Background. Both dietary phosphorus restriction and the ingestion of ammonium chloride (NH4Cl) given to rats on a high-phosphorus diet have been shown to preserve renal function in the azotaemic rat. ...Parathyroidectomy also has been reported to preserve renal function and, in addition, to prevent kidney hypertrophy in the remnant kidney model. Our goals were (i) to evaluate in azotaemic rats the effect of dietary phosphorus on renal function in a shorter time frame than previously studied and (ii) to determine whether NH4Cl administration (a) enhances the renoprotective effect of dietary phosphorus restriction and (b) improves renal function in the absence of parathyroid hormone (PTH). Methods. High (H; 1.2%), normal (N; 0.6%) and low (L; <0.05%) phosphorus diets (PD) were given for 30 days to 5/6 nephrectomized rats. In each dietary group, one-half of the rats were given NH4Cl in the drinking water. The six groups were HPD + NH4Cl, HPD, NPD + NH4Cl, NPD, LPD + NH4Cl and LPD. The effect of NH4Cl administration was also evaluated in 5/6 nephrectomized, parathyroidectomized (PTX) rats on NPD. Results. In each of the three dietary phosphorus groups, creatinine and urea clearances were greater (P<0.01) in rats receiving NH4Cl. Neither creatinine nor urea clearance was reduced by high dietary phosphorus. Urine calcium excretion was greatest in the LPD group and was increased (P ≤ 0.001) in all three groups by NH4Cl ingestion. An inverse correlation was present between plasma calcium and phosphorus in the parathyroid intact (r = −0.79, P<0.001) and PTX groups (r = −0.46, P = 0.02). In PTX rats, NH4Cl ingestion increased (P ≤ 0.01) creatinine and urea clearances and both an increasing plasma calcium concentration (r = 0.67, P<0.001) and urine calcium excretion (r = 0.73, P<0.001) increased urine phosphorus excretion. Conclusions. At 30 days of renal failure (i) NH4Cl ingestion increased creatinine and urea clearances, irrespective of dietary phosphorus; (ii) high urine calcium excretion, induced by dietary phosphorus restriction and NH4Cl ingestion, did not adversely affect renal function; (iii) high dietary phosphorus did not decrease renal function; (iv) the absence of PTH did not preserve renal function or prevent NH4Cl from improving renal function; and (v) both an increasing plasma calcium concentration and urine calcium excretion resulted in an increase in urine phosphorus excretion in PTX rats.
The concept that the PTH-calcium curve is representative of parathyroid function has been discussed. Comparisons of parathyroid function have been made between normal humans and hemodialysis patients ...and also between hemodialysis patients with different forms of renal osteodystrophy. From these comparisons, it is apparent that the magnitude of HPT is much greater in patients with renal failure than in normal humans, and as represented by the ratio of basal to maximal PTH, the parathyroid gland appears to be stimulated at basal serum calcium levels in hemodialysis patients. Similarly, based on an analysis of the PTH-calcium curve, we were able to determine that several differences in parathyroid function were present in hemodialysis patients with different forms of renal osteodystrophy. As compared to hemodialysis patients with LTAABD and aplastic bone disease, patients with osteitis fibrosa have a greater magnitude of hyperparathyroidism, a greater sensitivity of the parathyroid cell (slope), a higher set point of calcium, and greater PTH stimulation at basal serum calcium (ratio of basal to maximal PTH). Calcitriol treatment of hemodialysis patients with osteitis fibrosa resulted in a significant decrease in PTH throughout the PTH-calcium curve and also reduced the sensitivity (slope) of the PTH-calcium curve. The concept of hysteresis has been discussed as well as the role that the ambient basal serum calcium concentration may have on the determination of the PTH-calcium curve. Finally, the effect that successful renal transplantation has on HPT has been examined. In conclusion, we believe that the PTH-calcium curve provides a reliable assessment of parathyroid function, and as such, has considerable application for the study of parathyroid disorders in the clinical setting.
