Hypomorphic expression of parathyroid Bmal1 disrupts the internal parathyroid circadian clock and increases parathyroid cell proliferation in response to uremia.

The molecular circadian clock is an evolutionary adaptation to anticipate recurring changes in the environment and to coordinate variations in activity, metabolism and hormone secretion. Parathyroid hyperplasia in uremia is a significant clinical challenge. Here, we examined changes in the transcriptome of the murine parathyroid gland over 24 hours and found a rhythmic expression of parathyroid signature genes, such as Casr, Vdr, Fgfr1 and Gcm2. Overall, 1455 genes corresponding to 6.9% of all expressed genes had significant circadian rhythmicity. Biological pathway analysis indicated that the circadian clock system is essential for the regulation of parathyroid cell function. To study this, a novel mouse strain with parathyroid gland-specific knockdown of the core clock gene Bmal1 (PTHcre;Bmal1flox/flox) was created. Dampening of the parathyroid circadian clock rhythmicity was found in these knockdown mice, resulting in abrogated rhythmicity of regulators of parathyroid cell proliferation such as Sp1, Mafb, Gcm2 and Gata3, indicating circadian clock regulation of these genes. Furthermore, the knockdown resulted in downregulation of genes involved in mitochondrial function and synthesis of ATP. When superimposed by uremia, these PTHcre;Bmal1flox/flox mice had an increased parathyroid cell proliferative response, compared to wild type mice. Thus, our findings indicate a role of the internal parathyroid circadian clock in the development of parathyroid gland hyperplasia in uremia.

Salmonella pathogenicity island 1 knockdown confers protection against myocardial fibrosis and inflammation in uremic cardiomyopathy via down-regulation of S100 Calcium Binding Protein A8/A9 transcription.

BACKGROUND/AIM: Uremic cardiomyopathy (UCM) is a characteristic cardiac pathology that is commonly found in patients with chronic kidney disease. This study dissected the mechanism of SPI1 in myocardial fibrosis and inflammation induced by UCM through S100A8/A9. METHODS: An UCM rat model was established, followed by qRT-PCR and western blot analyses of SPI1 and S100A8/A9 expression in myocardial tissues. After alterations of SPI1 and S100A8/A9 expression in UCM rats, the blood specimens were harvested from the cardiac apex of rats. The levels of creatine phosphokinase-MB (CK-MB), blood creatinine, blood urea nitrogen (BUN), and inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-α [TNF-α]) were examined in the collected blood. Collagen fibrosis was assessed by Masson staining. The expression of fibrosis markers [transforming growth factor (TGF)-ß1, α-smooth muscle actin (SMA), Collagen 4a1, and Fibronectin], IL-6, IL-1ß, and TNF-α was measured in myocardial tissues. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were conducted to test the binding relationship between SPI1 and S100A8/A9. RESULTS: S100A8/A9 and SPI1 were highly expressed in the myocardial tissues of UCM rats. Mechanistically, SPI1 bound to the promoter of S100A8/A9 to facilitate S100A8/A9 transcription. S100A8/A9 or SPI1 knockdown reduced myocardial fibrosis and inflammation and the levels of CK-MB, blood creatinine, and BUN, as well as the expression of TGF-ß1, α-SMA, Collagen 4a1, Fibronectin, IL-6, TNF-α, and IL-1ß in UCM rats. CONCLUSION: SPI1 knockdown diminished S100A8/A9 transcription, thus suppressing myocardial fibrosis and inflammation caused by UCM.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Resveratrol Reduces Kidney Injury in a Rat Model of Uremia and is Associated with Increased Expression of Heat Shock Protein 70 (Hsp70).

