p-Cresol Sulfate Caused Behavior Disorders and Neurodegeneration in Mice with Unilateral Nephrectomy Involving Oxidative Stress and Neuroinflammation.

Protein-bound uremic toxins, such as p-cresol sulfate (PCS), can be accumulated with declined renal function and aging and is closely linked with central nervous system (CNS) diseases. In the periphery, PCS has effects on oxidative stress and inflammation. Since oxidative stress and inflammation have substantial roles in the pathogenesis of neurological disorders, the CNS effects of PCS were investigated in unilateral nephrectomized C57/BL/6 mice. Unlike intact mice, unilateral nephrectomized mice showed increased circulating levels of PCS after exogenous administration. Upon PCS exposure, the unilateral nephrectomized mice developed depression-like, anxiety-like, and cognitive impairment behaviors with brain PCS accumulation in comparison with the nephrectomy-only group. In the prefrontal cortical tissues, neuronal cell survival and neurogenesis were impaired along with increased apoptosis, oxidative stress, and neuroinflammation. Circulating brain-derived neurotrophic factors (BDNF) and serotonin were decreased in association with increased corticosterone and repressor element-1 silencing transcription factor (REST), regulators involved in neurological disorders. On the contrary, these PCS-induced changes were alleviated by uremic toxin absorbent AST-120. Taken together, PCS administration in mice with nephrectomy contributed to neurological disorders with increased oxidative stress and neuroinflammation, which were alleviated by PCS chelation. It is suggested that PCS may be a therapeutic target for chronic kidney disease-associated CNS diseases.

Excessive salt intake increases peritoneal solute transport rate via local tonicity-responsive enhancer binding protein in subtotal nephrectomized mice.

BACKGROUND: High peritoneal transport is associated with high mortality and technical failure in peritoneal dialysis (PD). Baseline peritoneal solute transport rate (PSTR) as measured by the peritoneal equilibration test (PET) within 6 months after PD initiation varies between patients. Sodium is reported to be stored in the skin or muscle of dialysis patients. This study investigated whether excessive salt intake in uremic mice caused peritoneal alterations without exposure to PD fluid. METHODS: Sham-operated (Sham) and subtotal nephrectomized (Nx) mice were randomly given tap water or 1% sodium chloride (NaCl)-containing water for 8 weeks. PET was then performed to evaluate peritoneal function. Human mesothelial cell line Met-5A was used for in vitro studies. RESULTS: We observed higher PSTR in Nx mice with 1% NaCl-containing drinking water (Nx + salt) compared with those with tap water (Nx + water), along with enhanced angiogenesis and inflammation in the peritoneum. Blockade of interleukin (IL)-6 signaling rescued peritoneal transport function in Nx + salt mice. In cultured Met-5A, additional NaCl in the medium upregulated IL-6 as well as vascular endothelial growth factor-A, associated with increased expression and nuclear translocation of tonicity-responsive enhancer binding protein (TonEBP). Knockdown of TonEBP lowered the induction caused by high tonicity. Peritoneal TonEBP expression was higher in Nx + salt mice, while removal of high-salt diet lowered TonEBP level and improved peritoneal transport function. CONCLUSIONS: Excessive dietary salt intake caused peritoneal membrane functional and structural changes under uremic status. TonEBP regulated hypertonicity-related inflammatory changes and might play a crucial role in high baseline peritoneal transport.

Effects of lactulose on renal function and gut microbiota in adenine-induced chronic kidney disease rats.

BACKGROUND: Constipation is frequently observed in patients with chronic kidney disease (CKD). Lactulose is expected to improve the intestinal environment by stimulating bowel movements as a disaccharide laxative and prebiotic. We studied the effect of lactulose on renal function in adenine-induced CKD rats and monitored uremic toxins and gut microbiota. METHODS: Wistar/ST male rats (10-week-old) were fed 0.75% adenine-containing diet for 3 weeks to induce CKD. Then, they were divided into three groups and fed as follows: control, normal diet; and 3.0- and 7.5-Lac, 3.0% and 7.5% lactulose-containing diets, respectively, for 4 weeks. Normal diet group was fed normal diet for 7 weeks. The rats were observed for parameters including renal function, uremic toxins, and gut microbiota. RESULTS: The control group showed significantly higher serum creatinine (sCr) and blood urea nitrogen (BUN) 3 weeks after adenine feeding than at baseline, with a 8.5-fold increase in serum indoxyl sulfate (IS). After switching to 4 weeks of normal diet following adenine feeding, the sCr and BUN in control group remained high with a further increase in serum IS. In addition, tubulointerstitial fibrosis area was increased in control group. On the other hand, 3.0- and 7.5-Lac groups improved sCr and BUN levels, and suppressed tubulointerstitial fibrosis, suggesting preventing of CKD progression by lactulose. Lac groups also lowered level of serum IS and proportions of gut microbiota producing IS precursor. CONCLUSION: Lactulose modifies gut microbiota and ameliorates CKD progression by suppressing uremic toxin production.

