Exercise-induced hypercalcemia and vasopressin-mediated bone resorption.

Our human observational study showed that elevated arginine vasopressin levels by heavy exercise, not catecholamines, were associated with elevated serum tartrate-resistant acid phosphatase 5b (TRACP-5b). The increase in serum calcium was positively associated with percent changes of TRACP-5b, implying the involvement of bone resorption in the pathogenesis of exercise-induced hypercalcemia. INTRODUCTION: It remains unclear whether enhanced bone resorption explains exercise-induced hypercalcemia. An experimental study demonstrated that arginine vasopressin (AVP) stimulated osteoclast activity. METHODS: We conducted a prospective observational study, enrolling 65 trained healthy male officers of the Japan Self-Defense Forces (34 and 31 in waves 1 and 2, respectively). Before and after a 5-h heavy exercise, we collected laboratory data including bone markers, symptoms, and ionized calcium (iCa; wave 2 only). As blood calcium levels change after exercise, we estimated calcium (corrected calcium) levels immediately after the exercise using the correlation between blood calcium and time from the end of exercise in another cohort. RESULTS: Body weight decreased by 6.9% after the exercise. Corrected post-exercise serum total calcium (tCa) and iCa levels were significantly higher than pre-exercise levels, and 18% of participants showed hypercalcemia defined as corrected tCa >10.4 mg/dL or iCa >1.30 mmol/L. Serum tartrate-resistant acid phosphatase 5b (TRACP-5b), plasma three fractions of catecholamines, and AVP elevated significantly (median 14.3 pg/mL), while procollagen type 1 N-terminal propeptide and whole parathyroid hormone showed significant decreases. Corrected tCa increase showed a non-linear positive association with percent changes of TRACP-5b (%ΔTRACP-5b) even after adjustment for confounders. In addition, %ΔTRACP-5b was not associated with catecholamines, but with post-exercise AVP levels after adjustment for pre-exercise TRACP-5b. Symptoms of nausea or vomiting (observed in 20%) were positively associated with corrected post-exercise iCa after adjustment for post-exercise blood pH. CONCLUSION: AVP elevation may explain bone resorption and the following hypercalcemia in the setting of heavy exercise.

Effects of raloxifene and alendronate on non-enzymatic collagen cross-links and bone strength in ovariectomized rabbits in sequential treatments after daily human parathyroid hormone (1-34) administration.

This study investigated the effects of raloxifene and alendronate to follow parathyroid hormone (PTH) on bone collagen and biomechanical properties in ovariectomized rabbits. Sequential treatments of raloxifene and alendronate after hPTH(1-34) treatment improved biomechanical properties with and without bone collagen improvement, respectively. INTRODUCTION: The standard sequential treatment to follow human parathyroid hormone (hPTH) (1-34) therapy for osteoporosis has yet to be determined. The objective of this study was to compare the effects of raloxifene and alendronate treatments to follow daily hPTH(1-34) treatment on non-enzymatic collagen cross-links, bone mass, and bone strength in ovariectomized (OVX) rabbits. METHODS: From 3 months after ovariectomy, seven month-old female New Zealand white rabbits were given either vehicle or hPTH(1-34) (8 µg/kg/day), once daily for 5 months. After hPTH(1-34) treatment, the hPTH(1-34)-treated animals were divided into two groups, and given raloxifene (10 mg/kg, daily) orally or alendronate (100 µg/kg, twice weekly) subcutaneously for 5 months. We evaluated bone mineral density (BMD), bone structural parameters, advanced glycation end product (AGE) content in collagen, and bone mechanical parameters including intrinsic parameters in the femur. RESULTS: Raloxifene (hPTH/RLX) and alendronate (hPTH/ALN) to follow hPTH(1-34) increased cortical thickness, maximum load, and maximum stress and decreased endocortical surface in the diaphysis, in addition to increasing total BMD in the distal metaphysis. Decreased trabecular AGE, pentosidine, and homocysteine contents and increased toughness and breaking energy were noted with hPTH/RLX treatment only. With hPTH/ALN treatment, no effects on non-enzymatic collagen cross-link AGEs were noted although increases in stiffness and elastic modulus were observed. CONCLUSION: These results suggest that sequential treatments with hPTH(1-34) and antiresorptive drugs (raloxifene and alendronate) have a beneficial effect on bone mass and biomechanical properties in OVX rabbits.

Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.

There are currently no effective therapies for progressive fibrotic diseases. Recent evidence has implicated overproduction of transforming growth factor-beta1 (TGF-beta1) as a major cause of tissue fibrosis. Furthermore, this evidence implies that inhibitors of TGF-beta1 may be clinically useful as antifibrotic agents. The proteoglycan decorin is a known inhibitor of TGF-beta1. In a rat model of glomerulonephritis we have shown that fibrosis is mediated by TGF-beta1. We report here that transfer of decorin cDNA into rat skeletal muscle increases the amount of decorin messenger RNA and protein present in skeletal muscle and decorin present in kidney, where it has a marked therapeutic effect on fibrosis induced by glomerulonephritis. Transfected glomerulonephritic rats showed a significant reduction in levels of glomerular TGF-beta1 mRNA and TGF-beta1 protein, extracellular matrix accumulation and proteinuria. These results demonstrate the potential of gene therapy as a novel treatment for fibrotic diseases caused by TGF-beta1.

Bcl-2 protects tubular epithelial cells from ischemia/reperfusion injury by dual mechanisms.

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.

Superagonistic CD28 antibody induces donor-specific tolerance in rat renal allografts.

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG-treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well-preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long-surviving recipients showed donor-specific tolerance, accepting secondary (donor-matched) Wistar cardiac allografts, but acutely rejecting third-party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA-treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor-specific tolerance. In conclusion, CD28 SA treatment successfully induces donor-specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.

Treatment adherence in renal transplant recipients: a questionnaire survey on immunosuppressants.

INTRODUCTION: To achieve a high graft survival rate, patient adherence to immunosuppressive therapy is critical. It is extremely difficult to establish the actual adherence status of transplant recipients; only a few surveys on the issue have been performed in Japan. METHODS: We conducted a questionnaire survey mainly on treatment adherence to calcineurin inhibitors among renal transplant recipients. RESULTS: The survey demonstrated some degree of nonadherence in a relatively high percentage of the patients. The adherence rate was significantly lower for the evening than the morning dose (McNemar test, P < .001). It significantly decreased with time following transplantation for both the morning and the evening doses (logistic regression analysis, P = .025 and <.001, respectively). CONCLUSIONS: Immunosuppressive treatment places a substantial burden on patients, some of whom cannot continue regular treatment at specified time points due to daily life restrictions after they have returned to work.

Glomerulosclerosis induced by in vivo transfection of transforming growth factor-beta or platelet-derived growth factor gene into the rat kidney.

Glomerulosclerosis, a final common lesion of various glomerular diseases, is characterized by mesangial cell proliferation and extracellular matrix (ECM) expansion. TGF-beta and PDGF are known to play a critical role in the regulation of ECM metabolism and mesenchymal cell proliferation, respectively. However, there is little evidence to demonstrate the direct role of each of these growth factors in the pathogenesis of glomerulosclerosis. Using an in vivo transfection technique, we could realize the selective overexpression of single growth factor in the kidney. The introduction of either TGF-beta or PDGF-B gene alone into the kidney induced glomerulosclerosis, although the patterns of action of these growth factors were different; TGF-beta affected ECM accumulation rather than cell proliferation and PDGF affected the latter rather than the former.

Diffusion Tensor Imaging MRI With Spin-Echo Sequence and Long-Duration Measurement for Evaluation of Renal Fibrosis in a Rat Fibrosis Model.

BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

Association between neuroleptic drug-induced extrapyramidal symptoms and dopamine D2-receptor polymorphisms in Japanese schizophrenic patients.