Alterations in phosphate homeostasis play an important role in the development of secondary hyperparathyroidism in renal failure. Until recently, it was accepted that phosphate retention only ...increased parathyroid hormone (PTH) secretion through indirect mechanisms affecting calcium regulation and calcitriol synthesis. However, recent in vitro studies have suggested that phosphate may directly affect PTH secretion. Our goal was to determine whether in vivo an intravenous phosphate infusion stimulated PTH secretion in the absence of changes in serum calcium. Three different doses of phosphate were infused intravenously during 120 minutes to increase the serum phosphate concentration in dogs. Sulfate was also infused intravenously as a separate experimental control. A simultaneous calcium clamp was performed to maintain a normal ionized calcium concentration throughout all studies. At the lowest dose of infused phosphate (1.2 mmol/kg), serum phosphate values increased to ∼3 mM, but PTH values did not increase. At higher doses of infused phosphate (1.6 mmol/kg and 2.4 mmol/kg), the increase in serum phosphate to values of ∼4 mM and 5 mM, respectively, was associated with increases in PTH, even though the ionized calcium concentration did not change. Increases in PTH were not observed until 30–60 minutes into the study. These increases were not sustained, since by 120 minutes PTH values were not different from baseline or controls despite the maintenance of marked hyperphosphatemia. During the sulfate infusion, serum sulfate values increased by ∼3‐fold, but no change in PTH values were observed. In conclusion, an acute elevation in serum phosphate stimulated PTH secretion in the intact animal, but the magnitude of hyperphosphatemia exceeded the physiologic range. Future studies are needed to determine whether PTH stimulation is more sensitive to phosphate loading in states of chronic phosphate retention. Moreover, the mechanisms responsible for the delay in PTH stimulation and the failure to sustain the increased PTH secretion need further evaluation.
Background. A variety of stimuli are involved in the pathogenesis of parathyroid gland hyperplasia in renal failure. Recently, it was shown that blocking the signal from the endothelin-1 (ET-1) ...receptor (ETAR/ETBR) by a non-selective receptor antagonist, bosentan, reduced parathyroid cell proliferation, parathyroid gland hyperplasia and parathyroid hormone (PTH) levels in normal rats on a calcium deficient diet. Our goal was to determine whether in 5/6 nephrectomized (NPX) rats with developing or established hyperparathyroidism, the endothelin receptor blocker, bosentan, reduced the increase in parathyroid cell proliferation, parathyroid gland hyperplasia and PTH values. Methods. High (HPD, 1.2%) or normal phosphorus diets (PD) (NPD, 0.6%) were given to 5/6 NPX rats for 15 days (NPX15). In each dietary group, one-half the rats were given bosentan (B) i.p. 100 mg/kg/day. The four groups of rats were: (1) NPX15-1.2% P; (2) NPX15-1.2% P+B; (3) NPX15-0.6% P; and (4) NPX15-0.6% P+B. In a second study in which hyperparathyroidism was already established in 5/6 NPX rats fed a HPD for 15 days, rats were divided into two groups in which one group was maintained on a HPD and the other group was changed to very low PD (VLPD, <0.05%) for an additional 15 days. In each dietary group, one-half the rats were given bosentan i.p. 100 mg/kg-day. The four groups of rats were: (1) NPX30-1.2% P; (2) NPX30-1.2% P+B; (3) NPX30-0.05% P and (4) NPX30-0.05% P+B. Parathyroid cell proliferation was measured by proliferating cell nuclear antigen (PCNA) staining and ET-1 expression by immunohistochemical techniques. Results. In the study of developing hyperparathyroidism, bosentan reduced ET-1 expression in the parathyroid glands of rats on the NPD and HPD (P<0.05). But only in rats on the NPD did bosentan result in a reduced increase in parathyroid gland weight (P<0.05). In the study of established hyperparathyroidism, in which 5/6 NPX rats were given a HPD for 15 days, bosentan started on day 15 reduced (P<0.05) ET-1 expression in rats maintained for 15 additional days on the HPD or the VLPD. On the VLPD, parathyroid gland weight was less (P<0.05) than that in rats on the HPD sacrificed at 15 or 30 days. Bosentan did not reduce parathyroid cell proliferation or parathyroid gland weight in rats maintained on the HPD or further reduce these parameters beyond that obtained with dietary phosphorus restriction. PTH values were lowest in the VLPD group, intermediate in the NPD group, and highest in the HPD group, but in none of the three groups did bosentan decrease PTH values. Conclusions. In azotemic rats with developing hyperparathyroidism, bosentan resulted in a reduced increase in parathyroid gland weight when dietary phosphorus content was normal. Despite a reduction in ET-1 expression in rats on a HPD with developing or established hyperparathyroidism, bosentan did not reduce the increase in parathyroid cell proliferation, parathyroid gland growth or PTH values. Thus, ET-1 blockade with bosentan did not prevent parathyroid gland growth in the azotemic rat.
