Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells.

Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.

2'-,5'-Oligoadenylate synthetase response ratio predicting virological response to PEG-interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis C.

OBJECTIVE: Although all the mechanisms of elimination of hepatitis C virus (HCV) by Interferon (IFN) have not been fully elucidated, the 2'-5'-oligoadenylate (2-5A) system is one of the mechanisms of the antiviral effect of IFN. Consequently, the measurement of 2'-5'-oligoadenylate synthetase (2-5AS) activity could be useful for the evaluation of IFN treatment. This retrospective study was aimed at assessing whether 2-5AS activity functions as a clinical marker of virological response to PEG-interferon-alpha2b (PEG-IFN) plus ribavirin therapy of chronic hepatitis C. METHODS: The 32 patients included in this study had high viral loads of serum HCV-RNA of genotype 1b with chronic hepatitis C. All the patients received a regimen of PEG-IFN plus ribavirin for 48 weeks, and were then divided into two groups: one group (effective group) with undetectable serum HCV-RNA levels at 24 weeks (n = 22) of therapy, the other group (ineffective group) with persistent presence of HCV-RNA in serum at 24 weeks (n = 10). The 2-5AS activity in serum was measured 2, 8 and 12 weeks before initial administration. RESULTS: The 2-5AS response ratio (measured value/measured value of baseline 2-5AS) at 2, 8 and 12 weeks after the administration in the effective group was significantly higher than that in the ineffective group. CONCLUSIONS: These results suggest that the ratio of 2-5AS is closely related to the antiviral effect, and that the measurement of 2-5AS response ratio may be a useful clinical parameter of virological response to PEG-IFN plus ribavirin therapy of chronic hepatitis C.

Insufficiency of PTH action on bone in uremia.

Abnormal bone turnover and mineral metabolism is observed in patients on dialysis. Secondary hyperparathyroidism (SHP) develops in response to mineral metabolism changes accompanying renal failure. As a factor of disease progression, the phenomenon of skeletal resistance to parathyroid hormone (PTH) is observed. With recent advances in the treatment of SHP, over-secretion of PTH can now be controlled. However, blood PTH levels 2 to 3 times higher than normal are considered necessary to maintain normal bone turnover in patients with renal failure. Various causes of skeletal resistance to PTH have been reported, including decrease in PTH receptor in osteoblasts, accumulation of 7-84 PTH fragment, and accumulation of osteoprotegerin. This skeletal resistance to PTH is not only a high-turnover bone accompanying SHP, but may also play a crucial role in the onset of low-turnover bone disease. We have produced a rat model of renal failure with normal level of PTH secretion and analyzed the bone of this model. Our results confirmed that bone turnover is lowered accompanying renal function impairment. We also found that this lowered bone turnover is improved by intermittent administration of PTH. In addition, PTH receptor gene expression is also decreased in low-turnover bone, as is observed in high-turnover bone disease. These findings confirm the presence of skeletal resistance to PTH in low-turnover bone accompanying renal failure. Control of calcium, phosphorus, and PTH levels with the target to maintain normal bone turnover is important in maintaining the quality of life of patients on dialysis.

Sp1-like activity mediates angiotensin-II-induced plasminogen-activator inhibitor type-1 (PAI-1) gene expression in mesangial cells.

Angiotensin II (Ang II) up-regulates plasminogen-activator inhibitor type-1 (PAI-1) expression in mesangial cells to enhance extracellular matrix formation. The proximal promoter region (bp -87 to -45) of the human PAI-1 gene contains several potent binding sites for transcription factors [two phorbol-ester-response-element (TRE)-like sequences; D-box (-82 to -76) and P-box (-61 to 54), and one Sp1 binding site-like sequence, Sp1-box 1 (-72 to -67)]. We studied this region to determine the transcription factor(s) that mediates Ang-II-induced transcriptional activation of the PAI-1 gene. Various double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to various sequences in the proximal promoter region were transfected to mesangial cells to examine the effects on Ang-II-induced PAI-1 mRNA expression. Transfection with the full-length decoy (bp -87 to -45, D-P-ODN) markedly attenuated Ang-II-induced PAI-1 mRNA expression by up to 70%. Transfection with D-ODN (-87 to -71) and P-ODN (-66 to -45), which correspond to each of the two TRE-like sequences, did not attenuate the expression. Gel-shift assays using nuclear extracts prepared from Ang-II-treated mesangial cells and D-P-ODN showed three specific complexes. The major complex was supershifted by anti-Sp1 antibody. The methylation-interference experiment demonstrated that human recombinant Sp1 bound to the so-called GT box (TGGGTGGGGCT, -78 to -69), which contains the Sp1-box 1. The complex that migrated with anti-Sp1 antibody was enhanced in the cells treated with Ang II. Further, D-Sp1-ODN (-85 to -63) containing the GT box attenuated up-regulation of PAI-1 mRNA expression induced by Ang II to a level (68+/-9% inhibition) comparable to D-P-ODN, whereas ODN with four mutations in the GT box had no effect. Our findings suggest that binding of Sp1 or an Sp1-like transcription factor to the GT box in the PAI-1 promoter up-regulates PAI-1 gene transcription in mesangial cells stimulated with Ang II. This transcription-factor binding site may be targeted to control Ang-II-dependent extracellular matrix formation by mesangial cells.

