Pathogenic mechanisms implicated in the intravascular coagulation in the lungs of BVDV-infected calves challenged with BHV-1.

Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (MΦs) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of MΦs in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNFα, IL-1α, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary MΦs. In this regard, the co-infected calves, in spite of presenting a greater number of IMΦs than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNFα and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.

Evaluation of serum concentrations of cortisol and sex hormones of adrenal gland origin after stimulation with two synthetic ACTH preparations in clinically normal dogs.

OBJECTIVE: To compare the adrenocortical response of healthy dogs to a commonly used dose of a nonadsorbed tetracosactide product (tetracosactide) with responses to 2 doses of a depot formulation of tetracosactide (depot tetracosactide). ANIMALS: 14 dogs. PROCEDURES: Dogs were randomly assigned to receive tetracosactide (5 mg/kg, IV) or depot tetracosactide (250 µg, IM, or 5 µg/kg, IM). Dogs received each treatment once with a 2-week interval between treatments. Blood samples were assayed for cortisol, progesterone, 17-hydroxyprogesterone, androstenedione, and estradiol concentrations. RESULTS: Serum cortisol concentrations were significantly higher than the preadministration (baseline) concentrations for all treatments 60 minutes after administration of ACTH. Peak cortisol concentration was detected 180 minutes after IM administration of 250 µg of the depot tetracosactide. Serum concentrations of progesterone, 17-hydroxyprogesterone, and androstenedione did not differ significantly from baseline concentrations after stimulation with the 5 µg/kg dose of depot tetracosactide. Adrenal gland progesterone response was significantly higher than baseline concentrations at 60 minutes after administration of the 250-µg dose of depot tetracosactide, and the 17-hydroxyprogesterone and androstenedione responses were significantly higher than baseline concentrations at 120 minutes. Compared with the response to tetracosactide, adrenocortical response was higher and more sustained following administration of the depot tetracosactide, except for androstenedione concentration, which had a nonsignificant response. CONCLUSIONS AND CLINICAL RELEVANCE: Except for androstenedione concentrations, a high dose of the depot tetracosactide (250 µg, IM) induced an adrenocortical response similar to that after administration of tetracosactide. Thus, depot tetracosactide may represent an alternative to the nonadsorbed tetracosactide product.

Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats.