Background. Recent evidence has shown that the assay for ‘intact’ parathyroid hormone (I‐PTH) not only reacts with 1–84 PTH but also with large non‐1–84 PTH fragments, most of which is probably 7–84 ...PTH. As a result, an assay specific for 1–84 PTH named ‘whole’ PTH (W‐PTH) has been developed. The present study was designed: (i) to determine whether the W‐PTH assay reliably measures PTH values in the dog; (ii) to evaluate differences between the W‐PTH and I‐PTH assays during hypo‐ and hypercalcaemia; and (iii) to assess the peripheral metabolism of W‐PTH and I‐PTH. Methods. In normal dogs, hypocalcaemia was induced by EDTA infusion and was followed with a 90 min hypocalcaemic clamp. Hypercalcaemia was induced with a calcium infusion. Results. I‐PTH and W‐PTH values increased from 36±8 and 13±3 pg/ml (P=0.01) at baseline to a maximum of 158±40 and 62±15 pg/ml (P=0.02 vs I‐PTH) during hypocalcaemia. The W‐PTH/I‐PTH ratio, 38±4% at baseline, did not change during the induction of hypocalcaemia, but sustained hypocalcaemia increased (P<0.05) this ratio. During hypercalcaemia, maximal suppression for I‐PTH was 2.0±0.5 and only 5.7±0.6 pg/ml for W‐PTH, due to a decreased sensitivity of the W‐PTH assay at values <5 pg/ml. The disappearance rate of PTH was determined in five additional dogs which underwent a parathyroidectomy (PTX). At 2.5 min after PTX, W‐PTH was metabolized more rapidly, with a value of 25±2% of the pre‐PTX value vs 30±3% for I‐PTH (P<0.05). Conclusions. (i) The W‐PTH/I‐PTH ratio is less in the normal dog than in the normal human, suggesting that the percentage of non‐1–84 PTH measured with the I‐PTH assay is greater in normal dogs than in normal humans; (ii) the lack of change in the W‐PTH/I‐PTH ratio during acute hypocalcaemia is different from the situation observed in humans; and (iii) the dog appears to be a good model to study I‐PTH and W‐PTH assays during hypocalcaemia.
In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy ...has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype.
This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2-3 studying a relationship between FGF23 and vascular dysfunction.
In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2-3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222.
In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.
ABSTRACT
In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)‐23. Abnormal regulation of the FGFR1‐Klotho receptor complex may cause a ...resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)‐1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a highphosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti‐FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of β‐cateniv together with a reduction in Klotho. Wnt/β‐catenin inhibition with Dkk‐1 prevented the P‐induced down‐regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/β‐catenin pathway.—Muñoz‐Castañeda, J. R., Herencia, C., Pendón‐Ruiz de Mier M. V., Rodriguez‐Ortiz, M. E., Diaz‐Tocados, J. M., Vergara, N., Martínez‐Moreno, J. M., Salmerón M. D., Richards, W. G., Felsenfeld, A., Kuro‐O, M., Almadén Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats. FASEB J. 31, 3858–3867 (2017). www.fasebj.org—Muñoz‐Castañeda, Juan R., Herencia, Carmen, Pendón‐Ruiz de Mier, Maria Victoria, Rodriguez‐Ortiz, Maria Encarnación, Diaz‐Tocados, Juan M., Vergara, Noemi, Martínez‐Moreno, Julio M., Salmerón, Maria Dolores, Richards, William G., Felsenfeld, Arnold, Kuro‐O, Makoto, Almadén, Yolanda, Rodríguez, Mariano Differential regulation of renal Klotho and FGFR1 in normal and uremic rats. FASEB J. 31, 3858–3867 (2017)
Objective-To evaluate the effects of metabolic acidosis and changes in ionized calcium (Ca(2+)) concentration on Pao2 in dogs. Animals-33 anesthetized dogs receiving assisted ventilation. ...Procedure-Normal acid-base status was maintained in 8 dogs (group I), and metabolic acidosis was induced in 25 dogs. For 60 minutes, normocalcemia was maintained in group I and 10 other dogs (group II), and 10 dogs were allowed to become hypercalcemic (group III); hypocalcemia was then induced in groups I and II. Groups II and IV (5 dogs) were treated identically except that, at 90 minutes, the latter underwent parathyroidectomy. At intervals, variables including Pao2, Ca(2+) concentration, arterial blood pH (pHa), and systolic blood pressure were assessed. Results-In group II, Pao2 increased from baseline value (96 +/- 2 mm Hg) within 10 minutes (pHa, 7.33 +/- 0.001); at 60 minutes (pHa, 7.21 +/- 0.02), Pao2 was 108 +/- 2 mm Hg. For the same pHa decrease, the Pao2 increase was less in group III. In group I, hypocalcemia caused Pao2 to progressively increase (from 95 +/- 2 mm Hg to 104 +/- 3 mm Hg), which correlated (r = -0.66) significantly with a decrease in systolic blood pressure (from 156 +/- 9 mm Hg to 118 +/- 10 mm Hg). Parathyroidectomy did not alter Pao2 values. Conclusions and Clinical Relevance-Induction of hypocalcemia and metabolic acidosis each increased Pao2 in anesthetized dogs, whereas acidosis-induced hypercalcemia attenuated that increase. In anesthetized dogs, development of metabolic acidosis or hypocalcemia is likely to affect ventilatory control.
