INTRODUCTION

Parathyroid glands (PTG) have a major role in the regulationand maintenance of calcium (Ca2+) and phosphorus (Pi)homeostasis. Small decreases in serum Ca2+ and increases inserum Pi concentrations stimulate PTG to synthesize and secreteparathyroid hormone (PTH). In bone and kidneys PTH binds toits receptors in order to maintain normal levels of Ca2+ and Pi (1).PTH is one of numerous factors responsible for regulation of theabundance of sodium phosphate cotransporter type 2a (NaPi 2a)besides 1.25(OH)2 vitamin D3, glucocorticoids and fibroblastgrowth factor 23 (FGF23) actions, or dietary phosphate intake(2). NaPi 2a is located in the epithelial cells of kidney proximaltubules, on the apical brush border membrane (BBM), and it isresponsible for reabsorbing most of the Pi from primary urine.

The andropause, the culminating phase of ageing in men, isassociated with a decline in serum testosterone level and anincreased incidence of cardiovascular issues, benign and malignantprostate diseases, and osteoporosis (3). During ageing PTG volumeand serum PTH level increase in both rats and humans, which bothcontribute to bone loss and osteoporosis (4, 5). PTH affirmed boneloss, together with its opposite effect on NaPi 2a expression,induces phosphaturia and disturbance of mineral homeostasis (6,2). Vanderschueren et al. (7) suggested that orchidectomized (Orx)

male rats, as an animal model of the osteoporosis, very well reflectssome skeletal changes caused by androgen deficiency inhypogonadal men. Both estrogens (especially in aromatasedeficient subjects) and androgens have very important roles in bonemetabolism (8-10), but using steroid hormones to treatosteoporosis has undesirable side effects, such as an increased riskof cardiovascular disease and prostate cancer (11). Taking intoaccount the potentially harmful aspects of hormone replacementtherapy, increasing emphasis is being placed on alternative, plant-originated therapeutics for osteoporosis.

Genistein is a soybean isoflavone, recognized as perspectivein osteoporosis treatment (12), and has structural similarityto17β-estradiol. Kuiper et al. (13) showed that genistein binds toestrogen receptors (ERs) α and β, with higher binding affinityfor ERβ. According to Carrillo-Lopes et al. (14), there are noERs in PTG, but estrogen regulation of PTG function could beindirect and may require FGF23, a PTH suppressing andphosphaturic hormone secreted by osteoblasts and osteocytes(15-17). Besides its estrogenic effect, the tyrosine kinaseinhibiting role of genistein as well as its’ effect on theextracellular-signal-regulated kinase (ERK1/2) pathways arewell known (18-20). These findings affirm the concept ofgenistein potential effect on the NaPi 2a cotransporterexpression (21).

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2013, 64, 3, 361-368www.jpp.krakow.pl

J. PANTELIC*1, V. AJDZANOVIC1, I. MEDIGOVIC1, M. MOJIC2, S. TRIFUNOVIC1, V. MILOSEVIC1, B. FILIPOVIC1

GENISTEIN AFFECTS PARATHYROID GLAND AND NaPi 2a COTRANSPORTERIN AN ANIMAL MODEL OF THE ANDROPAUSE

1Department of Cytology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia;2Department of Immunology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia

This study aimed to examine the effects of genistein on the structural and functional changes in parathyroid glands(PTG) and sodium phosphate cotransporter 2a (NaPi 2a) in orchidectomized rats. Sixteen-month-old Wistar rats weredivided into sham-operated (SO), orchidectomized (Orx) and genistein-treated orchidectomized (Orx+G) groups.Genistein (30 mg/kg/day) was administered subcutaneously for 3 weeks, while the controls received vehicle alone. PTGwas analyzed histomorphometrically, while the expressions of NaPi 2a mRNA/protein levels from kidneys weredetermined by real time PCR and Western blots. Serum and urine parameters were determined biochemically. The PTGvolume in Orx rats was increased by 30% (p<0.05), compared to the SO group. Orx+G treatment increased the PTGvolume by 35% and 75% (p<0.05) respectively, comparing to Orx and SO animals. Orchidectomy led to increment ofserum PTH by 27% (p<0.05) compared to the SO group, Orx+G decreased it by 18% (p<0.05) comparing to Orxanimals. NaPi 2a expression in Orx animals was reduced in regards to its abundance in SO animals, although it wasincreased in Orx+G group compared to the Orx. Phosphorus urine content of Orx animals was raised by 12% (p<0.05)compared to that for the SO group, while Orx+G induced a 17% reduction (p<0.05) in regards to Orx animals. Our studyshows that Orx increases PTG volume and serum PTH level, while protein expression of NaPi 2a is reduced. Applicationof genistein attenuates the orchidectomy-induced changes in serum PTH level, stimulates the expression of NaPi 2a andreduces urinary Pi excretion, implying potential beneficial effects on andropausal symptoms.