The aim of the present study is to examine the relationship between dopamine D2-receptor gene (DRD2) polymorphisms (Taq1A, Taq1B, -141C Ins/Del) and the risk of extrapyramidal adverse effects (EPS), assessed according to the Drug-Induced Extra-Pyramidal Symptoms Scale (DIEPSS), or the maintenance dose of antipsychotics in schizophrenic patients. The DIEPSS score was significantly higher in patients bearing the -141C Del allele than in those without it. Taq1A and Taq1B restriction fragment length polymorphisms (RFLPs) did not significantly affect the DIEPSS score. On the other hand, maintenance doses of neuroleptics and antiparkinsonian drugs were significantly higher in patients with the B1 allele of Taq1B RFLP than in those without it, while the Taq1A RFLP and -141C Ins/Del polymorphisms were not significantly related to the maintenance doses. In conclusion, the risk of EPS may be increased in patients with the -141C Del allele of the DRD2 gene. In these patients, antipsychotics should be administered with caution.

Noninvasive measurement of cerebral blood flow with (99m)Tc-hexamethylpropyleneamine oxime single-photon emission computed tomography and 1-point venous blood sampling.

BACKGROUND AND PURPOSE: The arterial and venous blood concentration of technetium 99m-labeled hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) reaches an equilibration more rapidly than other CBF tracers. We hypothesized that (99m)Tc radioactivity of a venous sample at equilibrium, which is similar to that of an arterial sample, would allow estimation of the integrated input function for the clinical measurement of CBF by use of single-photon emission CT. METHODS: In 53 patients with stable cerebrovascular disease, the radioactivity of a venous sample 5 minutes after injection of (99m)Tc-HMPAO was correlated with 5-minute arterial blood radioactivity and the first 5 minutes of the integrated arterial curves of the lipophilic tracer. The measured CBF values were compared with those of xenon 133. RESULTS: The radioactivity of 5-minute venous blood was almost equivalent to that of 5-minute arterial blood (r(2) = 0.987; y = 0.993x + 1.63; P : <0.0001). The correlation between the venous blood radioactivity and the integrated arterial lipophilic fraction was excellent (r(2) = 0.935, P : <0.0001). A strong correlation was obtained between (99m)Tc-HMPAO and (133)Xe CBF values (r(2) = 0.825, P : <0.0001). CBF values were reproducible (coefficient of variation, 8.6%). CONCLUSIONS: This approach is fast, simple, and an alternative to continuous blood sampling in clinical quantitative (99m)Tc-HMPAO CBF studies.

Quantitation of rCBF by 99mTc-hexamethylpropyleneamine oxime single photon emission computed tomography combined with 133Xe CBF.

A simple, noninvasive method of measuring CBF that uses single photon emission computed tomography (SPECT) of 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) and whole-brain CBF obtained by 133Xe clearance technique was developed. SPECT data were normalized to the count density of HMPAO uptake in the whole brain and then converted to the absolute units of CBF by multiplying average CBF in the whole brain obtained by 133Xe. Mean CBF values in healthy volunteers (n = 12) were 49 +/- 7 and 30 +/- 5 ml 100 g-1 min-1 for gray matter and white matter, respectively, with a global flow value of 45 +/- 5 ml 100 g-1 min-1. The mean flow value was 19 +/- 7 ml 100 g-1 min-1 for the core of the infarct and 31 +/- 5 ml 100 g-1 min-1 for the contralateral region (n = 13). CBF values were reproducible for all brain regions. The method was convenient to use and suitable for the routine measurement of regional CBF in normal and pathologic states.

Effects of hematocrit variations on cerebral blood flow and oxygen transport in ischemic cerebrovascular disease.