Association of TGF-beta signaling in angiotensin II-induced PAI-1 mRNA upregulation in mesangial cells: role of PKC.

This study aimed to identify the intracellular signaling pathway in angiotensin II (Ang II)-induced upregulation of plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in cultured rat glomerular mesangial cells, and to examine the interaction between Ang II and TGF-beta signaling. Ang II-induced upregulation of PAI-1 mRNA expression was prevented by a protein kinase C (PKC) inhibitor, bisindorylmaleimide I. While phorbol 12-myristate 13-acetate (PMA) upregulated the PAI-1 mRNA expression, a calcium ionophore, ionomycin, had little effect. Mesangial cells pretreated with PMA for 24 h to downregulate PKC demonstrated attenuated response to Ang II. A protein tyrosine kinase inhibitor, genistein, completely blocked both Ang II- and PMA-induced PAI-1 mRNA expression. Transforming growth factor-beta1 (TGF-beta1) alone induced the expression, and in the presence of Ang II, TGF-beta1 superinduced PAI-1 mRNA expression to a higher extent. Both bisindorylmaleimide I and genistein suppressed the Ang II plus TGF-beta1-induced PAI-1 mRNA upregulation to the basal level, while downregulation of PKC attenuated the synergistic upregulation of PAI-1 mRNA expression to the level comparable to TGF-beta1 alone. These data suggest that, in rat mesangial cells, (1) PKC and protein tyrosine kinase(s) are involved in the Ang II signaling cascade, (2) protein tyrosine kinase(s) works downstream from PKC in the cascade, and (3) there is an interaction between the Ang II and TGF-beta signal pathways downstream from PKC. In in vivo settings, local activation of renin-angiotensin and TGF-beta systems in the glomeruli may synergistically augment PAI-1 expression, promote mesangial matrix accumulation and progression of glomerular injury.

Role for "uremic toxin" in the progressive loss of intact nephrons in chronic renal failure.

We studied the effect on the progression of glomerular sclerosis of two different experimental maneuvers, peritoneal dialysis and oral adsorbent, which remove circulating substances in different fashions. Munich-Wistar rats with established glomerular sclerosis, verified by renal biopsy analysis at seven weeks after subtotal nephrectomy, were treated for four weeks with either peritoneal dialysis (PD) or oral charcoal adsorbent (AST-120). Treatment was initiated at eight weeks. Rats were paired in treatment and control groups according to the similarity in the degree of sclerosis determined at biopsy with a minimum of 50 glomeruli analyzed. Systolic blood pressure and BUN and creatinine clearance, measured at seven to eight weeks, were not different among groups. In Group 2 rats, PD was performed with 1.5% dextrose for eight one-hour cycles, six days per week, while Group 1 control rats had zero indwelling time of the dialysate. Group 4 rats received AST-120, an oral adsorbent charcoal, mixed 5% by weight with standard rat chow and given ad libitum from 8 to 12 weeks after subtotal nephrectomy, while control Group 3 rats received only rat chow. Whole kidney GFR at 12 weeks was significantly higher in Group 2 PD versus Group 1 control (0.50 +/- 0.08 vs. 0.30 +/- 0.05 ml/min, P less than 0.05). There was no statistical difference for BUN and whole kidney creatinine or inulin clearance in Group 4 AST-120 treated versus Group 3 control rats. Light microscopic studies in autopsy specimens revealed that both PD and AST-120 attenuated progression of glomerular sclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)