K e y w o r d s : andropause, genistein, parathyroid gland, NaPi 2a, orchidectomy, serum calcium, osteoblasts

In light of the above mentioned we assume that genisteincould modulate the activity of PTG and NaPi 2a and thus makean impact on homeostasis of Ca2+ and Pi. The aim of this studywas to detect and analyze the structure and function of PTG andNaPi 2a cotransporter, key regulators in Ca2+ and Pi homeostasisin middle-aged Orx rats, as an animal model of the andropause,before and after treatment with genistein.

MATERIAL AND METHODS

Animals and diets

The experiment was performed on middle-aged (16-month-old at the time of sacrificing) Wistar male rats, bred in theInstitute for Biological Research, Belgrade, Serbia, underconstant laboratory conditions (22±2°C, 12/12h light/dark cycle).Animals were fed a soy-free diet prepared in cooperation with theDepartment of Nutrition, School of Veterinary Medicine, andINSHRA PKB (Belgrade, Serbia), according to Picherit et al.(22), with corn oil as the fat source. The diet composition ispresented in Table 1. Casein and crystalline cellulose werepurchased from Alfa Aesar, Johnson Mattehey Gmbh & Co.KG(Karlsruhe, Germany). Carbohydrate, corn oil, vitamin/mineralmix, calcium carbonate and dibasic calcium phosphate were fromINSHRA PKB, Belgrade, Serbia. DL-methionine was obtainedfrom Sigma Chemical Company (St. Louise, Missouri, USA).Food and water were available ad libitum.

Table 1. The soy-free diet composition.

Ingredient per 100 g of dietCasein 23.5 gCornstarch 45 gSucrose 20 gCorn oil 2 gFiber (crystalline cellulose) 3.7 gVitamin/mineral mix 1.5 gCalcium phosphate dibasic 1.8 gCalcium carbonate 1.0 gDL-methionine 1.5 g

Experimental design

Initially, animals (n=24) were divided into two groups.Under ketamine anesthesia (ketamine hydrochloride; RichterPharma, Wels, Austria; 15 mg/kg b.w.) the first group of animals(n=8) was sham-operated (SO), while the second group (n=16)was bilaterally orchidectomized (Orx). During the 2 weeks ofrecovery period animals were fed with soy-free diet that lasteduntil the end of the experiment (5 weeks in total). Orx animalswere divided into two groups of eight animals each. One groupof Orx rats was given genistein subcutaneously (s.c.) (Orx+G;30mg/kg b.w.) every day, for 3 weeks. Genistein (LCLaboratories, MA, USA) was dissolved in a mixture of sterileolive oil and absolute ethanol (ratio 9:1). The dose of genisteinwas selected based on our previous studies as the effective onein the treatment of some andropausal symptoms (23-25).

The other Orx group and the SO group were treated with themixture of sterile olive oil and absolute ethanol, following thesame regime and they served as controls. Before sacrifice, urinesamples were collected for Ca2+ and Pi analyses. The rats weredecapitated 24 h after the last injection. Blood samples were thencollected from the trunk and the separated serum samples werestored at –80°C until analyzed. The experimental protocols wereapproved by the Local Animal Care Committee of the Institute

for Biological Research (Belgrade, Serbia) in conformity withthe recommendations provided in the European Convention forthe Protection of Vertebrate Animals used for Experimental andOther Scientific Purposes (ETS no. 123. Appendix A).

Histological and electron microscopy analysis

Thyroid-parathyroid tissue was excised from six animals pergroup and fixed in Bouin’s solution for 48 h. After dehydrationthrough a series of alcohols of increasing concentration, the tissuewas embedded in paraplast. The parathyroid glands were cutserially, using a rotational microtome (Leica, Germany), at 3 µmthickness. The sections were stained with hematoxylin-eosin andmounted with DPX (Sigma-Aldrich, Co., USA).

Thyroid-parathyroid glands were removed from two animalsper group after decapitation and immediately cut into slices.Tissue slices were immersed in 4% glutaraldehyde solution in0.1 M phosphate buffer (PB) (pH 7.4) for 24 h, and postfixedwith 1% OsO4 in the same buffer for 1 h. Samples weredehydrated in a graded ethanol series and embedded in Aralditeand Harderner resin. An LKB ultramicrotome III (type 8802A;Sweden) with a Diatome ultra 45° diamond knife (Diatome,Switzerland) was used for cutting ultrathin sections. Grids withthin sections were stained with uranyl acetate and lead citrate,and examined under a MORGAGNI 268 (FEI Company, USA)transmission electron microscope.