BACKGROUND This study aimed to investigate the effects of resveratrol on kidney function in a rat model of uremia and the expression of heat shock proteins. MATERIAL AND METHODS The rat model of uremia was developed by 5/6 nephrectomy of Sprague-Dawley rats. The Hsp70 inhibitor MKT-077, a rhodacyanine dye, was used. The study groups included rats with sham surgery (the sham group), the rat model of uremia (the model group), the solvent-treated control group (the control group), the rat model treated with resveratrol group (the resveratrol group), the rat model treated with MKT-077 (the MKT-077 group), and the resveratrol+MKT-077 group. Kidney tissues were studied histologically. Renal cell apoptosis was detected by the TUNEL method. Expression of p53, Bax, and Bcl-2 mRNA and protein were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively. RESULTS Compared with the sham group, the expression levels of heat shock proteins Hsp70, Hsp90, Hsp27, Hsp25, Hsp40, and Hsp60 in the kidney of the rat model group increased to different degrees. Compared with the model group, the Hsp70 levels in the resveratrol group were significantly increased (p<0.05). Compared with the model group, treatment with MKT-077 reduced the survival rate of rats, which was increased following resveratrol treatment. Compared with the resveratrol group, renal function in the resveratrol+MKT-077 group was significantly reduced (p<0.05). CONCLUSIONS In a rat model of uremia, resveratrol reduced renal injury and improved both renal function and survival, which were associated with increased expression of Hsp70.

The Redox-Sensitive Na/K-ATPase Signaling in Uremic Cardiomyopathy.

In recent years, Na/K-ATPase signaling has been implicated in different physiological and pathophysiological conditions, including cardiac hypertrophy and uremic cardiomyopathy. Cardiotonic steroids (CTS), specific ligands of Na/K-ATPase, regulate its enzymatic activity (at higher concentrations) and signaling function (at lower concentrations without significantly affecting its enzymatic activity) and increase reactive oxygen species (ROS) generation. On the other hand, an increase in ROS alone also regulates the Na/K-ATPase enzymatic activity and signaling function. We termed this phenomenon the Na/K-ATPase-mediated oxidant-amplification loop, in which oxidative stress regulates both the Na/K-ATPase activity and signaling. Most recently, we also demonstrated that this amplification loop is involved in the development of uremic cardiomyopathy. This review aims to evaluate the redox-sensitive Na/K-ATPase-mediated oxidant amplification loop and uremic cardiomyopathy.

DLX5 gene regulates the Notch signaling pathway to promote glomerulosclerosis and interstitial fibrosis in uremic rats.

Uremia largely results from the accumulation of organic waste products normally cleared by the kidneys, which commonly accompanies kidney failure and chronic kidney disease. However, genetic investigations in a uremia remain largely unclear. This study aimed to determine the expression patterns of distal-less homeobox 5 (DLX5) in uremia rat model and further to study its effects on glomerulosclerosis and interstitial fibrosis. Uremic expression chip was applied to screen differentially expressed genes in uremia. Next, we used small interfering RNA-mediated RNA interference to specifically silence DLX5 in experimental uremic rats to understand the regulatory mechanism of DLX5. To understand effect of Notch1 signaling pathway in uremia, we also treated experimental uremic rats with γ-secretase inhibitor (GSI), an inhibitor of Notch1 signaling pathway. The expression of fibronectin (FN), laminin (LN), transforming growth factor-ß1 (TGF-ß1), Hes1, Hes5, and Jagged2 was determined. The semiquantitative assessment was applied to verify the effects of DLX5 on glomerulosclerosis. In the uremic expression chip, we found that DLX5 was upregulated in uremia samples, and considered to regulate the Notch signaling pathway. We found that small interfering RNA-mediated DLX5 inhibition or Notch1 signaling pathway inhibitory treatment relieved and delayed the kidney injury and glomerulosclerosis in uremia. Meanwhile, inhibition of DLX5 or Nothch1 signaling pathway reduced expression of FN, LN, Nothch1, TGF-ß1, Hes1, Hes5, and Jagged2. Intriguingly, we discovered that Notch1 signaling pathway was inhibited after silencing DLX5. In conclusion, these findings highlight that DLX5 regulates Notch signaling, which may, in turn, promote complications of uremia such as kidney fibrosis, providing a novel therapeutic target for treating uremia.

Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium.

(1) The beneficial effects of hydrogen sulfide (H2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H2S biosynthesis, previously used as a marker for H2S production, is dramatically increased in circulation in uremia, while H2S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H2S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine-ß-synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca2+ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid.

RANKL/OPG system regulation by endogenous PTH and PTH1R/ATF4 axis in bone: Implications for bone accrual and strength in growing rats with mild uremia.

Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), and parathyroid hormone (PTH) play a central role in the regulation of bone turnover in chronic kidney disease (CKD), but their influence on bone mineral density (BMD) and strength remains unclear, particularly in children. We studied the clinical significance of OPG and RANKL in relation to PTH, femur weight, BMD, and bone biomechanical properties in growing rats after one month (CKD-1) and three months (CKD-3) of surgically-induced mild CKD. Gene expression of parathyroid hormone 1 receptor (PTH1R) and activating transcription factor 4 (ATF4), major regulators of anabolic PTH response in bone, was also determined. Serum PTH and bone PTH1R/ATF4 expression was elevated in CKD-3 compared with other groups, and it positively correlated with femur weight, BMD, and the biomechanical properties of the femoral diaphysis reflecting cortical bone strength. In contrast, bone RANKL/OPG ratios were decreased in CKD-3 rats compared with other groups, and they were inversely correlated with PTH and the other abovementioned bone parameters. However, the PTH-PTH1R-ATF4 axis exerted an unfavorable effect on the biomechanical properties of the femoral neck. In conclusion, this study showed for the first time an inverse association between serum PTH and the bone RANKL/OPG system in growing rats with mild CKD. A decrease in the RANKL/OPG ratio, associated with PTH-dependent activation of the anabolic PTH1R/ATF4 pathway, seems to be responsible for the unexpected, beneficial effect of PTH on cortical bone accrual and strength. Simultaneously, impaired biomechanical properties of the femoral neck were observed, making this bone site more susceptible to fractures.

Circulating MicroRNA-125b Predicts the Presence and Progression of Uremic Vascular Calcification.

OBJECTIVE: Vascular calcification (VC) is a major cause of mortality in patients with end-stage renal diseases. Biomarkers to predict the progression of VC early are in urgent demand. APPROACH AND RESULTS: We identified circulating, cell-free microRNAs as potential biomarkers using in vitro VC models in which both rat and human aortic vascular smooth muscle cells were treated with high levels of phosphate to mimic uremic hyperphosphatemia. Using an Affymetrix microRNA array, we found that miR-125b and miR-382 expression levels declined significantly as biomineralization progressed, but this decline was only observed for miR-125b in the culture medium. A time-dependent decrease in aortic tissue and serum miR-125b levels was also found in both ex vivo and in vivo renal failure models. We examined the levels of circulating, cell-free miR-125b in sera from patients with end-stage renal diseases (n=88) and found an inverse association between the severity of VC and the circulating miR-125b level, irrespective of age or mineral-related hormones (odds ratio, 0.71; P=0.03). Furthermore, serum miR-125b levels on enrollment can predict VC progression years later (for high versus low, odds ratio, 0.14; P<0.01; for the highest versus lowest tertile and middle versus lowest tertile, odds ratio, 0.55 and 0.13; P=0.3 and <0.01, respectively). The uremic VC prediction efficacy using circulating miR-125b levels was also observed in an independent cohort (n=135). CONCLUSIONS: The results suggest that serum miR-125b levels are associated with VC severity and serve as a novel predictive marker for the risk of uremia-associated calcification progression.

Uremic Serum Impairs Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stromal Cells.

Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD) is characterized by an increased fracture risk. Bone marrow mesenchymal stromal cells (BMSCs) may be involved in the pathogenesis of bone disease and, in view of their promising potential applications in bone tissue engineering, the effect of uremia on BMSCs regenerative potential represents a central issue. The present study evaluated in vitro the effect of a serum pool from hemodialysis patients on BMSCs to observe its influence on osteogenic differentiation. Besides alterations in spatial organization and cytotoxicity along with hyperproliferation, gene expression analysis suggested an impairment in the osteogenic differentiation. More importantly, Receptor activator of nuclear factor kappa-B ligand (RANKL) was upregulated with a mild reduction in osteoprotegerin levels. In summary, uremic environment seems to impair BMSCs osteogenic differentiation. Moreover BMSCs themselves may enhance osteoclastogenesis, feasibly contributing to the altered bone remodeling in CKD-MBD patients. J. Cell. Physiol. 232: 2201-2209, 2017. © 2016 Wiley Periodicals, Inc.

Role of Small Interfering RNA Silencing Protein Kinase C-α Gene on the Occurrence of Ultrafiltration Failure in Peritoneal Dialysis Rats.

This study aims to explore the effect of PKC-α gene silencing on the occurrence of ultrafiltration failure (UFF) in peritoneal dialysis (PD) rats. Forty-eight male SD rats were collected to establish 5/6 renal resection uremic and uremic PD rats models. Rats were assigned into control, sham operation, uremia, PD-2 W (peritoneal dialysis for 2 weeks), PD-4 W (peritoneal dialysis for 4 weeks), negative control (NC) (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg blank plasmid into abdominal cavity) and PKC-α siRNA (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg PKC-α siRNA into abdominal cavity) groups. CD34 staining was performed to determine microvessel density (MVD) for peritoneal tissues. The mRNA and protein expression of PKC-α in peritoneal tissue were detected by qRT-PCR and Western blot. Compared with the control group, MVD, the mRNA and protein expression of PKC-α were significantly increased in rats of the uremia, PD-2 W, PD-4 W, NC, and PKC-α siRNA groups. Compared with the uremia group, MVD, the mRNA and protein expression of PKC-α were increased, the changes observed in the PD-4 W and NC groups were better obvious than in the PD-2 W group. In comparison with the PD-4 W and NC groups, MVD, the mRNA and protein expression of PKC-α in rats were decreased in the PKC-α siRNA group. PKC-α gene has a high expression in uremic PD rats, and PKC-α gene silencing is able to increase UF while decrease MVD and glucose transport in peritoneal tissues thus reversing UFF in PD rats. J. Cell. Biochem. 118: 4607-4616, 2017. © 2017 Wiley Periodicals, Inc.

Cilostazol inhibits uremic toxin-induced vascular smooth muscle cell dysfunction: role of Axl signaling.

Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, and vascular smooth muscle cell (VSMC) dysfunction plays a pivotal role in uremic atherosclerosis. Axl signaling is involved in vascular injury and is highly expressed in VSMCs. Recent reports have shown that cilostazol, a phosphodiesterase type 3 inhibitor (PDE3), can regulate various stages of the atherosclerotic process. However, the role of cilostazol in uremic vasculopathy remains unclear. This study aimed to identify the effect of cilostazol in VSMCs in the experimental CKD and to investigate whether the regulatory mechanism occurs through Axl signaling. We investigated the effect of P-cresol and cilostazol on Axl signaling in A7r5 rat VSMCs and the rat and human CKD models. From the in vivo CKD rats and patients, aortic tissue exhibited significantly decreased Axl expression after cilostazol treatment. P-cresol increased Axl, proliferating of cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and matrix metalloproteinase-2 (MMP-2) expressions, decreased caspase-3 expression, and was accompanied by increased cell viability and migration. Cilostazol significantly reversed P-cresol-induced Axl, downstream gene expressions, and cell functions. Along with the increased Axl expression, P-cresol activated PLCγ, Akt, and ERK phosphorylation and cilostazol significantly suppressed the effect of P-cresol. Axl knockdown significantly reversed the expressions of P-cresol-induced Axl-related gene expression and cell functions. Cilostazol with Axl knockdown have additive changes in downstream gene expression and cell functions in P-cresol culture. Both in vitro and in vivo experimental CKD models elucidate a new signal transduction of cilostazol-mediated protection against uremic toxin-related VSMCs dysfunction and highlight the involvement of the Axl signaling and downstream pathways.

