarum AD3 as an effective probiotic strain for acetaminophen induced uremic patient. Methods Plasma uremic profiles, oxidative stress marker, DNA fragmentation assay of kidney tissue and kidney histological studies were investigated on acetaminophen-induced uremic rats (Wister strain albino male). Limited fecal matter analysis and HPLC of intestinal fluid were also performed. Results In APAP treated rats, plasma urea, creatinine (Cr), glutamate oxaloacetate transaminase (GOT) and malonaldehyde (MDA) level elevated significantly compared to negative control. However, level of plasma urea, creatinine, GOT and MDA in tested rats were significantly lower in comparison to uremic control (p Conclusion The urease positive probiotic L. plantarum AD3 strain have the antioxidative and antiuremic efficacy in acetaminophen induced experimental rats.

Establishment of colonic dialysis model in uremic rats by right nephrectomy and left partial nephrectomy

Conventional treatments for patients suffering with end-stage renal disease (ESRD) has several disadvantages, highlighting the importance of other reproducible modalities such as colonic dialysis (CD). The aim was to establish a CD model in uremic rats and evaluate the effect of two different peritoneal dialysis (PD) solutions. Thirty-two male Wistar rats were randomly divided into four groups. After right nephrectomy and left partial nephrectomy, a Malone antegrade continence enema (MACE) stoma was created. Seven days after the procedure, blood sampling was performed. In group I (N=8) no postoperative intervention was performed. In group II (N=8), CD was started through the MACE stoma by a low osmolar PD solution. Rats of group III (N=8) underwent the same procedure with a high osmolar PD solution. Rats of group IV (N=8) underwent CD without nephrectomy in order to evaluate the feasibility of this technique. Mannitol and activated charcoal were also added to both PD solutions. Weekly blood sampling was performed in order to evaluate the plasma creatinine and blood urea nitrogen (BUN) level. In rats of the control group, the respective mean±SD creatinine level was 1.5±0.04mg/dL, 7 days after the surgical procedure, but a lower creatinine level was found in groups II and III (0.8±0.02 and 0.5±0.03, respectively). Despite the fact that the creatinine level was in steady low states after regular CD in group II (1±0.05) and group III (0.6±0.02), it remained at higher levels in the control group (1.7±0.08) 2 weeks postoperatively. Rats of group I did not survive until the third postoperative week, while the creatinine level was still lower in group III than in group II (0.6±0.02 vs. 1.1±0.03). Similar results were obtained for the BUN level at these timepoints. The mean±SD survival period was 11±2, 20±3, and 33±2 days in the animals of groups I, II, and III, respectively. To the best of our knowledge, this is the first study of CD establishment in a rat model. Unfortunately, the amount of protein loss, elevation of blood glucose levels, and electrolyte disturbance were not evaluated in the current study because of the limited amount of blood samples. Disturbance of these factors might be a cause of mortality in experimental groups undergoing CD while a significant decrease in BUN and creatinine levels was obtained. CD with an efficient PD solution through a MACE stoma may be a valuable option when conventional methods are not available.

Exogenous BMP7 in aortae of rats with chronic uremia ameliorates expression of profibrotic genes, but does not reverse established vascular calcification.

Hyperphosphatemia and vascular calcification are frequent complications of chronic renal failure and bone morphogenetic protein 7 (BMP7) has been shown to protect against development of vascular calcification in uremia. The present investigation examined the potential reversibility of established uremic vascular calcification by treatment of uremic rats with BMP7. A control model of isogenic transplantation of a calcified aorta from uremic rats into healthy littermates examined whether normalization of the uremic environment reversed vascular calcification. Uremia and vascular calcification were induced in rats by 5/6 nephrectomy, high phosphate diet and alfacalcidol treatment. After 14 weeks severe vascular calcification was present and rats were allocated to BMP7, vehicle or aorta transplantation. BMP7 treatment caused a significant decrease of plasma phosphate to 1.56 ± 0.17 mmol/L vs 2.06 ± 0.34 mmol/L in the vehicle group even in the setting of uremia and high phosphate diet. Uremia and alfacalcidol resulted in an increase in aortic expression of genes related to fibrosis, osteogenic transformation and extracellular matrix calcification, and the BMP7 treatment resulted in a decrease in the expression of profibrotic genes. The total Ca-content of the aorta was however unchanged both in the abdominal aorta: 1.9 ± 0.6 μg/mg tissue in the vehicle group vs 2.2 ± 0.6 μg/mg tissue in the BMP7 group and in the thoracic aorta: 71 ± 27 μg/mg tissue in the vehicle group vs 54 ± 18 μg/mg tissue in the BMP7 group. Likewise, normalization of the uremic environment by aorta transplantation had no effect on the Ca-content of the calcified aorta: 16.3 ± 0.6 μg/mg tissue pre-transplantation vs 15.9 ± 2.3 μg/mg tissue post-transplantation. Aortic expression of genes directly linked to extracellular matrix calcification was not affected by BMP7 treatment, which hypothetically might explain persistent high Ca-content in established vascular calcification. The present results highlight the importance of preventing the development of vascular calcification in chronic kidney disease. Once established, vascular calcification persists even in the setting when hyperphosphatemia or the uremic milieu is abolished.