Immunofluorescent studies

Kidneys were fixed in formalin solution at room temperaturefor 48 h, embedded in paraplast, and sectioned at 3 µm. Forimmunofluorescence staining, sections were deparafinised anddehydrated, while antigen retrieval was performed in 0.1 Mcitrate buffer solution pH 6.0. Sections were washed in PBS andpretreated with blocking normal donkey serum (Dako,Denmark) diluted in PBS (1:10). After blocking, they wereincubated overnight at room temperature with rabbit anti-ratNaPi 2a antibody (1:100; kindly donated by Dr Jurg Biber,Institute of Physiology, University of Zurich, Zurich,Switzerland). After rinsing in PBS, the sections were covered for2 h at room temperature with secondary antibody Alexa Fluor555 donkey anti-rabbit IgG (1:200; Molecular Probes, Inc.,USA). Finally, they were rinsed five times in PBS, and incubatedwith DAPI (1:1000, Molecular Probes, Inc., USA) for 5 minutesand rinsed six times in PBS. Sections were cover slipped usingMowiol 4-88 (Sigma-Aldrich Co., USA). Twenty transverseimmunofluorescently stained kidney sections were analyzed peranimal, using Carl Zeiss AxioVision microscope (Zeiss,Germany), containing an ApoTome software module forgenerating optical sections through fluorescence samples.

Real time PCR

Total RNA was isolated from rat kidney cortex (5 animalsper group were used) using TRIzol Reagent (Life Technologies,USA) according to the manufacturer’s instructions. RNA wasquantified by spectrophotometry, and cDNA was synthesizedusing reagents from cDNA Reverse Transcription kit (AppliedBiosystems, USA). PCR amplification of cDNA was performedin a real-time PCR machine ABI Prism 7000 (AppliedBiosystems) with SYBRGreen PCR master mix (AppliedBiosystems) as indicated: 2 minutes at 50°C for dUTPactivation, 10 minutes at 95°C for initial denaturation of cDNA,followed by 40 cycles, each consisting of 15 s of denaturation at95°C and 60 s at 60°C for primer annealing and chain extension.Primer pairs were the following: NaPi 2a, forward, 5’-GCCACTTCTTCTTCAACATC-3’; reverse, 5’-

362

CACACGAGGAGGTAGAGG-3’; cyclo A forward 5’-CAAAGTTCCAAAGACAGCAGAAAA-3’; reverse, 5’-CCACCCTGGCACATGAAT-3’. The expression level of eachgene was calculated using formula 2–(Cti–Cta), where Cti is thecycle threshold value of the gene of interest and Cta was thecycle threshold value of cyclophilin A. All of the data werecalculated from triplicate reactions. RNA data are presented asaverage relative levels versus cyclophilin A±S.D.

Western blot analysis

Immunoblot analyses were performed on isolated BBMvesicles from rat kidney cortex (6 animals per group were used)using Mg2+ precipitation technique as previously described (26).BBM proteins were solubilized in Laemmli sample buffer, andSDS-PAGE was performed on 12% polyacrylamide gels.Proteins were transferred electrophoretically to polyvinylidenedifluoride membranes at 5 mA/cm2 with a semidry blottingsystem (Fastblot B43; Bio-Rad, Goettingen, Germany). Themembranes were blocked with 5% BSA in PBS with 0.1%Tween 20 for overnight, followed by incubation with rabbit anti-rat NaPi 2a primary antibody (1:2000), and rabbit anti- rat βactin (Abcam, Cambridge, USA) overnight at 4°C. Afterwashing, blots were incubated with secondary antibody (1:000;ECL donkey anti-rabbit horse-radish peroxidase-linked; GEHealthcare, Chalfont St. Giles, Buckinghamshire, UK) for 1 h atroom temperature. Antibody binding was detected using achemiluminescence detection system (ECL; GE Healthcare).

Stereological measurements

The volume of PTG was estimated using Cavalieri’s principle(27) with a newCAST stereological software package (VIS-Visiopharm Integrator System, version 2.12.1.0; Visiopharm;Denmark). Every 30th section from each of the tissue blocks wasanalyzed. The PTG volume (Vptg) was calculated by the formula:

where a(p) is the area associated with each sampling point(10956.52 µm2); d

–is the mean distance between two consecutively

studied sections (90 µm); n is the number of sections studied for

each PTG; and ΣPi is the sum of points hitting a given target. Thepercentages of chief cells and interstitium (blood vessels andconnective tissue) were determined for every sampled section.

Biochemical analyses

Serum PTH concentration was measured in duplicate sampleswithout dilution, using a Rat Intact PTH ELISA Kit(Immunotopics, Inc., San Clemente, CA, USA), within a singleassay. The intra-assay coefficient of variation (CV) was 2.4%. Thelowest concentration of rat intact PTH measurable by this kit was1.6 pg/mL (assay sensitivity). Serum concentrations of Pi andCa2+, and urinary concentration of Pi were determined on a Hitachi912 analyzer (Roche Diagnostics GmbH, Mannheim, Germany).