BACKGROUND: The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors. We have previously shown that N-terminal parathyroid hormone-related protein (PTHrP) enhanced the secretory PTH response to low calcium in vivo and in vitro in rat parathyroid glands. N-terminal PTHrP fragments are equipotent to N-terminal PTH as ligands for the PTH/PTHrP receptor that is demonstrated in parathyroid tissue. This supports the possibility that the parathyroid cells respond to PTH/PTHrP receptor ligands and as such are target for an autoregulatory action of PTH and PTHrP. Our aim was to search for the PTH/PTHrP receptor in rat parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary hyperparathyroidism (HPT) in uremic rats. METHODS: PTH secretion was examined during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia both with and without PTHrP. Five groups, each of six normal rats, received a bolus of increasing doses of 0.1, 1.0, 10, and 100 microg of PTHrP 1-40, or vehicle only. Chronic renal failure (CRF) was induced by 5/6 nephrectomy. One group of 12 CRF rats received a standard diet, while another CRF group of 18 rats received a high phosphorus diet to induce more severe HPT. After 8 weeks of uremia, the rats were infused with EGTA and PTHrP 1-40 or vehicle. The presence of the PTH/PTHrP receptor in the rat parathyroid glands was examined by reverse transcription-polymerase chain reaction (RT-PCR) technique. PTH was measured by a rat PTH assay that does not cross-react with PTHrP. RESULTS: In a dose-related manner, PTHrP enhanced the PTH response to hypocalcemia in normal rats. A similar rate of decrease of plasma Ca++ was induced by EGTA in all experimental groups. In CRF rats, plasma creatinine (0.99 +/- 0.10 mmol/L vs. 0.33 +/- 0.01 mmol/L, P < 0.001) and plasma PTH (226 +/- 32 pg/mL vs. 69 +/- 16 pg/mL, P < 0.001) levels were significantly increased. The CRF rats on high phosphorus diet had significant hypocalcemia (Ca++, 1.04 +/- 0.02 mmol/L vs. 1.28 +/- 0.03 mmol/L, P < 0.001), hyperphosphatemia (3.48 +/- 0.3 mmol/L vs. 2.25 +/- 0.1 mmol/L, P < 0.001) and severe secondary HPT, PTH (984 +/- 52 pg/mL vs. 226 +/- 32 pg/mL, P < 0.001) compared to CRF rats on a standard phosphorus diet. The maximal PTH response to hypocalcemia was enhanced in CRF rats (maximum PTH 382 +/- 58 pg/mL vs. 196 +/- 29 pg/mL in normal rats, P < 0.01) and further enhanced by PTHrP 1-40 to 826 +/- 184 pg/mL (P < 0.01). The secretory capacity of the parathyroid glands in response to low Ca++ was severely diminished in uremia. In CRF rats given a high phosphorus diet, the basal PTH levels were at the upper part of the calcium/PTH curve, and the induction of more marked hypocalcemia did not stimulate PTH secretion further (maximum PTH 1475 +/- 208 pg/mL vs. basal 1097 +/- 69 pg/mL, NS). PTHrP, however, further enhanced the maximal PTH levels significantly (maximum PTH 3142 +/- 296 pg/mL, P < 0.01). The presence of the PTH/PTHrP receptor in the rat parathyroid glands was confirmed by RT-PCR technique. CONCLUSION: PTHrP enhanced significantly, in a dose-related manner, the low Ca++-stimulated PTH secretion in normal rats. The PTH/PTHrP receptor is present in rat parathyroid glands. The impaired secretory capacity of the parathyroid glands in uremic rats is significantly enhanced by PTHrP. An autocrine/paracrine role in the parathyroid glands of the PTH/PTHrP receptor targeting peptides, PTHrP and PTH, is suggested. Thus, it is hypothesized that PTH during hypocalcemia might have a positive auto-feedback regulatory role on its own secretion.

Effects of uremic ultrafiltrate on the regulation of the parathyroid cell cycle by calcitriol.

BACKGROUND: Calcitriol (CTR) is used in the treatment of hyperparathyroidism secondary to renal failure because it decreases parathyroid hormone (PTH) synthesis and parathyroid cell proliferation. Previous studies in tissues other than parathyroids have demonstrated that uremic factors affect the action of CTR on the target cells. We questioned whether the uremic milieu interferes with the inhibition of parathyroid cell proliferation by CTR. METHODS: Studies were performed in vitro using freshly excised normal dog parathyroid tissue incubated for 24 hours with and without CTR and in the presence of either total uremic ultrafiltrate (UUF) from uremic patients or high-pressure liquid chromatography (HPLC)-derived fractions (hydrophilic compounds eluting early and hydrophobic compounds eluting late) of this UUF (F1 to F4). Parathyroid cell proliferation was assessed by flow cytometry. RESULTS: The addition of CTR 10-8 and 10-7 mol/L to parathyroid tissue produced an inhibition of the proliferation that was prevented in the presence of UUF. In a medium containing CTR 10-8 mol/L, the addition of F1, F2 and F3, but not F4, prevented the CTR-induced inhibition of parathyroid cell proliferation. With CTR 10-7 mol/L, the inhibition of proliferation was observed even in the presence of F1, F2 and also F4, but was prevented by F3. Uric acid (7 mg/dL), indoxyl sulfate (5 mg/dL) and p-cresol (1.4 mg/dL), which coeluted with F1, F2 and F4, respectively, did not interfere with the inhibitory action of CTR 10-7 mol/L; however, the addition of phenol (0.14 mg/dL), which coeluted with F3, prevented the CTR-induced inhibition of parathyroid cell proliferation. CONCLUSIONS: The presence of uremic toxins prevents the inhibition of parathyroid cell proliferation induced by calcitriol.

Regulation of parathyroid vitamin D receptor expression by extracellular calcium.

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl(2) or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.

Persistent downregulation of calcium-sensing receptor mRNA in rat parathyroids when severe secondary hyperparathyroidism is reversed by an isogenic kidney transplantation.