Kidney fibroblast growth factor 23 does not contribute to elevation of its circulating levels in uremia.

Fibroblast growth factor 23 (FGF23) secreted by osteocytes is a circulating factor essential for phosphate homeostasis. High plasma FGF23 levels are associated with cardiovascular complications and mortality. Increases of plasma FGF23 in uremia antedate high levels of phosphate, suggesting a disrupted feedback regulatory loop or an extra-skeletal source of this phosphatonin. Since induction of FGF23 expression in injured organs has been reported we decided to examine the regulation of FGF23 gene and protein expressions in the kidney and whether kidney-derived FGF23 contributes to the high plasma levels of FGF23 in uremia. FGF23 mRNA was not detected in normal kidneys, but was clearly demonstrated in injured kidneys, already after four hours in obstructive nephropathy and at 8 weeks in the remnant kidney of 5/6 nephrectomized rats. No renal extraction was found in uremic rats in contrast to normal rats. Removal of the remnant kidney had no effect on plasma FGF23 levels. Well-known regulators of FGF23 expression in bone, such as parathyroid hormone, calcitriol, and inhibition of the FGF receptor by PD173074, had no impact on kidney expression of FGF23. Thus, the only direct contribution of the injured kidney to circulating FGF23 levels in uremia appears to be reduced renal extraction of bone-derived FGF23. Kidney-derived FGF23 does not generate high plasma FGF23 levels in uremia and is regulated differently than the corresponding regulation of FGF23 gene expression in bone.

Signaling of Serum Amyloid A Through Receptor for Advanced Glycation End Products as a Possible Mechanism for Uremia-Related Atherosclerosis.

OBJECTIVE: Cardiovascular disease is the leading cause of death in patients with end-stage renal disease. Serum amyloid A (SAA) is an acute phase protein and a binding partner for the multiligand receptor for advanced glycation end products (RAGE). We investigated the role of the interaction between SAA and RAGE in uremia-related atherogenesis. APPROACH AND RESULTS: We used a mouse model of uremic vasculopathy, induced by 5 of 6 nephrectomy in the Apoe(-/-) background. Sham-operated mice were used as controls. Primary cultures of Ager(+/+) and Ager(-/-) vascular smooth muscle cells (VSMCs) were stimulated with recombinant SAA, S100B, or vehicle alone. Relevance to human disease was assessed with human VSMCs. The surface area of atherosclerotic lesions at the aortic roots was larger in uremic Apoe(-/-) than in sham-operated Apoe(-/-) mice (P<0.001). Furthermore, atherosclerotic lesions displayed intense immunostaining for RAGE and SAA, with a pattern similar to that of α-SMA. Ager transcript levels in the aorta were 6× higher in uremic animals than in controls (P<0.0001). Serum SAA concentrations were higher in uremic mice, not only after 4 weeks of uremia but also at 8 and 12 weeks of uremia, than in sham-operated animals. We investigated the functional role of RAGE in uremia-induced atherosclerosis further, in animals lacking RAGE. We found that the induction of uremia in Apoe(-/-) Ager(-/-) mice did not accelerate atherosclerosis. In vitro, the stimulation of Ager(+/+) but not of Ager(-/-) VSMCs with SAA or S100B significantly induced the production of reactive oxygen species, the phosphorylation of AKT and mitogen-activated protein kinase-extracellular signal-regulated kinases and cell migration. Reactive oxygen species inhibition with N-acetyl cysteine significantly inhibited both the phosphorylation of AKT and the migration of VSMCs. Similar results were obtained for human VSMCs, except that the phosphorylation of mitogen-activated protein kinase-extracellular signal-regulated kinases, rather than of AKT, was subject to specific redox-regulation by SAA and S100B. Furthermore, human aortic atherosclerotic sections were positively stained for RAGE and SAA. CONCLUSIONS: Uremia upregulates SAA and RAGE expression in the aortic wall and in atherosclerotic lesions in mice. Ager(-/-) animals are protected against the uremia-induced acceleration of atherosclerosis. SAA modulates the functions of murine and human VSMCs in vitro in a RAGE-dependent manner. This study, therefore, identifies SAA as a potential new uremic toxin involved in uremia-related atherosclerosis through interaction with RAGE.