Review of pentosidine and pyrraline in food and chemical models: formation, potential risks and determination.

Pyrraline and pentosidine are advanced Maillard reaction products derived from the reaction of glucose with the lysine amino group on proteins. They have been implicated in uremia, diabetes, and related complications, including inflammation, retinopathy, and nephropathy. This review focuses on the formation mechanism, human potential risks, and detections of pentosidine and pyrraline and lays the foundation for further study of pentosidine and pyrraline. © 2017 Society of Chemical Industry.

Effects of growth hormone treatment on growth plate, bone, and mineral metabolism of young rats with uremia induced by adenine.

BackgroundIn a model of growth retardation secondary to chronic kidney disease (CKD) induced by adenine, this study explores the effects of growth hormone (GH) therapy on growth plate and mineral metabolism.MethodsWeaning female rats receiving a 0.5% adenine diet during 21 days, untreated (AD) or treated with GH (ADGH) for 1 week, were compared with control rats receiving normal diet, either ad libitum or pair-fed with AD animals. AD and ADGH rats had similarly elevated serum concentrations of urea nitrogen, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23).ResultsUremia induced by adenine caused growth retardation and disturbed growth cartilage chondrocyte hypertrophy. We demonstrated marked expression of aquaporin 1 in the growth plate, but its immunohistochemical signal and the expression levels of other proteins potentially related with chondrocyte enlargement, such as Na-K-2Cl cotransporter, insulin-like growth factor 1 (IGF-1), and IGF-1 receptor, were not different among the four groups of rats. The distribution pattern of vascular endothelial growth factor was also similar. AD rats developed femur bone structure abnormalities analyzed by micro-computerized tomography.ConclusionGH treatment accelerated longitudinal growth velocity, stimulated the proliferation and enlargement of chondrocytes, and did not modify the elevated serum PTH or FGF23 concentrations or the abnormal bone structure.

Acute and subacute oral toxicity of periodate salts in rats.

Periodate salts are being developed as potential replacements for perchlorate due to potential health hazards associated with exposure to perchlorate. The aim of this study was to investigate acute and subacute effects of periodate salts in rats. Acute oral toxicity of potassium and sodium periodate was determined using the Sequential Stage-Wise Probit method. The LD50 for potassium periodate was 732 (95% CI = 539-838, slope = 13.4) and 685 mg/kg (95% CI = 580-809, slope = 10.6) for females and males, respectively. The LD50 for sodium periodate was 318 (95% CI = 292-347, slope = 24.3) and 741 mg/kg (95% CI = 704-779, slope = 31.2) for females and males, respectively. In the subacute study, rats were administered sodium periodate at five doses (1/16 LD50 up to LD50) or distilled water for 14-days via oral gavage. Female rats in the 318 mg/kg-day group and male rats in the 185, 370, and 741 mg/kg-day groups exhibited moribundity, kidney toxicity, uremia, and a stress response. BMDL10s of 17.2 and 33.7 mg/kg-day were derived for females and males, respectively. Comparison with the NOAEL for perchlorate-induced thyroid toxicity in rats (0.009 mg/kg-day) suggests sodium periodate is less toxic than perchlorate on a subacute basis.

Uremic Toxicity of Advanced Glycation End Products in CKD.

Advanced glycation end products (AGEs), a heterogeneous group of compounds formed by nonenzymatic glycation reactions between reducing sugars and amino acids, lipids, or DNA, are formed not only in the presence of hyperglycemia, but also in diseases associated with high levels of oxidative stress, such as CKD. In chronic renal failure, higher circulating AGE levels result from increased formation and decreased renal clearance. Interactions between AGEs and their receptors, including advanced glycation end product-specific receptor (RAGE), trigger various intracellular events, such as oxidative stress and inflammation, leading to cardiovascular complications. Although patients with CKD have a higher burden of cardiovascular disease, the relationship between AGEs and cardiovascular disease in patients with CKD is not fully characterized. In this paper, we review the various deleterious effects of AGEs in CKD that lead to cardiovascular complications and the role of these AGEs in diabetic nephropathy. We also discuss potential pharmacologic approaches to circumvent these deleterious effects by reducing exogenous and endogenous sources of AGEs, increasing the breakdown of existing AGEs, or inhibiting AGE-induced inflammation. Finally, we speculate on preventive and therapeutic strategies that focus on the AGE-RAGE axis to prevent vascular complications in patients with CKD.