Experimental severe secondary hyperparathyroidism (HPT) is reversed within 1 wk after reversal of uremia by an isogenic kidney transplantation (KT) in the uremic rats. Abnormal parathyroid hormone (PTH) secretion in uremia is related to downregulation of CaR and vitamin D receptor (VDR) in the parathyroid glands (PG). The aim of this investigation was to examine the expression of CaR and VDR genes after reversal of uremia and HPT in KT rats. 5/6 nephrectomized rats were kept on a normal or high-phosphorus (hP) diet for 8 wk to induce severe HPT (n = 8 in each group). In another group of seven uremic hP rats, uremia was reversed by an isogenic KT and PG were harvested within 1 wk posttransplant. Plasma urea, creatinine, total calcium, phosphorus, and PTH levels were measured. Parathyroid CaR and VDR mRNA were measured by quantitative PCR. Uremic hP rats had significantly elevated levels of creatinine, urea, and phosphorus (P < 0.001) and developed significant hypocalcemia (plasma calcium 1.83 +/- 0.2 mmol/L; P < 0.001) compared with normal control rats. After KT, the levels were normalized from day 3 to 7: creatinine from 0.117 +/- 0.016 to 0.050 +/- 0.002 mmol/L; urea from 23 +/- 4 to 7 +/- 0.3 mmol/L; phosphorus from 3.9 +/- 0.6 to 1.5 +/- 0.06 mmol/L; calcium from 1.8 +/- 0.2 to 2.5 +/- 0.02 mmol/L. Plasma PTH levels fell from 849 +/- 224 to a normal level of 38 +/- 9 pg/ml (P < 0.01). In uremic rats on a standard diet, CaR mRNA was similar to that of normal control rats, whereas VDR mRNA was significantly decreased. In uremic rats kept on hP diet, CaR mRNA was significantly decreased to 26 +/- 7% of control rats (P = 0.01) and VDR mRNA reduced to 36 +/- 11% (P < 0.01). In KT, previously hP uremic rats, both CaR mRNA and VDR mRNA remained severely reduced (CaR, 39 +/- 7%; VDR, 9 +/- 3%; P < 0.01) compared with normal rats. In conclusion, circulating plasma PTH levels normalized rapidly after KT, despite persisting downregulation of CaR and VDR gene expression. This indicates that upregulation of CaR mRNA and VDR mRNA is not necessary to induce the rapid normalization of PTH secretion from hyperplastic parathyroid glands.

Pathogenesis of refractory secondary hyperparathyroidism.

Calcitriol is currently used to reduce parathyroid hormone (PTH) levels in uremic patients. However, a significant number of patients fail to respond to calcitriol therapy. The data suggest that a poor response to calcitriol can be anticipated in patients with severe hyperparathyroidism (with a high basal PTH levels) and uncontrolled serum phosphate. The abnormal parathyroid response to calcitriol in uremic patients with severe parathyroid hyperplasia may be attributed, to a large extent, to the development of nodular hyperplasia as a result of clonal transformation from a diffuse polyclonal hyperplasia. The factors involved in the development of polyclonal parathyroid hyperplasia, at earlier stages of secondary hyperparathyroidism, appear to be the same factors that stimulate PTH secretion and synthesis: hypocalcemia, hyperphosphatemia and low serum calcitriol levels. Studies performed in vitro using parathyroid tissue from uremic patients who required parathyroidectomy demonstrate that in nodular hyperplasia there is an abnormal response to calcium and calcitriol, which suggests that there are factors intrinsic to the hyperplastic cell (such as decrease in calcium sensor receptors and vitamin D receptors) responsible for an abnormal regulation of parathyroid function. Accumulation of phosphate is a key factor in the pathogenesis of secondary hyperparathyroidism and a poor response to calcitriol treatment is associated with the failure to control the serum phosphorus. High phosphate stimulates PTH secretion as demonstrated by in vivo and in vitro studies. In addition, animal studies strongly suggest that phosphate increases parathyroid cell proliferation. There are growth-related genes potentially involved in uremic hyperparathyroidism; however, changes in the expression of these genes may be the consequence rather than the cause of parathyroid hyperplasia.