The contribution of hematocrit (Ht) changes on cerebral blood flow (CBF) and brain oxygenation in ischemic cerebrovascular disease is still controversial. In the present study, effects of Ht variations of CBF and oxygen delivery were investigated in patients with ischemic cerebrovascular disease. CBF was measured by the Xe-133 intracarotid injection method in 27 patients, whose diagnoses included completed stroke, reversible ischemic neurological deficit, and transient ischemic attack. Ht values in the patients ranged from 31 to 53%. There was a significant inverse correlation between CBF and Ht in these Ht ranges. Oxygen delivery, i.e., the product of arterial oxygen content and CBF, increased with Ht elevation and reached the maximum level in the Ht range of 40-45% and then declined. The CBF-Ht and oxygen transport-Ht relations observed in our study were similar to those in the glass-tube model studies by other workers rather than to those in intact animal experiments. From these results, it is conceivable that in ischemic cerebrovascular disease, the vasomotor adjustment was impaired in such a manner that the relations among Ht, CBF, and oxygen delivery were different from those in healthy subjects. Further, an "optimal hematocrit" for brain oxygenation was also discussed.

Free radical generation during brief period of cerebral ischemia may trigger delayed neuronal death.

We investigated the pathogenic role of free radical formation in ischemic neuronal death using radical scavenger, superoxide dismutase. Cerebral ischemia was produced in the gerbil by bilateral common carotid occlusion for 5 min, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. The effects of free superoxide dismutase and a derivatized superoxide dismutase, pyran copolymer conjugated superoxide dismutase, on early ischemic damages, detected sensitively by the immunohistochemical reaction for microtubule associated protein 2, and a subsequent delayed neuronal death after restoration of blood flow were investigated. Preischemic treatment by pyran conjugated superoxide dismutase showed clear protective effects against both the neuronal damages detected by immunohistochemistry after 5 min ischemia and the delayed neuronal necrosis after one week of recovery, although no clear beneficial effects were observed when this drug was administered just before the recirculation or free superoxide dismutase was used. These results strongly suggest that free radical generation during brief period of ischemia plays a pivotal role in triggering the ischemic neuronal damages causing delayed neuronal death at the selectively vulnerable areas of the brain.

Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage--immunohistochemical investigation of dendritic damage.

We investigated the neuronal distribution of microtubule-associated protein 2 in gerbil brain and monitored the progression of ischemic damage immunohistochemically by using this protein as a dendritic marker. The reaction for microtubule-associated protein 2 in normal gerbil brain clearly visualized neuronal soma and dendrites but other structures such as axonal bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, we could detect the ischemic lesions as early as 3 min after right common carotid occlusion at the subiculum-CA1 region of the ipsilateral hippocampus as faint loss of the reaction in the dendrites. After ischemia for 30 min, the ischemic lesions were clearly detected as loss of the reaction in the nerve cell bodies, dendrites and the neuropil in the hippocampus, cerebral cortex, thalamus and the caudoputamen. Although the mechanism for prompt disappearance of the immunohistochemical reaction for microtubule-associated protein 2 is not clear, the present investigation suggests that dendrites in the vulnerable regions may be quite susceptible to ischemic stress and that the immunohistochemical procedure for microtubule-associated protein 2 may be very useful for demonstration of dendritic damage in various pathophysiological states of the central nervous system.

Cerebral blood flow imaging with technetium-99m-HMPAO SPECT in a patient with chronic subdural hematoma: relationship with neuropsychological test.

We report the relationship between cerebral blood flow (CBF) and neuropsychologic tests in a patient with a chronic subdural hematoma suffering from severe dementia and left hemiparesis. Regional CBF was quantified using 99mTc-HMPAO SPECT and 133Xe-CBF. CBF-SPECT could detect the hematoma which was isodense by CT scan and the neuropsychological test improved remarkably with the increase in CBF after surgery. We conclude that if there is a strong clinical suspicion of subdural hematoma and CT scan is not diagnostic then CBF-SPECT may be valuable in localizing the hematoma and monitoring the effect of operation.

Cerebral blood flow and magnetic resonance imaging in locked-in syndrome.