The fibroblast growth factor receptor mediates the increased FGF23 expression in acute and chronic uremia.

Serum FGF23 is markedly elevated in chronic kidney disease and has been associated with poor long-term outcomes. FGF23 expression is increased by activation of the FGF receptor 1 (FGFR1) in rats with normal renal function and in vitro in bone-derived osteoblast-like cells. We studied the regulation of FGF23 by FGFR1 in vivo in acute and chronic uremia in mice and rats. Folic acid-induced acute kidney injury increased calvaria FGF23 mRNA and serum FGF23 and parathyroid hormone (PTH) levels at 6 h. The FGFR1 receptor inhibitor PD173074 prevented the folic acid-induced increase in both FGF23 mRNA and serum levels but had no effect on serum PTH levels. A more prolonged uremia due to an adenine high-phosphorus diet for 14 days resulted in high levels of FGF23 mRNA and serum FGF23 and PTH. PD173074 decreased serum FGF23 and mRNA levels with no effect on PTH in the adenine high phosphorus-induced uremic rats. Therefore, a derangement in FGF23 regulation starts early in the course of acute kidney injury, is in part independent of the increase in serum PTH, and involves activation of FGFR1. It is possible that FGFR1 in the osteocyte is activated by locally produced canonical FGFs, which are increased in uremia. This is the first demonstration that activation of FGFR1 is essential for the high levels of FGF23 in acute and chronic experimental uremia.

[High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1].

Objective: To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis. Methods: Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-ß1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF in peritoneal membrane and HPECs. Results:In vivo, compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly increased (all P<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). In vitro, the mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all P<0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). Conclusion: High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.

Chronic kidney disease induced by adenine: a suitable model of growth retardation in uremia.

Growth retardation is a major manifestation of chronic kidney disease (CKD) in pediatric patients. The involvement of the various pathogenic factors is difficult to evaluate in clinical studies. Here, we present an experimental model of adenine-induced CKD for the study of growth failure. Three groups (n = 10) of weaning female rats were studied: normal diet (control), 0.5% adenine diet (AD), and normal diet pair fed with AD (PF). After 21 days, serum urea nitrogen, creatinine, parathyroid hormone (PTH), weight and length gains, femur osseous front advance as an index of longitudinal growth rate, growth plate histomorphometry, chondrocyte proliferative activity, bone structure, aorta calcifications, and kidney histology were analyzed. Results are means ± SE. AD rats developed renal failure (serum urea nitrogen: 70 ± 6 mg/dl and creatinine: 0.6 ± 0.1 mg/dl) and secondary hyperparathyroidism (PTH: 480 ± 31 pg/ml). Growth retardation of AD rats was demonstrated by lower weight (AD rats: 63.3 ± 4.8 g, control rats: 112.6 ± 4.7 g, and PF rats: 60.0 ± 3.8 g) and length (AD rats: 7.2 ± 0.2 cm, control rats: 11.1 ± 0.3 cm, and PF rats: 8.1 ± 0.3 cm) gains as well as lower osseous front advances (AD rats: 141 ± 13 µm/day, control rats: 293 ± 16 µm/day, and PF rats: 251 ± 10 µm/day). The processes of chondrocyte maturation and proliferation were impaired in AD rats, as shown by lower growth plate terminal chondrocyte height (21.7 ± 2.3 vs. 26.2 ± 1.9 and 23.9 ± 1.3 µm in control and PF rats) and proliferative activity index (AD rats: 30 ± 2%, control rats: 38 ± 2%, and PF rats: 42 ± 3%). The bone primary spongiosa structure of AD rats was markedly disorganized. In conclusion, adenine-induced CKD in young rats is associated with growth retardation and disturbed endochondral ossification. This animal protocol may be a useful new experimental model to study growth in CKD.

Spironolactone ameliorates arterial medial calcification in uremic rats: the role of mineralocorticoid receptor signaling in vascular calcification.