Statistical analysis

STATISTICA® version 6.0 (StatSoft, Inc) was used for thestatistical analysis. All results were expressed as mean ±S.D.Differences between the groups were assessed by one-wayanalyses of variance (ANOVA) followed by Duncan’s multiplerange tests for post hoc comparisons between groups. Values ofp<0.05 were considered statistically significant.

RESULTS

Parathyroid glands volume

In sham-operated (SO) middle aged male rats PTG volumewas 0.14±0.01 mm3. The value was 30% (p<0.05) greater in Orxrats than in the SO group (Fig. 1A). After treatment withgenistein (Orx+G) the PTG volume was increased by 35% and75% (p<0.05) respectively, when compared with values for theOrx and SO animals (Fig. 1A). The volume density of the PTGchief cells was raised by 2% (p<0.05) in the Orx group ofanimals when compared with the SO group, while treatmentwith genistein (Orx+G) induced a 4% decrease of chief cellvolume (p<0.05), when compared to Orx animals (Fig. 1B).After genistein treatment the presence of interstitium in PTG(Orx+G) was increased by 20% (p<0.05), in comparison to theOrx group of animals (Fig. 1B).

363

Fig. 1. [A] - The volume of PTG in sham-operated (SO), orchidectomized (Orx) and orchidectomized rats treated with genistein(Orx+G) group. All values are presented as mean ±S.D.; ap<0.05 versus SO, bp<0.05 versus Orx. [B] - Relative representation of chiefcells and interstitium in sham-operated (SO), orchidectomized (Orx) and orchidectomized rats treated with genistein (Orx+G). Allvalues are presented as mean ±S.D.; ap<0.05 versus SO, bp<0.05 versus Orx.

Histological findings in the parathyroid glands

The parathyroid glands (PTG) are located laterally to thethyroid gland lobes. They typically possess an oval shape and aresurrounded with a connective tissue capsule. These glands arecomposed of one cell type, the chief cells, densely packed in cordsor clusters around and along capillaries, with spherical to oval orelongated nuclei. PTG in the sham-operated (SO) rats had anapparent connective tissue capsule, while the chief cells (Fig. 2A,white arrows) were separated with a delicate stroma of connectivetissue and blood vessels (Fig. 2A, black arrows). In comparisonwith this, PTG in the Orx group were larger, with numerous chief

cells (Fig. 2B, white arrows) and noticeable interstitium (Fig. 2B,black arrows). After genistein treatment (Orx+G) PTGs were evenlarger, with massive interstitium (black arrows), in relation to theglands in Orx animals (Fig. 2C).

Ultrastructural observations in the parathyroid glands

Ultrathin sections of PTG in the SO group showedcompactly arranged chief cells, with numerous interdigitationsof the cell membrane. Rough endoplasmatic reticulum (RER;white arrows) and the Golgi complex (black arrows) weremoderately developed. Mitochondria (white arrow head) were

364

Fig. 2. Sections of PTG from [A] sham-operated (SO), [B] orchidectomized (Orx) and [C] orchidectomized rats treated with genistein(Orx+G). Chief cells (white arrows) and interstitium (black arrows) are clearly visible. Hematoxylin-eosin staining, scale bar – 100 µm.

Fig. 3. Ultrathin sections of PTG chief cells from [A] sham-operated (SO), [B] orchidectomized (Orx) and [C] orchidectomized ratstreated with genistein (Orx+G). RER (white arrows), Golgi complex (black arrows) and mitochondria (white arrow head) are markedas the representative structures. Scale bar – 500 nm.

Fig. 4. Localization of NaPi 2a cotransporter in epithelial cells of proximal tubules. [A] In the sham-operated group of animals (SO)NaPi 2a was strongly labeled in the brush border membrane of proximal tubule epithelial cells, [B] NaPi 2a staining intensity in theapical domain of proximal tubule cells was decreased after orchidectomy (Orx). The signal was also detected in the subapical regionof the cells, [C] After genistein treatment (Orx+G) the intensity of NaPi 2a labeling was increased. Paraffin sections,immunofluorescence, scale bar – 10 µm.

dispersed throughout the cytoplasm, while the nuclei wereelongated and located near the apical domain of the cells (Fig.3A). Interdigitations of the plasma membrane in Orx animalswere more numerous than in the chief cells of SO animals. TheRER and Golgi complex were more developed than in the SOgroup, with abundant mitochondria and larger centrally locatednuclei (Fig. 3B). Ultrastructural micrographs of chief cells fromOrx animals treated with genistein (Orx+G) showed lessprominent plasma membrane interdigitations. The RER andGolgi complex were poorly represented with fewer and smallermitochondria, in comparison to the Orx animals (Fig. 3C).

