total intracellular PTH was the non-PTH (1-84), most likely A novel mechanism for skeletal resistance in uremia.PTH 7-84. Background. In treating secondary hyperparathyroidism, Conclusion. In patients with chronic renal failure, the presthe target level of serum intact parathyroid hormone (I-PTH) ence of high circulating levels of non-1-84 PTH fragments should be three to five times normal to prevent adynamic bone (most likely 7-84 PTH) detected by the "intact" assay and the disease. In circulation, there is a non-(1-84) PTH-truncated antagonistic effects of 7-84 PTH on the biological activity of fragment, likely 7-84, which, in addition to PTH 1-84, is mea-1-84 PTH explain the need of higher levels of "intact" PTH sured by most I-PTH immunoradiometric (IRMA) assays, givto prevent adynamic bone disease. ing erroneously high I-PTH values. We have developed a new IRMA assay in which the labeled antibody recognizes only the first six amino acids of the PTH molecule. Thus, this new IRMA assay (Whole PTH) measures only the biologically active 1-84 Parathyroid hormone (PTH), a single-chain polypep-PTH molecule. tide of 84 amino acids [1], plays a critical role in the Methods. Using this new IRMA assay (Whole PTH) and the Nichols "intact" PTH assay, we compared the ability of regulation of mineral metabolism. Ionized calcium, caleach assay to recognize human PTH (hPTH) 1-84 and hPTH citriol, and phosphorus are the three major regulators 7-84 and examined the percentage of non-1-84 PTH in circulaof PTH homeostasis in humans. tion and in parathyroid glands. Possible antagonistic effects of The human PTH gene is located on the short arm of the 7-84 PTH fragment on the biological activity of 1-84 PTH chromosome 11. The coding region spans more than 4 in rats were also tested. Results. In 28 uremic patients, PTH values measured with kb and consists of three exons. The first exon contains the Nichols assay, representing a combined measurement of the 5Ј untranslated region of the PTH transcript. Theboth hPTH 1-84 and hPTH 7-84, were 34% higher than with coding sequence spans exons 2 and 3. The spliced cytothe Whole assay (hPTH 1-84 only); the median PTH was 523 plasmic transcript is 772 bases long. The primary translaversus 318 pg/mL (P Ͻ 0.001). Similar results were found in tion product, pre-pro-PTH (115 amino acids), is formed 14 renal transplant patients. In osteoblast-like cells, ROS 17.2, 1-84 PTH (10 Ϫ8 mol/L) increased cAMP from 18.1 Ϯ 1.25 to in the rough endoplasmic reticulum of parathyroid chief 738 Ϯ 4.13 mmol/well. Conversely, the same concentration of cells [2] and is converted within seconds to pro-PTH (90 7-84 PTH had no effect. In parathyroidectomized rats fed a amino acids) [3]. In the Golgi apparatus, pro-PTH is calcium-deficient diet, 7-84 PTH was not only biologically inacconverted to intact PTH (I-PTH; 84 amino acids) approxtive, but had antagonistic effects on 1-84 PTH in bone. Plasma calcium was increased (0.65 mg/dL) two hours after 1-84 PTH imately 15 minutes after the biosynthesis of the original tre...