Co-Expression Analysis of Blood Cell Genome Expression to Preliminary Investigation of Regulatory Mechanisms in Uremia.

BACKGROUND Uremia involves a series of clinical manifestations and is a common syndrome that occurs in nearly all end-stage kidney diseases. However, the exact genetic and/or molecular mechanisms that underlie uremia remain poorly understood. MATERIAL AND METHODS In this case-control study, we analyzed whole-genome microarray of 75 uremia patients and 20 healthy controls to investigate changes in gene expression and cellular mechanisms relevant to uremia. Gene co-expression network analysis was performed to construct co-expression networks using differentially expressed genes (DEGs) in uremia. We then determined hub models of co-expressed gene networks by MCODE, and we used miRNA enrichment analysis to detect key miRNAs in each hub module. RESULTS We found nine co-expressed hub modules implicated in uremia. These modules were enriched in specific biological functions, including "proteolysis", "membrane-enclosed lumen", and "apoptosis". Finally, miRNA enrichment analysis to detect key miRNAs in each hub module found 15 miRNAs that were specifically targeted to uremia-related hub modules. Of these, miRNA-21-3p and miRNA-210-3p have been identified in other studies as being important for uremia. CONCLUSIONS In summary, our study connected biological functions, genes, and miRNAs that underpin the network modules that can be used to elucidate the molecular mechanisms involved in uremia.

ADAM17, a New Player in the Pathogenesis of Chronic Kidney Disease-Mineral and Bone Disorder.

The triad composed by α-Klotho, fibroblast growth factor-23, and its receptor are involved in the pathogenesis of chronic kidney disease-mineral and bone disorder. A disintegrin and metalloproteinase 17 (ADAM17) is a metalloproteinase causing the proteolytic shedding of α-Klotho from the cell membrane, and its role in chronic kidney disease-mineral and bone disorder is not yet known. We studied the circulating levels of the above-mentioned mediators in patients with secondary hyperparathyroidism due to uremia, compared to control subjects, as well as in patients with primary hyperparathyroidism. We also measured the immunofluorescence pattern of the relevant tissue proteins in specimens obtained from patients undergoing parathyroid surgery for secondary compared to primary hyperparathyroidism. Results showed that α-Klotho tissue levels are reduced, in the presence of increased ADAM17 tissue levels. In addition, we showed increased serum levels of the main product of ADAM17 proteolytic activity, tumor necrosis factor-α. Thus, we found a paradoxical situation, in secondary compared to primary hyperparathyroidism, that is, that in the face of increased tumor necrosis factor-α in circulation, both soluble and tissue α-Klotho are reduced significantly, despite increased tissue ADAM17. In conclusion, tissue and serum levels of α-Klotho seem to have become independent from the regulation induced by ADAM17, which constitutes therefore another tassel in the impaired α-Klotho-FGF23 receptor axis present in uremia.

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.

MicroRNA-34b/c inhibits aldosterone-induced vascular smooth muscle cell calcification via a SATB2/Runx2 pathway.