Immunofluorescent appearance of NaPi 2a

Kidneys sections of all experimental groups wereimmunofluorescently stained with the specific antibody for

NaPi 2a. Kidney sections of SO rats NaPi 2a showed a strongsignal in the microvilli of the BBM in proximal tubule cells(Fig. 4A). After Orx, the intensity of the NaPi 2a signal inBBM was reduced, in comparison with SO animals (Fig. 4B).Besides in the apical membrane, NaPi 2a was also localized inthe subapical domain of epithelial cells of proximal tubules inOrx animals (Fig. 4B). Treatment with genistein (Orx+G)increased the abundance of NaPi 2a in the brush border of theproximal tubules, in comparison with the Orx group ofanimals (Fig. 4C).

NaPi 2a exspression levels in epithelial cells of proximaltubules

Analysis of NaPi 2a mRNA level reveled that orchidectomyslightly decreased NaPi 2a mRNA compared to SO group ofanimals, while genistein administration led to an increase inNaPi 2a mRNA expression level in comparison with Orxanimals (Fig. 5A).

365

Fig. 5. [A] Real-time PCR analyses for NaPi 2a mRNA in control (SO), orchidectomised (Orx) and orchidectomized rats treated withgenistein (Orx+G), data are presented as relative expression of mRNA. [B] The abundance of NaPi 2a in the BBM in control (SO),orchidectomized (Orx) and orchidectomized rats treated with genistein (Orx+G), determined by a Western blot analysis. Densitometricanalysis of data from a representative of three experiments was presented as fold increase relative to (against) β-actin and therespective control groups. All values are presented as mean ±S.D., ap<0.05 versus SO, bp<0.05 versus Orx.

Fig. 6. Serum concentration of PTH in sham-operated (SO),orchidectomized (Orx) and orchidectomized rats treated withgenistein (Orx+G). All values are presented as mean ±S.D.,ap<0.05 versus SO, bp<0.05 versus Orx.

Fig. 7. Serum Ca2+ and Pi levels, in sham-operated (SO),orchidectomized (Orx) and orchidectomized rats treated withgenistein (Orx+G). All values are presented as mean ±S.D.,ap<0.05 versus SO, bp<0.05 versus Orx.

Immunoblots of BBM preformed with NaPi 2a antibodyshowed decrease expression of NaPi 2a (p<0.05) cotransporterin Orx animals in comparison with SO control group (Fig. 5B).Treatment with genistein significantly elevated expression ofNaPi 2a (p<0.05) compared to Orx group (Fig. 5B).

Biochemical findings

Serum PTH concentration in SO rats was 62.5±5.68 ng/L.Orchidectomy induced a 27% increase of serum PTH (p<0.05),when compared to SO animals (Fig. 6). After treatment withgenistein (Orx+G) serum PTH concentration was 18% lower(p<0.05) than in the Orx group (Fig. 6). In SO animals serumCa2+ was 2.33±0.05mmol/L and serum Pi 2.04±0.07 mmol/L.After Orx, concentrations were decreased by 5% (Ca2+) and 10%(Pi) (p<0.05) respectively, in comparison with the SO group(Fig. 7). After genistein treatment (Orx+G) serum concentrationof Ca2+ and Pi were 7% and 12% (p<0.05) higher respectively,than for Orx animals (Fig. 7). In SO animals urine Pi

concentration was 35.15±1.85 mmol/L. Orchidectomy increasedthe Pi concentration in urine by 12% (p<0.05), in comparisonwith the SO control (Fig. 8). After genistein treatment (Orx+G)urinary Pi concentration was 17% lower (p<0.05), than for theOrx group (Fig. 8).

DISCUSSION

It has been established that PTG has a central role inregulating mineral and bone metabolism (28). Pertinent to this,the increased level of PTH during ageing (4), together with theage-related decline in serum testosterone, contribute to bone lossand osteoporosis. Hormone replacement therapy in elderlypeople may cause hyperphosphaturia (29) and increase the riskof cancer development (11), so finding alternative therapeuticsolutions in osteoporosis treatment is definitely needed.Accumulating evidence suggests that soy isoflavones mayrepresent a promising alternative remedy for aging symptoms inboth genders (30, 31), but their role in PTG regulation has notbeen previously studied. Therefore, we have used an animal

model of the andropause to explore the effects of the soyisoflavone, genistein, on the structure and function of PTG andNaPi 2a cotransporter in BBM of proximal tubules.