Vascular calcification (VC) is a critical complication in patients with chronic kidney disease (CKD). The effects of spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, on VC have not been fully investigated in CKD. The present in vivo study determined the protective effects of SPL on VC in CKD rats. Rats were divided into a control group and four groups of rats with adenine-induced CKD. Three groups were treated with 0, 50, and 100 mg·kg(-1)·day(-1) SPL for 8 wk, and one group was treated with 100 mg·kg(-1)·day(-1) SPL for the last 2 wk of the 8-wk treatment period. After 8 wk, CKD rats developed azotemia and hyperphosphatemia, with increases in the expression of serum and glucocorticoid-regulated kinase-1 and sodium-phosphate cotransporter, in inflammation and oxidative stress level, in osteogenic signaling and apoptosis, and in aortic calcification, compared with control rats. SPL dose dependently decreased these changes in the aortas, concomitant with improvements in renal inflammation, tubulointerstitial nephritis, and kidney function. SPL neither lowered blood pressure level nor induced hyperkalemia. Treatment of CKD rats for the last 2 wk with 100 mg·kg(-1)·day(-1) SPL attenuated VC compared with CKD rats with the same degree of kidney function and hyperphosphatemia. In conclusion, SPL dose dependently inhibits the progression of VC by suppressing MR signaling, local inflammation, osteogenic transition, and apoptosis in the aortas of CKD rats.

Role of local versus systemic vitamin D receptors in vascular calcification.

OBJECTIVE: Calcitriol and various analogs are commonly used to suppress secondary hyperparathyroidism in chronic kidney disease but may also exacerbate vascular calcification. Although this could be because of increased intestinal calcium and phosphate absorption, direct effects through vitamin D receptors (VDRs) on vascular smooth muscle have also been proposed. APPROACH AND RESULTS: The role of these receptors was investigated by examining gene regulation in rat aortas treated with calcitriol ex vivo and in vivo and by transplanting aortas from VDR-null (VDR(-/-)) mice into wild-type mice before induction of uremia and treatment with calcitriol. In cultured rat aortas, calcitriol increased the expression of mRNA for CYP24A1 but not mRNA for any bone-related or calcification-related genes. Gene expression in aortas in vivo was not altered by doses of calcitriol that promote calcification. Calcitriol markedly increased aortic calcification in uremic mice and this did not differ between VDR(-/-) aortic allografts and VDR(+/+) recipient aortas. CONCLUSIONS: Calcitriol promotes vascular calcification through a systemic action rather than through a direct vascular action.

Therapeutic potential of different commercially available synbiotic on acetaminophen-induced uremic rats.

BACKGROUND: Currently kidney disease appears a foremost problem across the world. Acetaminophen is a commonly used antipyretic agent, which in high doses, causes uremia and used for experimentally induction of kidney disease. Bacteriotherapy affords a promising approach to mitigate uremic toxins by ingestion of urease positive bacteria, probiotics and symbiotic able to catabolize uremic solutes within the gut. The present study evaluates the effect of seven commercial symbiotic on kidney disease. METHODS: Fifty-four albino male rats were randomly divided into nine groups. Control group (Group-I) received distilled water interperitoneally for 7 days. Positive control group (Group-II) received 500 mg/kg acetaminophen interperitoneally for 7 days. Commercially available seven symbiotic combinations at a dose of 10(9)cells/day for 3 weeks was administered to the tested groups (Group III-IX) after receiving 500 mg/kg/day acetaminophen interperitoneally for 7 days. Blood, kidney, liver and stool samples were collected after scarification for biochemical tests and DNA fragmentation assay of kidney tissue, kidney histological studies. Limited fecal analysis was conducted. RESULT: Blood urea nitrogen and toxicity indicators were increased, and antioxidant enzymes were decreased in Group-II. Blood urea nitrogen, toxicity indicators, glomerular necrosis, DNA damage of kidney tissue were reduced, and antioxidant enzymes were increased significantly in the treated Groups IV and IX (p < 0.05) in response to Group-II. Number of pathogenic bacteria decreased in synbiotic treated groups than Group I and II. CONCLUSION: The study demonstrated that some of commercial symbiotic combination can reduce the sever effect of kidney disease.

The uremic toxicity of indoxyl sulfate and p-cresyl sulfate: a systematic review.