Dietary phosphorus (P) restriction is known to ameliorate secondary hyperparathyroidism in renal failure patients. In early renal failure, this effect may be mediated by an increase in 1,25-(OH) 2 D 3 , whereas in advanced renal failure, P restriction can act independent of changes in 1,25-(OH) 2 D 3 and serum ionized calcium (ICa). In this study, we examined the effects of dietary P on serum PTH, PTH mRNA, and parathyroid gland (PTG) hyperplasia in uremic rats. Normal and uremic rats were maintained on a low (0.2%) or high (0.8%) P diet for 2 mo. PTG weight and serum PTH were similar in both groups of normal rats and in uremic rats fed the 0.2% P diet. In contrast, there were significant increases in serum PTH (130 Ϯ 25 vs. 35 Ϯ 3.5 pg/ml, P Ͻ 0.01), PTG weight (1.80 Ϯ 0.13 vs. 0.88 Ϯ 0.06 g/gram of body weight, P Ͻ 0.01), and PTG DNA (1.63 Ϯ 0.24 vs. 0.94 Ϯ 0.07 g DNA/gland, P Ͻ 0.01) in the uremic rats fed the 0.8% P diet as compared with uremic rats fed the 0.2% P diet. Serum ICa and 1,25-(OH) 2 D 3 were not altered over this range of dietary P, suggesting a direct effect of P on PTG function. We tested this possibility in organ cultures of rat PTGs. While PTH secretion was acutely (30 min) regulated by medium calcium, the effects of medium P were not evident until 3 h. During a 6-h incubation, PTH accumulation was significantly greater in the 2.8 mM P medium than in the 0.2 mM P medium (1,706 Ϯ 215 vs. 1,033 Ϯ 209 pg/ g DNA, P Ͻ 0.02); the medium ICa was 1.25 mM in both conditions. Medium P did not alter PTH mRNA in this system, but cycloheximide (10 g/ml) abolished the effect of P on PTH secretion. Thus, the effect of P is posttranscriptional, affecting PTH at a translational or posttranslational step. Collectively, these in vivo and in vitro results demonstrate a direct action of P on PTG function that is independent of ICa and 1,25-(OH) 2 D 3 . ( J. Clin. Invest. 1996. 97:2534-2540.)

Vascular calcification is associated with cardiovascular disease, the most common cause of death in chronic kidney disease (CKD). Patients with CKD are treated with vitamin D receptor activators (VDRAs); therefore, we determined if this treatment affects vascular calcification. Uremic rats were given vehicle, calcitriol, paricalcitol, or doxercalciferol three times a week for 1 month. Calcitriol significantly increased the serum calcium-phosphate product and aortic calcium content. Paricalcitol had no effect but the same dose of doxercalciferol significantly increased the calcium-phosphate product and the aortic calcium content, the latter being confirmed by von Kossa staining. To see if the increased aortic calcium was due to an increased serum calcium-phosphate product or to a differential effect of the two VDRAs, we lowered the dose of doxercalciferol and increased the dose of paricalcitol. A lower doxercalciferol did not increase the calcium-phosphate product but increased the aortic calcium content. A higher dose of paricalcitol still had no effect. Doxercalciferol treatment increased the mRNA and protein expression of the bone-related markers Runx2 and osteocalcin in the aorta, whereas paricalcitol did not. Hence, different VDRAs have different effects on vascular calcification in uremic rats. The effects are independent of the serum calcium-phosphate product suggesting independent mechanisms.

Monotherapy with angiotensin-converting enzyme inhibitors has been shown to be beneficial in suppressing the progression of experimentally induced kidney diseases. Whether such therapy provides additional benefits when combined with vitamin D or an analog of vitamin D has not been established. Rats were made uremic by 5/6 nephrectomy and treated as follows: Uremic ؉ vehicle (UC), uremic ؉ enalapril (30 mg/L in drinking water; E), uremic ؉ paricalcitol (19-nor; 0.8 g/kg, three times a week), and uremic ؉ enalapril ؉ paricalcitol (E ؉ 19-nor). A group of normal rats served as control (NC). BP was significantly elevated in the UC and 19-nor groups compared with the NC group but was indistinguishable from normal in the E and E ؉ 19-nor groups. The decrease in creatinine clearance and the increase in the excretion of urinary protein that were observed in the UC group were ameliorated by the use of E alone or by E ؉ 19-nor (P < 0.05 versus UC). The glomerulosclerotic index was significantly decreased in both the 19-nor (P < 0.01) and E ؉ 19-nor groups (P < 0.01) compared with the UC group. Tubulointerstitial volume was significantly decreased in both the E (P < 0.05) and E ؉ 19-nor groups (P < 0.01) compared with the UC group. Both macrophage infiltration (ED-1-positive cells) and production of the chemokine monocyte chemoattractant protein-1 were significantly blunted in E ؉ 19-nor compared with E group. TGF-␤1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E ؉ 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor-treated rats (5.5-fold versus NC). When rats were treated with E ؉ 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-␤ signaling pathway, and this effect is amplified when BP is controlled via renin-angiotensin system blockade.

In early uremia, vitamin D suppression of high P-induced PT hyperplasia and high dietary Ca arrest of PT growth involve induction of PT p21 and prevention of increases in TGF-alpha.