Under our experimental conditions, orchidectomyperformed in middle-aged rats with a view to emphasize theandropausal symptoms, induced a significant increase of PTGvolume, chief cells percentage and serum PTH concentration.Ultrastructural analysis revealed numerous interdigitations ofchief cells membranes, well developed RER and Golgi complex,and a large number of mitochondria in Orx animals whencompared to SO rats. These characteristics suggest increasedactivity of the PTH producing PTG chief cells and confirmearlier studies (32). Furthermore, Orx was previously found tocause hypocalcaemia in middle-aged male rats (33), presumablyby promoting PTH secretion. In our experimental conditions,subcutaneous genistein treatment led to significantly decreasedserum PTH, while PTG volume was increased. The proportionof interstitial tissue in the PTG was significantly elevated, whichobviously contributed to the increase in PTG volume. Also,genistein administration resulted in less prominentinterdigitations on chief cell membranes, together with poorlydeveloped RER and Golgi complex, and fewer mitochondria.The exact mechanism by which genistein treatment influence onlow PTH serum level, in the milieu of almost entirely withoutandrogens, remains to be clarified. It should be considered thatgenistein might bind to ERs, with a higher binding affinity forERβ (13), but the presence of ERs in PTG is still a controversialissue (34, 14). It is possible that the suppressing effect ofgenistein on PTG activity is achieved through an indirectmechanism. In one study, Ben-Dov et al. (16) showed thatFGF23 has an inhibitory role in the synthesis and secretion ofPTH through binding to the FGFR-Klotho receptor complex andactivation of the MAPK signaling pathway. In addition, Carrillo-Lopez et al. (14) suggest that the factor involved in the possibleindirect effect of estrogen on PTG could be FGF23. Namely, theauthors demonstrated in vitro that estradiol increases FGF23levels in osteoblast-like cells in a concentration and time-dependent manner. The presence of ERs in bone is welldocumented (35), as well as the importance of steroid hormonesfor bone metabolism, so some possibility arises that genistein,which is structurally similar to estradiol, might inducestimulation of FGF23 synthesis and secretion by binding to ERsin bone cells.

Consistent with our previous findings (33, 36), Orx induceda significant decrease in serum Ca2+ and Pi levels, together withincreased excretion of Ca2+ and Pi in urine. However, this studyfor the first time showed that genistein treatment of Orx ratssignificantly increased serum Ca2+ and Pi concentrations, andregulated the reabsorption of Pi from primary urine in kidneytubules. Our findings indicated that the phosphaturia occurringafter Orx was due to diminished gene and protein expression ofthe NaPi 2a co-transporter in epithelial cells of proximal tubules.Genistein treatment reduced Pi excretion in urine and recoveredNaPi 2a expression in BBM. Coherently, NaPi 2a mRNA levelswere increased after genistein administration. Kempson et al.(37) showed that PTH decreased NaPi 2a protein content on theapical domain of epithelial cells of kidney proximal tubules,while Bacic et al. (38) observed that acute application of PTHinduced withdrawal of NaPi 2a via receptor-mediatedendocytosis. The augmented expression of NaPi 2a in genisteintreated andropausal male rats in our study could have resultedfrom reduced PTH inhibition, since the level of this hormonedeclined significantly after genistein application. Moreover, thePTH and FGF23 induced downregulation of NaPi 2a is mediatedthrough the ERK1/2 signaling pathway (21, 39). The tyrosinekinase inhibiting role of genistein suggests possible interferencein the ERK1/2 signaling pathway and diminished PTH and

366

Fig. 8. Urine Pi concentration, in sham-operated (SO),orchidectomized (Orx) and orchidectomized rats treated withgenistein (Orx+G). All values are presented as mean ±S.D.,ap<0.05 versus SO, bp<0.05 versus Orx.

FGF23 induced inhibition of Pi uptake. Besides the inhibitoryrole of PTH in the regulation of NaPi 2a expression,glucocorticoids may also have some suppressing effect in thesame field (40). Ajdzanovic et al. (24) demonstrated thatgenistein treatment inhibited corticosterone production andsecretion in an animal model of the andropause, so a diminishedglucocorticoid effect may be involved as well. Decreasedcorticosterone production, after genistein application in vitro,was also observed (41). However, we cannot exclude a possibledirect mechanism of genistein action on the proximal tubulecells. Faroqui et al. (42) showed that estradiol has a significantimpact on downregulation of NaPi 2a in the renal proximaltubule, but this effect probably was not mediated through ERα.Since genistein preferentially binds to ERβ, besides ERα (13),its effect may be mediated via such a mechanism. Rogers et al.(43) demonstrated that androgen deprivation induced significantelevation of ERβ content in the kidney cortex of male rats, whileERα remained the same. Additional molecular studies areneeded to elucidate possible mechanisms of genistein action onthe regulation of NaPi 2a expression in BBM.

In summary, this is the first report evaluating genisteineffects on PTG and the functionally related NaPi 2acotransporter in kidney tubules. Our results showed that Orx,emphasizing andropausal symptoms, led to elevation of PTGvolume and serum PTH level, but also decreased expression ofthe NaPi 2a cotransporter in epithelial cells of proximal tubules.Genistein administration reduced the elevated serum PTH level,stimulated expression of NaPi 2a in the apical domain ofproximal tubule cells and decreased urinary Pi excretion in Orxrats. These data credibly suggest that the soy isoflavone,genistein, inhibits the function of PTG and stimulates the NaPi2a cotransporter activity, in an animal model of the andropause.

