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Background Although hydrochlorothiazide ( HCTZ ) is a well‐established first‐line antihypertensive in the United States, <50% of HCTZ treated patients achieve blood pressure ( BP ) control. Thus, identifying biomarkers that could predict the BP response to HCTZ is critically important. In this study, we utilized metabolomics, genomics, and lipidomics to identify novel pathways and biomarkers associated with HCTZ BP response. Methods and Results First, we conducted a pathway analysis for 13 metabolites we recently identified to be significantly associated with HCTZ BP response. From this analysis, we found the sphingolipid metabolic pathway as the most significant pathway ( P =5.8E‐05). Testing 78 variants, within 14 genes involved in the sphingolipid metabolic canonical pathway, with the BP response to HCTZ identified variant rs6078905, within the SPTLC 3 gene, as a novel biomarker significantly associated with the BP response to HCTZ in whites (n=228). We found that rs6078905 C‐allele carriers had a better BP response to HCTZ versus noncarriers (∆ SBP /∆ DBP : −11.4/−6.9 versus −6.8/−3.5 mm Hg; ∆ SBP P =6.7E‐04; ∆ DBP P =4.8E‐04). Additionally, in blacks (n=148), we found genetic signals in the SPTLC 3 genomic region significantly associated with the BP response to HCTZ ( P <0.05). Last, we observed that rs6078905 significantly affects the baseline level of 4 sphingomyelins (N24:2, N24:3, N16:1, and N22:1; false discovery rate <0.05), from which N24:2 sphingomyelin has a significant correlation with both HCTZ DBP ‐response ( r =−0.42; P =7E‐03) and SBP ‐response ( r =−0.36; P =2E‐02). Conclusions This study provides insight into potential pharmacometabolomic and genetic mechanisms underlying HCTZ BP response and suggests that SPTLC 3 is a potential determinant of the BP response to HCTZ . Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 00246519.

Unilateral sciatic neurectomy (USN) resulted in cortical osteopenia in tibiae from the sciatic nerve-sectioned limb of growing rats. The bone deficit resulted from decreased periosteal addition; there were no changes in the indexes of bone resorption. The periosteal bone formation rate was reduced in the nerve-sectioned limb within 7 days of sciatic neurectomy, and this decrease persisted for at least 56 days. Steady-state mRNA levels for bone proteins were determined in periosteum isolated from tibiae and femurs 7 and 14 days after sciatic nerve section. Nerve section resulted in decreased levels of mRNA for osteocalcin, alkaline phosphatase, and possibly the prepro-alpha (I)-subunit of type I collagen (collagen). The effects were more pronounced in tibiae than femurs, corresponding to the greater degree of immobility induced by USN in the former bone. The results demonstrate that decreased bone formation precedes establishment of disuse cortical osteopenia in growing rats with no evidence for a change in bone resorption. Furthermore, the decreased bone formation is associated with, and may be due to, reduced mRNA levels for matrix proteins and other important bone proteins.

Renal plasma flow, glomerular filtration rate, and cardiac output are traditionally indexed for body surface area by expressing these traits as per-surface-area ratios. Indexing is intended to remove interindividual variation attributable to differences in body size. Regression is an alternative method commonly used to adjust other biological traits for the effects of a covariate, such as body surface area. The purpose of this study was to compare the indexing and regression methods of adjusting renal plasma flow, glomerular filtration rate, and cardiac output for interindividual differences in body surface area. We estimated renal plasma flow by the clearance of p-aminohippurate, glomerular filtration rate by clearance of inulin, and cardiac output by thoracic electrical impedance in a sample of 78 unrelated females and 78 unrelated males (ages 20-49.9 yr) from the general population of Rochester, MN. The indexing method created negative dependencies of renal plasma flow and cardiac output on body surface area and failed to eliminate the positive dependency of glomerular filtration rate on body surface area. Moreover, indexing obscured differences in mean renal plasma flow between females and males and created differences in mean cardiac output between the genders. In contrast, the regression method consistently eliminated dependencies of each trait on body surface area and did not lead to inappropriate inferences about mean differences in these traits between females and males. We conclude that the indexing method of adjusting renal plasma flow, glomerular filtration rate, and cardiac output for interindividual differences in body surface area should be abandoned and replaced by use of the regression method.

The ability of bone to respond to increased loading as a function of age was tested by use of three-point bending and histomorphometry. The hindlimbs of male Fischer 344 rats of three age groups (young = 4 mo, adult = 12 mo, and old = 22 mo; n = 10 per age group) were progressively overloaded by training the rats to depress a lever high on the side of a cage while wearing a weighted backpack. This squatlike movement required full extension of the hindlimbs. Exercised (Exer) rats performed 50 repetitions three times per week for 9 wk. Pack weight was gradually increased to 65% of body weight. Controls ( n = 10 per age group) performed the same exercise without additional weight. Neither the mechanical properties of the femur nor histomorphometry in the proximal tibia was significantly affected in young or adult rats. However, old Exer rats were found to have significantly smaller medullary areas and a decreased trabecular spacing than their age-matched controls. These results suggest a greater sensitivity to increased loading in aged rats.