Effects of fasting, feeding, and bisphosphonate administration on serum calcitriol levels in phosphate-deprived rats.
In a recent study, we showed in phosphate-deprived rats that morning feeding ...decreased serum phosphate and increased serum calcium values as compared with similar rats fasted overnight, and high doses of bisphosphonates did not reduce the magnitude of hypercalcemia. In the present study, we evaluated in phosphate-deprived rats whether serum calcitriol values were: (1) affected by the differences in serum phosphate induced by morning feeding and overnight fasting, (2) correlated with changes in serum phosphate levels, and (3) influenced by bisphosphonate administration.
Four groups of rats were studied: (1) low-phosphate diet (LPD; P < 0.05%), (2) LPD + the bisphosphonate pamidronate (APD), (3) normal diet (ND; P 0.6%), and (4) ND + APD. Both diets contained 0.6% calcium. In rats receiving APD, high doses (0.8 mg/kg) were given subcutaneously four times during the study. On day 11, rats were sacrificed after an overnight fast or two to four hours after morning feeding.
In the fed phosphate-deprived rats (LPD and LPD + APD), serum phosphate levels were less (P < 0.05) and serum calcium levels were greater (P < 0.05) than in similar rats fasted overnight. In rats on the ND (ND and ND + APD), no differences were observed between fed and fasted rats. In phosphate-deprived rats, serum calcitriol levels were greater (LPD, P < 0.05) or tended to be greater (LPD + APD, P = 0.10) in the fed than in the fasted groups. In APD-treated rats, serum calcitriol values were greater than in rats not given APD whether rats were (1) fed or fasted, or (2) on an LPD or ND. An inverse correlation was present between serum phosphate and serum calcitriol (r = -0.58, P = 0.001). In a stepwise regression model in which serum calcitriol was the dependent variable and independent variables were APD administration and serum calcium, phosphate, and PTH, serum phosphate (P = 0.003) had an inverse and APD (P < 0.001) administration a direct effect on serum calcitriol (r2 = 0.59).
Calcitriol synthesis is rapidly inducible in rats during chronic phosphate deprivation, and the increase in serum calcitriol values is best attributed to feeding-induced decreases in serum phosphate. APD administration independently increases serum calcitriol levels in rats on normal and phosphate-deprived diets. Finally, whether our results in the rat are applicable to the clinical setting should be evaluated because in previous human studies of dietary phosphate restriction, serum calcitriol measurements were performed the morning after an overnight fast.
Background. Whether calcitriol administration, which is used to treat secondary hyperparathyroidism in dialysis patients, induces regression of parathyroid‐gland hyperplasia remains a subject of ...interest and debate. If regression of the parathyroid gland were to occur, the presumed mechanism would be apoptosis. However, information on whether high doses of calcitriol can induce apoptosis of parathyroid cells in hyperplastic parathyroid glands is lacking. Consequently, high doses of calcitriol were given to azotaemic rats and the parathyroid glands were evaluated for apoptosis. Methods. Rats were either sham‐operated (two groups) or underwent a two‐stage 5/6 nephrectomy (three groups). For the first 4 weeks, all rats were given a high (1.2%) phosphorus (P) diet to stimulate parathyroid gland growth and then were changed to a normal (0.6%) P diet for 2 weeks. At week 7, three of the five groups were given high doses of calcitriol (500 pmol/100 g body weight) intraperitoneally every 24 h during 72 h before sacrifice. The five groups during week 7 were: (i) normal renal function (NRF)+0.6% P diet; (ii) NRF+0.6% P+calcitriol; (iii) renal failure (RF)+0.6% P; (iv) RF+1.2% P+calcitriol; and (v) RF+0.6% P+calcitriol. Parathyroid glands were removed at sacrifice and the TUNEL stain was performed to detect apoptosis. Results. At sacrifice, the respective serum calcium values in calcitriol‐treated groups (groups 2, 4, and 5) were 15.52±0.26, 13.41±0.39 and 15.12±0.32 mg/dl. In group 3, PTH was 178±42 pg/ml, but in calcitriol‐treated groups, PTH values were suppressed, 8±1 (group 2), 12±2 (group 4), and 7±1 pg/ml (group 5). Despite, the severe hypercalcaemia and marked PTH suppression in calcitriol‐treated groups, the percentage of apoptotic cells in the parathyroid glands was very low (range 0.08±0.04 to 0.25±0.20%) and not different among the five groups. Conclusions. We found no evidence in hyperplastic parathyroid glands that apoptosis could be induced in azotaemic rats by the combination of high doses of calcitriol and severe hypercalcaemia despite the marked reduction in PTH levels that was observed.
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