Acknowledgements: This work was supported by theMinistry of Education, Science and Technological Developmentof the Republic of Serbia, grant number 173009. The authorswish to thank Dr Anna Nikolic for language correction of themanuscript. The authors express their gratitude to the late DrDana Brunner for her guidance and contribution, and to Dr JurgBiber, Institute of Physiology, University of Zurich, Zurich,Switzerland, for tremendous support.

Conflict of interests: None declared.

REFERENCES

1. Lavi-Moshayoff V, Wasserman G, Meir T, Silver J, Naveh-Many T. PTH increases FGF23 gene expression andmediates the high-FGF23 levels of experimental kidneyfailure: a bone parathyroid feedback loop. Am J PhysiolRenal Physiol 2010; 299: F882-F889.

2. Biber J, Hernando N, Forster I, Murer H. Regulation ofphosphate transport in proximal tubules. Pflugers Arch EurJ Physiol 2009; 458: 39-52.

3. Vance ML. Andropause. Growth Horm IGF Res 2003; 13:S90-S92.

4. Halloran B, Uden P, Duh QY, et al. Parathyroid glandvolume increases with postmaturational aging in the rat. AmJ Physiol Endocrinol Metab 2002; 282: E557-E563.

5. Meyer R, Schreckenberg R, Kraus D, Kretschmer F, SchulzR, Schluter KD. Cardiac effects of osteostatin in mice.J Physiol Pharmacol 2012; 63: 17-22.

6. Bikle D. Hormonal regulation of bone mineral homeostasis.Touch Briefings 2008; 70-74.

7. Vanderschueren D, Van Herck E, Suiker AM, Visser WJ,Schot LPC, Bouillon R. Bone and mineral metabolism in

aged male rats: short- and long-term effects of androgendeficiency. Endocrinology 1992; 130: 2906-2916.

8. Bilezikian JP, Morishima A, Bell J, Grumbach MM.Increased bone mass as a result of estrogen therapy in aman with aromatase deficiency. New Engl J Med 1998;339: 599-603.

9. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK,Bouillon R, Ohlsson C. Androgens and bone. Endocr Rev2004; 25: 389-425.

10. Matsumoto C, Inada M, Toda K, Miyaura C. Estrogen andandrogen play distinct roles in bone turnover in male micebefore and after reaching sexual maturity. Bone 2006; 38:220-226.

11. Moutsatsou P. The spectrum of phytoestrogens in nature: ourknowledge is expanding. Hormones 2007; 6: 173-193.

12. Bitto A, Burnett BP, Polito F, et al. Effects of genisteinaglycone in osteoporotic, ovariectomized rats: a comparisonwith alendronate, raloxifene and oestradiol. Br J Pharmacol2008; 155: 896-905.

13. Kuiper GJM, Carlsson B, Grandien K, et al. Comparison ofthe ligand binding specificity and transcript tissuedistribution of estrogen receptors α and β. Endocrinology1997; 138: 863-870.

14. Carrillo-Lopez N, Roman-Garcia P, Rodriguez-Rebollar A,Fernandez-Martin JL, Naves-Diaz M, Cannata-Andia JB.Indirect regulation of PTH by estrogens may require FGF23.J Am Soc Nephrol 2009; 20: 2009-2017.

15. Fukumoto S, Yamashita T. Fibroblast growth factor-23 is thephosphaturic factor in tumor-induced osteomalacia and maybe phosphatonin. Curr Opin Nephrol Hypertens 2002; 11:385-389.

16. Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al. Theparathyroid is a target organ for FGF23 in rats. J ClinInvestig 2007; 117: 4003-4008.

17. Silver J, Naveh-Many T. Phosphate and the parathyroid.Kidney Int 2009; 75: 898-905.

18. Akiyama T, Ishida J, Nakagawa S, et al. Genistein, a specificinhibitor of tyrosine specific protein kinases. J Biol Chem1987; 262: 5592-5595.

19. Yan GR, Yin XF, Xiao CL, Tan ZL, Xu SH, He QY.Identification of novel signaling components in genistein-regulated signaling pathways by quantitativephosphoproteomics. J Proteomics 2011; 75: 695-707.

20. Lederer ED, Sohi SS, MCleish KR. Parathyroid hormonestimulates extracellular signal-regulated kinase (ERK)activity throught two independent signal transductionpathways: role of ERK in sodium-phosphate cotransport.J Am Soc Nephrol 2000; 11: 222-231.

21. Bacic D, Schulz N, Biber J, Kaissling B, Murer H, WagnerCA. Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Picotransporter in mouse kidney. Pflugers Arch Eur J Physiol2003; 446: 52-60.

22. Picherit C, Coxam V, Bennetau-Pelissero C, et al. Daidzeinis more efficient than genistein in preventing ovariectomy-induced bone loss in rats. J Nutr 2000; 130: 1675-1681.

23. Ajdzanovic V, Sosic-Jurjevic B, Filipovic B, et al. Genistein-induced histomorphometric and hormone secreting changesin the adrenal cortex in middle-aged rats. Exp Biol Med2009; 234: 148-156.