A growing number of publications supports a biologic effect of the protein-bound uremic retention solutes indoxyl sulfate and p-cresyl sulfate. However, the use of unrealistically high free concentrations of these compounds and/or inappropriately low albumin concentrations may blur the interpretation of these results. Here, we performed a systematic review, selecting only studies in which, depending on the albumin concentration, real or extrapolated free concentrations of indoxyl sulfate and p-cresyl sulfate remained in the uremic range. The 27 studies retrieved comprised in vitro and animal studies. A quality score was developed, giving 1 point for each of the following criteria: six or more experiments, confirmation by more than one experimental approach, neutralization of the biologic effect by counteractive reagents or antibodies, use of a real-life model, and use of dose-response analyses in vitro and/or animal studies. The overall average score was 3 of 5 points, with five studies scoring 5 of 5 points and six studies scoring 4 of 5 points, highlighting the superior quality of a substantial number of the retrieved studies. In the 11 highest scoring studies, most functional deteriorations were related to uremic cardiovascular disease and kidney damage. We conclude that our systematic approach allowed the retrieval of methodologically correct studies unbiased by erroneous conditions related to albumin binding. Our data seem to confirm the toxicity of indoxyl sulfate and p-cresyl sulfate and support their roles in vascular and renal disease progression.

Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC.

[The specific features of kidney injury due to abuse of saluretics and uncontrollable fasting in anorexia nervosa].

The paper describes a case of severe preranal acute renal failure that was induced by the uncontrolled long-term use of furosemide and that was reversible after infusion therapy. Another case is a female patient with anorexia nervosa and end-stage uremia progressing to chronic tubulointerstitial nephritis. Some problems of the pathogenesis of kidney injury and its diagnostic difficulties in anorexia nervosa and diuretic abuse are discussed.

Kruppel-like factor 4 contributes to high phosphate-induced phenotypic switching of vascular smooth muscle cells into osteogenic cells.

Hyperphosphatemia in chronic kidney disease is highly associated with vascular calcification. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. In the present study, we determined whether Krüppel-like factor 4 (Klf4) and phosphorylated Elk-1, transcriptional repressors of SMC differentiation marker genes activated by intimal atherogenic stimuli, contributed to this process. Rat aortic SMCs were cultured in the medium with normal (0.9 mmol/liter) or high (4.5 mmol/liter) phosphate concentration. Results showed that high phosphate concentration induced SMC calcification. Moreover, high phosphate decreased expression of SMC differentiation marker genes including smooth muscle α-actin and SM22α, whereas it increased expression of osteogenic genes, such as Runx2 and osteopontin. High phosphate also induced Klf4 expression, although it did not phosphorylate Elk-1. In response to high phosphate, Klf4 selectively bound to the promoter regions of SMC differentiation marker genes. Of importance, siRNA-mediated knockdown of Klf4 blunted high phosphate-induced suppression of SMC differentiation marker genes, as well as increases in expression of osteogenic genes and calcium deposition. Klf4 was also induced markedly in the calcified aorta of adenine-induced uremic rats. Results provide novel evidence that Klf4 mediates high phosphate-induced conversion of SMCs into osteogenic cells.

High glucose concentration does not modulate the formation of arterial medial calcification in experimental uremic rats.

High phosphate-induced phenotypic switching of smooth muscle cells (SMCs) into osteogenic cells is critical for the formation of arterial medial calcification in chronic kidney disease. Because vascular calcification is also prevalent in type 2 diabetes, we examined whether glucose concentration affects high phosphate-induced SMC phenotypic switching and calcification. First, the formation of arterial medial calcification was compared among 4 groups: adenine-fed uremic rats, streptozotocin-injected hyperglycemic rats, adenine-fed and streptozotocin-injected uremic/hyperglycemic rats, and control rats. Calcification was obvious in uremic and uremic/hyperglycemic rats, whereas it was undetectable in the others. Aortic calcium contents were significantly elevated in uremic and uremic/hyperglycemic rats, but they were not different between the two groups. Moreover, hyperglycemia had no effects on the reduced expression of SMC differentiation markers including smooth muscle α-actin and SM22α and on the increased expression of osteogenic markers, such as Runx2, in uremic rats. Second, cultured SMCs were incubated in the medium with various concentrations of phosphate (0.9-4.5 mmol/l) and glucose (5-50 mmol/l), and calcium deposition was measured. Although high phosphate dose-dependently increased calcium contents, they were unaffected by glucose concentration. Results suggest that glucose concentration does not directly modulate high phosphate-induced SMC phenotypic switching and arterial medial calcification.

Thalidomide suppresses inflammation in adenine-induced CKD with uraemia in mice.