The cerebral blood flow (CBF) of a patient suffering from locked-in syndrome (LiS) was examined before and after the onset using 99mTc-hexamethylpropyleneamine oxime single-photon emission computed tomography (SPECT) and the intravenous 133Xe injection method. The mean CBF during the locked-in state was 32.2 ml/100 g/min, a 42% reduction from the asymptomatic stage. SPECT showed profound reductions of perfusion in the bilateral cerebral cortices, subcortical regions and in the cerebellum, with a less marked reduction in the frontal cortices. On Day 49, the patient showed some minimal voluntary return with a moderate increase in mean CBF of 40.2 ml/100 g/min. The relative CBF values in the cerebral cortices and subcortical regions were restored, but the bilateral cerebellar hypoperfusion remained unchanged. SPECT and CBF are useful for a better characterization of the brain pathophysiology in LiS.

Cerebral perfusion imaging with albumin microspheres tagged with Tc-99m and In-111 in cases with internal carotid occlusion.

Cerebral perfusion imaging with dual-tracer (Tc-99m and In-111) human albumin microspheres (HAM scintigraphy) was performed in 15 cases with unilateral occlusion of the internal carotid artery, for the diagnosis and evaluation of collateral circulation patterns. After injection of Tc-99m microspheres into one common carotid artery and In-111 HAMs into the other, two perfusion images, one for each carotid artery, were clearly differentiated by appropriate pulse-height discrimination. With this method, diagnosis of internal carotid artery occlusion was definitely made in eight patients, suspected in six, and missed in one. The collateral perfusion areas from the contralateral ICA and ipsilateral external carotid artery were well demonstrated by this method, and the scintigraphic results agreed well with the angiographic findings in all cases. Dual-tracer HAM scintigraphy is capable of adding information about collaterals at the capillary level to the anatomic information obtained by angiography.

Platelet accumulation in carotid atherosclerotic lesions: semiquantitative analysis with indium-111 platelets and technetium-99m human serum albumin.

To evaluate platelet accumulation in carotid atherosclerotic lesions semiquantitatively, a dual-tracer technique was applied, using In-111 platelets and Tc-99m human serum albumin. With this approach, we investigated the ratio of radioactivity in In-111 platelets deposited on the vascular wall to those circulating in the blood pool, platelet accumulation index ( PAI ). This study included 12 normal subjects and 25 patients with ischemic cerebrovascular disease (CVD). Angiographic abnormalities were observed at 34 of 50 carotid bifurcations in the CVD patients. The mean PAI value was significantly higher at the carotid bifurcations with angiographic abnormality than at the normal ones (p less than 0.001). Furthermore, elevations of mean PAI were prominent at the lesions with severe stenosis or ulceration. The degree of platelet accumulation was well demonstrated by this technique, which can also yield information on thrombogenicity and efficiency of antiplatelet therapy in carotid atherosclerotic disease.

A new apparatus for brain imaging: four-head rotating gamma camera single-photon emission computed tomograph.

The development of a new rotating gamma camera SPECT device for imaging the brain was undertaken with the objective of achieving the highest full volume imaging spatial and temporal resolution performance. For this purpose, four rectangular gamma camera detectors were arranged as close to the head as possible, and united in a block to insure detector head registration and alignment as well as to enable rotation stability at high speeds. Phantom and clinical studies performed demonstrated 42 sequential, 4-mm thick transaxial images acquired in one scan and with sufficient volume to permit the entire cerebrum and cerebellum to be imaged with high sensitivity. The central field of view reconstructed spatial resolution measured 7.0 mm full width of half maximum utilizing the high-resolution collimator, and tomographic images of arbitrary planes including sagittal and coronal demonstrated equally high resolution. The high sensitivity and high speed rotational acquisition capability of the device permits dynamic SPECT studies to be carried out in the analysis of rapidly varying radiotracer concentrations.

Iodine-131-labeled fibronectin: potential agent for imaging atherosclerotic lesion and thrombus.

Fibronectin is known to interact with fibrin, collagen, etc. We have labeled fibronectin with 131I, and measured its accumulation in the deendothelialized lesion in the rabbit aorta to evaluate it as a candidate for imaging atherosclerotic lesions and thrombi. Accumulation of [131I] fibronectin in the deendothelialized lesion was apparent at 48 hr, and increased at 72 hr after injection of the agent. Our results indicate that radiolabeled fibronectin may be a useful tracer for imaging early atherosclerotic lesion and thrombus.