Increasing evidence shows that aldosterone and specific microRNAs (miRs) contribute to vascular smooth muscle cell (VSMC) calcification. In this study, we aim to explore the mechanistic links between miR-34b/c and aldosterone in VSMC calcification. VSMC calcification models were established both in vitro and in vivo. First, the levels of aldosterone, miR-34b/c and special AT-rich sequence-binding protein 2 (SATB2) were measured. Then, miR-34b/c mimics or inhibitors were transfected into VSMCs to evaluate the function of miR-34b/c. Luciferase reporter assays were used to demonstrate whether SATB2 was a direct target of miR-34b/c. Aldosterone and SATB2 were found to be markedly upregulated during VSMC calcification, whereas miR-34b/c expression was downregulated. Treatment with the mineralocorticoid receptor (MR) antagonist eplerenone inhibited VSMC calcification. In aldosterone-induced VSMC calcification, miR-34b/c levels were downregulated and SATB2 protein was upregulated. Furthermore, miR-34b/c overexpression alleviated aldosterone-induced VSMC calcification as well as inhibited the expression of SATB2 protein, whereas miR-34b/c inhibition markedly enhanced VSMC calcification and upregulated SATB2 protein. In addition, luciferase reporter assays showed that SATB2 is a direct target of miR-34b/c in VSMCs. Overexpression of SATB2 induced Runx2 overproduction and VSMC calcification. Therefore, miR-34b/c participates in aldosterone-induced VSMC calcification via a SATB2/Runx2 pathway. As miR-34b/c appears to be a negative regulator, it has potential as a therapeutic target of VSMC calcification.

Parathyroid-specific deletion of dicer-dependent microRNAs abrogates the response of the parathyroid to acute and chronic hypocalcemia and uremia.

MicroRNAs (miRNAs) down-regulate gene expression and have vital roles in biology but their functions in the parathyroid are unexplored. To study this, we generated parathyroid-specific Dicer1 knockout (PT-Dicer(-/-) ) mice where parathyroid miRNA maturation is blocked. Remarkably, the PT-Dicer(-/-) mice did not increase serum parathyroid hormone (PTH) in response to acute hypocalcemia compared with the >5-fold increase in controls. PT-Dicer(-/-) glands cultured in low-calcium medium secreted 5-fold less PTH at 1.5 h than controls. Chronic hypocalcemia increased serum PTH >4-fold less in PT-Dicer(-/-) mice compared with control mice with no increase in PTH mRNA levels and parathyroid cell proliferation compared with the 2- to 3-fold increase in hypocalcemic controls. Moreover, uremic PT-Dicer(-/-) mice increased serum PTH and FGF23 significantly less than uremic controls. Therefore, stimulation of the parathyroid by both hypocalcemia and uremia is dependent upon intact dicer function and miRNAs. In contrast, the PT-Dicer(-/-) mice responded normally to activation of the parathyroid calcium-sensing receptor (Casr) by both hypercalcemia and a calcimimetic that decreases PTH secretion, demonstrating that they are dicer-independent. Therefore, miRNAs are essential for the response of the parathyroid to both acute and chronic hypocalcemia and uremia, the major stimuli for PTH secretion.

Loss of miR-125b contributes to upregulation of CYP24 in uraemic rats.

AIM: Abnormal upregulation of CYP24 contributes to vitamin D insufficiency and resistance to vitamin D therapy in chronic kidney disease (CKD), because human CYP24 is a key enzyme involved in the inactivation of 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3; calcitriol) and 1,25(OH)2D3. There are multiple mechanisms regulating CYP24 in a variety of types of tissues and diseases. An increasing body of evidence suggests that microRNA-125b (miR-125b) plays an important role in post-transcriptional regulation of CYP24 mRNA. METHODS: We sought to test hypothesis that abnormal elevation of CYP24 in CKD is a consequence of loss of miR-125b in CKD in a uraemia rat model. RESULTS: We found that expression of miR-125b was significantly inhibited in uraemic rats coupled with increased CYP24 at both protein and mRNA levels compared with normal controls. In NRK-52 kidney cells, we further found that miR-125b antagomirs increased CYP24 but miR-125b mimics decreased CYP24, and luciferase assay confirmed that CYP24 is a direct target of miR-125b. Vitamin D status exerted no significant effects on expression of both miR-125b and CYP24 in uraemic rats. CONCLUSION: These results suggest that modulation of miR-125b may be used for treatment of Vitamin D insufficiency in CKD.