This review of the peer-reviewed literature focuses on the effects of spaceflight on bone. Studies performed in humans and laboratory animals have revealed abnormalities in bone and mineral metabolism that suggest that long-duration spaceflight will have detrimental effects on the skeleton. However, because of large gaps in our knowledge, it is not presently possible to estimate the magnitude of the health risk, individual variations in risk, effective countermeasures, or mechanism(s) of action. Specific recommendations are made for future research to ascertain risk and develop appropriate countermeasures.

Bone loss occurs as a consequence of exposure to microgravity. Using the hindlimb-unloaded rat to model spaceflight, this study had as its purpose to determine whether skeletal unloading and cephalic fluid shifts alter bone blood flow. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal structures of the forelimbs and head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7 days, and 28 days of hindlimb unloading (HU). Femoral and tibial perfusion were reduced with 10 min of HU, and blood flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of femora (−11%, P < 0.05) and tibiae (−6%, P < 0.1) was lowered with 28 days of HU. In contrast, blood flow to the skull, mandible, clavicle, and humerus was increased with 10 min HU but returned to control levels with 7 days HU. Mandibular (+10%, P < 0.05), clavicular (+18%, P < 0.05), and humeral (+8%, P < 0.1) mass was increased with chronic HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.

Unilateral sciatic neurectomy (USN) resulted in cortical osteopenia in tibiae from the sciatic nerve-sectioned limb of growing rats. The bone deficit resulted from decreased periosteal addition; there were no changes in the indexes of bone resorption. The periosteal bone formation rate was reduced in the nerve-sectioned limb within 7 days of sciatic neurectomy, and this decrease persisted for at least 56 days. Steady-state mRNA levels for bone proteins were determined in periosteum isolated from tibiae and femurs 7 and 14 days after sciatic nerve section. Nerve section resulted in decreased levels of mRNA for osteocalcin, alkaline phosphatase, and possibly the prepro-alpha (I)-subunit of type I collagen (collagen). The effects were more pronounced in tibiae than femurs, corresponding to the greater degree of immobility induced by USN in the former bone. The results demonstrate that decreased bone formation precedes establishment of disuse cortical osteopenia in growing rats with no evidence for a change in bone resorption. Furthermore, the decreased bone formation is associated with, and may be due to, reduced mRNA levels for matrix proteins and other important bone proteins.

In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-α(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

Westerlind, Kim C., James D. Fluckey, Scott E. Gordon, William J. Kraemer, Peter A. Farrell, and Russell T. Turner.Effect of resistance exercise training on cortical and cancellous bone in mature male rats. J. Appl. Physiol. 84(2): 459–464, 1998.—The effect of resistance training on tibial cancellous and cortical bone was evaluated in rats by using static histomorphometry and Northern analysis. Five-month-old male Sprague-Dawley rats were randomly assigned to exercise (Ex; n = 8) or control (Con; n = 4) groups. Animals were operantly conditioned to press two levers, facilitating full extension and flexion of the hindlimbs (“squats”), while wearing an unweighted vest. After an 8-wk familiarization period, Ex animals performed 3 sessions/wk for 17–19 sessions with progressively increased amounts of weight applied to the vest. Con rats completed the same exercise protocol without applied resistance. No difference in cross-sectional, medullary, or cortical bone area was observed between Ex and Con rats in the tibial diaphysis. In contrast, the cancellous bone area in the proximal tibial metaphysis was significantly larger in trained rats. Trabecular number, trabecular thickness, and the percentage of cancellous bone covered by osteoid were significantly greater in the Ex animals compared with Con animals. In addition, steady-state mRNA levels for osteocalcin for the Ex group were 456% those expressed in the Con group. The data demonstrate that resistance training increases cancellous bone area in sexually mature male rats and suggest that it does so, in part, by stimulating bone formation.

The decrease in cancellous bone formation after estrogen treatment is generally thought to be coupled with a prior decrease in bone resorption. To test the possibility that estrogen has rapid tissue-specific actions on bone metabolism, we determined the time course (1–32 h) effects of diethylstilbestrol on steady-state mRNA levels for immediate-response genes, extracellular matrix proteins, and signaling peptides in the proximal tibial metaphysis and uterus by using Northern blot and RNase protection assays. The regulation of signaling peptides by estrogen, although tissue specific, followed a similar time course in bone and uterus. The observed rapid decreases in expression of insulin-like growth factor I, a growth factor associated with bone formation; decreases in mRNA levels for bone matrix proteins; evidence for reduced bone matrix synthesis; failure to detect rapid increases in mRNA levels for signaling peptides implicated in mediating the inhibitory effects of estrogen on bone resorption (interleukin-1 and -6) as well as other cytokines that can increase bone resorption; and the comparatively long duration of the bone remodeling cycle in rats indicate that estrogen can decrease bone formation by a mechanism that does not require a prior reduction in bone resorption.