Homocysteine Aggravates Intestinal Epithelial Barrier Dysfunction in Rats with Experimental Uremia

Background/Aims: Previous studies have shown that homocysteine (Hcy) is an important intestinal-derived uremic toxin. However, whether Hcy is involved in the epithelial barrier dysfunction observed in uremia remains unclear. This study aimed to investigate the effect of Hcy on intestinal permeability and intestinal barrier structure and function in adenine-induced uremic rats. Methods: Sprague-Dawley rats were divided into five groups: normal control (group NC), Hcy (group H), uremia (group U), uremia + Hcy (group UH), and uremia + Hcy + VSL#3 (group UHV). Experimental uremia was induced by intragastric adenine administration, and Hcy was injected subcutaneously. The animal models were assessed for renal function and pathological tissue staining. The pathological changes of intestinal tissue were observed by hematoxylin and eosin staining and electron microscopy. The serum and intestinal tissue levels of Hcy, interleukin (IL)-6, tumor necrosis factor (TNF)-α, superoxide dismutase (SOD), and malondialdehyde (MDA) as well as serum endotoxin and intestinal permeability were assessed. The levels of the tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) were assessed by western blotting. Results: Blood analyses and renal pathology indicated that experimental uremia was induced successfully. Pathological damage to intestinal structure was most obvious in group UH. Serum and tissue Hcy, serum endotoxin, and intestinal permeability were significantly elevated in group UH. The protein levels of claudin-1, occludin, and ZO-1 were decreased to various degrees in group UH compared with groups NC, H, and U. The serum and tissue levels of IL-6, TNF-α, and MDA were significantly increased, while SOD activity was markedly decreased. Supplementation with the probiotic VSL#3 improved these parameters to various degrees and up-regulated the abundance of tight junction proteins, which indicated a role for Hcy in the increase of intestinal permeability and destruction of the epithelial barrier in uremia. Conclusion: Hcy aggravates the increase of intestinal permeability and destruction of the epithelial barrier by stimulating inflammatory and oxidative damage. Probiotic administration can ameliorate this damage by reducing the levels of Hcy-induced inflammation and oxidation.

Vitamin E protects against extraskeletal calcification in uremic rats fed high fat diets

BackgroundHigh fat diets are implicated in the pathogenesis of metabolic syndrome, obesity and renal disease. Previous studies have revealed that high fat diets promote vascular calcification in uremic rats. Moreover, vitamin E has been shown to prevent uremic calcifications in genetically obese Zucker rats fed standard diet. The objective of this study was to investigate the influence of vitamin E supplementation on the development of extraskeletal calcifications in non-obese (wild type) uremic rats fed high fat diets.MethodsWistar rats (n = 32) were preconditioned by feeding either a normal (NF) or high fat (HF) diet for 45 days and subsequently were subjected to 5/6 nephrectomy (Nx). Just before performing the first Nx step, a blood sample (Pre-Nx) was obtained. After Nx rats were switched to a diet with 0.9% phosphorus and supplemented with calcitriol. Also, after Nx, half of the rats from each group (NF and HF) were treated with vitamin E (VitE) in the diet (30,000 mg/kg) and the other half were maintained on basic VitE requirements (27 mg/kg). Thus, rats were allotted to four experimental groups: Nx-NF (n = 8), Nx-NF-VitE (n = 8), Nx-HF (n = 8) and Nx-HF-VitE (n = 8). At the time of sacrifice (day 66), blood and tissue samples were obtained.ResultsFeeding a HF diet for 45 days did not increase body weight but elicited hyperglycemia, hypertriglyceridemia, an increase in plasma fibroblast growth factor 23 and a reduction in plasma calcitriol concentrations. After Nx, rats fed HF diet showed substantial extraskeletal calcification with aortic calcium content that was higher than in rats fed NF diet. Supplementation with VitE significantly (p < 0.05) reduced aortic (from 38.4 ± 8.8 to 16.5 ± 1.4 mg/g), gastric (from 5.6 ± 2.7 to 1.2 ± 0.4 mg/g) and pulmonary (from 1.8 ± 0.3 to 0.3 ± 0.2 mg/g) calcium content in rats on HF diets.ConclusionsUremic rats fed HF diets developed more severe extraosseous calcifications than their normocaloric-fed counterparts and dietary VitE supplementation protected against uremic calcifications in rats fed HF diets. Thus, eating energy-rich foods should be discouraged in patients with renal disease and their deleterious effect may be ameliorated with adequate antioxidant supply.