24. Ajdzanovic V, Sosic-Jurjevic B, Filipovic B, et al. Genisteinaffects the morphology of pituitary ACTH cells anddecreases circulating levels of ACTH and corticosterone inmiddle-aged male rats. Biol Res 2009; 42: 13-23.

25. Sosic-Jurjevic B, Filipovic B, Ajdzanovic V, et al.Subcutaneously administrated genistein and daidzeindecrease serum cholesterol and increase triglyceride levels

367

in male middle-aged rats. Exp Biol Med 2007; 232: 1222-1227.

26. Biber J, Stieger B, Stange G, Murer H. Isolation of renalproximal tubular brush-border membranes. Nat Protoc 2007;2: 1356-1359.

27. Gundersen HJ, Jensen EB. The efficiency of systematicsampling in stereology and its prediction. J Microsc 1987;147: 229-263.

28. Galitzer H, Ben-Dov I, Lavi-Moshayoff V, Naveh-Many T,Silver J. Fibroblast growth factor 23 acts on the parathyroidto decrease parathyroid hormone secretion. Curr OpinNephrol Hypertens 2008; 17: 363-367.

29. Uemura H, Irahara M, Yoneda N, et al. Close correlationbetween estrogen treatment and renal phosphatereabsorption capacity. J Clin Endocrinol Metab 2000; 85:1215-1219.

30. Adlercreutz H, Mazur W. Phyto-oestrogens and WesternDiseases. Ann Med 1997; 29: 95-120.

31. Mezesova L, Bartekova M , Javorkova V, Vlkovicova J,Breier A, Vrbjar N. Effect of quercetin on kinetic propertiesof renal NA, K-ATPase in normotensive and hypertensiverats. J Physiol Pharmacol 2010; 61: 593-598.

32. Coleman R, Silbermann M. Ultrastructure of parathyroidglands in triamcinolone-treated mice. J Anat 1978; 126:181-192.

33. Filipovic B, Sosic-Jurjevic B, Ajdzanovic V, et al. The effectof orchidectomy on thyroid C cells and bonehistomorphometry in middle-aged rats. Histochem Cell Biol2007; 128: 153-159.

34. Naveh-Many T, Almogi G, Livni N, Silver J. Estrogenreceptors and biologic response in rat parathyroid tissue andC cells. J Clin Investig 1992; 90: 2434-2438.

35. Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T. Expressionof estrogen receptor beta in rat bone. Endocrinology 1997;138: 4509-4512.

36. Filipovic B, Sosic-Jurjevic B, Ajdzanovic V, et al. Daidzeinadministration positively affects thyroid C cells and bonestructure in orchidectomized middle-aged rats. OsteoporosisInt 2010; 2: 1609-1616.

37. Kempson SA, Lotscher M, Kaissling B, Biber J, Murer H,Levi M. Parathyroid hormone action on phosphatetransporter mRNA and protein in rat renal proximal tubules.Am J Physiol 1995; 268: F784-F791.

38. Bacic D, LeHir M, Biber J, Kaissling B, Murer H, Wagner CA.The renal Na+/phosphate cotransporter NaPi-IIa is internalizedvia the receptor-mediated endocytic route in response toparathyroid hormone. Kidney Int 2006; 69: 495-503.

39. Yamashita T, Konishi M, Miyake A, Inui K, Itoh N.Fibroblast growth factor (FGF)-23 inhibits renal phosphatereabsorption by activation of the mitogen-activated proteinkinase pathway. J Biol Chem 2002; 277: 28265-28270.

40. Levi M, Shayman JA, Abe A, et al. Dexamethasonemodulates rat renal brush border membrane phosphatetransporter mRNA and protein abundance andglycoshingolipid composition. J Clin Investig 1995; 96:207-216.

41. Kaminska B, Ciereszko R, Kiezun M, Dusza L. In vitroeffects of genistein and daidzein on the activity ofadrenocortical steroidogenic enzymes in mature female pigs.J Physiol Pharmacol 2013; 64: 103-108.

42. Faroqui S, Levi M, Soleimani M, Amlal H. Estrogendownregulates the proximal tubule type IIa sodiumphosphate cotransporter causing phosphate wasting andhypophosphatemia. Kidney Int 2008; 73: 1141-1150.

43. Rogers JL, Mitchell AR, Maric C, Sandberg K, Myers A,Mulroney SE. Effect of sex hormones on renal estrogen andangiotensin type 1 receptors in female and male rats. AmJ Physiol Regul Integr Comp Physiol 2007; 292: R794-R799.

R e c e i v e d : February 21, 2013A c c e p t e d : May 29, 2013

Author’s address: Dr. Jasmina Pantelic, Institute forBiological Research “Sinisa Stankovic”, 142 Despot StefanBlvd, 11060 Belgrade, Serbia.E-mail: jasminap@ibiss.bg.ac.rs

368