BACKGROUND: Persistent systemic inflammation has been widely recognized in patients with chronic kidney disease (CKD), and is associated with increased risk of morbidity and mortality. Intervention therapies aiming for the blockade of inflammatory cytokines are considered attractive approaches for CKD patients with signs of chronic inflammation. In this context, thalidomide, due to its potent anti-inflammatory and immunomodulatory properties, may represent an alternative strategy of treatment. In the present study, we developed an experimental model of CKD with uraemia in mice, induced by a diet rich in adenine, which causes progressive renal dysfunction, resembling the human uraemic features. Inflammatory parameters were analysed in this model of CKD and the potential beneficial effects of thalidomide as an anti-inflammatory drug was also investigated. METHODS: C57/BL-6 mice were fed with an adenine-containing diet during a period of 6 weeks. Thirty mice were divided into three groups: Control group (animals receiving normal diet), ADE group (mice receiving adenine-containing diet) and ADE + TLD group (CKD mice receiving thalidomide, 30 mg/kg/day, by gavage). Besides biochemical and histopathological changes, local and systemic inflammatory parameters were also analysed, including expression of cytokines interleukin (IL)-1ß, tumour necrosis factor-α, IL-6, IL-4 and IL-10 in kidney samples by real-time RT-PCR and quantification of serum levels of cytokines. Finally, the electrophoretic mobility shift assay (EMSA) for NF-κB was also examined. RESULTS: Adenine-fed mice developed advanced CKD characterized by a marked increase in serum urea, creatinine, phosphorus and intact parathyroid hormone (iPTH) levels. In addition, histological changes of tubulointerstitial injury, characterized by deposition of crystals in the kidney, accompanied by tubular dilatation, degeneration of proximal tubular epithelium with loss of the brush border, inflammatory cellular infiltration, foreign-body granuloma formation and interstitial fibrosis were also evident. By immunohistochemistry, Mac-2- and α-SMA-positive cells were identified in the tubulointerstitial compartment. Treatment with thalidomide significantly reduced serum urea, creatinine, phosphorus and iPTH levels and protected against tubulointerstitial injury. Local and systemic inflammation in the mice model of adenine-induced CKD was confirmed by the findings of significantly high expression of cytokine mRNA levels and NF-κB activation in the kidney tissue as well as marked increased serum levels of inflammatory cytokines. Thalidomide treatment significantly reduced gene expression of these cytokines and the activation of the NF-κB in the renal tissue and the circulating levels of cytokines. CONCLUSIONS: Dietary adenine caused advanced CKD with uraemia in mice providing a useful experimental model to study molecular and morphological changes associated with this disease. The negative impact of inflammation in this CKD model was overcome by the marked anti-inflammatory effects of thalidomide, promoting renal protection.

Restoration of bone mineralization by cinacalcet is associated with a significant reduction in calcitriol-induced vascular calcification in uremic rats.

The present study investigated to what extent normalization of bone turnover goes along with a reduction of high-dose calcitriol-induced vascular calcifications in uremic rats. Five groups of male Sprague-Dawley rats were studied: sham-operated controls (n = 7), subtotally nephrectomized (SNX) uremic (CRF) animals (n = 12), CRF + calcitriol (vitD) (0.25 µg/kg/day) (n = 12), CRF + vitD + cinacalcet (CIN) (10 mg/kg/day) (n = 12), and CRF + vitD + parathyroidectomy (PTX) (n = 12). Treatment started 2 weeks after SNX and continued for the next 14 weeks. High-dose calcitriol treatment in hyperparathyroid rats went along with the development of distinct vascular calcification, which was significantly reduced by >50 %, in both CIN-treated and PTX animals. Compared to control animals and those of the CRF group, calcitriol treatment either in combination with CIN or PTX or not was associated with a significant increase in bone area comprising ±50 % of the total tissue area. However, whereas excessive woven bone accompanied by a dramatically increased osteoid width/area was seen in the CRF + vitD group, CIN treatment and PTX resulted in significantly reduced serum PTH level, which was accompanied by a distinct reduction of both the bone formation rate and the amount of osteoid. These data indicate that less efficient calcium and phosphorus incorporation in bone inherent to the severe hyperparathyroidism in vitamin D-treated uremic rats goes along with excessive vascular calcification, a process which is partially reversed by CIN treatment in combination with a more efficacious bone mineralization, thus restricting the availability of calcium and phosphate for being deposited in the vessel wall.