BACKGROUND
human  <cit>  and animal studies  <cit>  have implicated y-chromosome  loci as influencing the onset of male hypertension.
in the spontaneously hypertensive rat strain , the shr yc contains a locus which increases blood pressure  approximately  <dig> mmhg compared to the normotensive wistar kyoto  y chromosome.
the yc is only one genetic component of the total shr hypertension.
in shr males, the presences of testosterone and androgen receptors through puberty are required for development of the hypertension and associated sympathetic nervous system potentiation, including the yc component  <cit> .
in examining phenotypes that mapped to the yc and differed between shr and wky, our laboratory demonstrated an earlier rise of testosterone  levels in shr males leading into puberty  <cit>  and this phenotype mapped to the shr yc.
because of the unique biology of the mammalian yc the blood pressure phenotype and the t timing phenotype cannot be separated using linkage.
with the interaction of t and the development of the yc hypertension, it is possible the two phenotypes are related and a single genetic locus is responsible for both phenotypes.
in further support of a physiological t-sns interaction  <cit> , we have previously reported that t increases the storage and release of norepinephrine in isolated kidney studies more in shr, as compared to the wky males  <cit> .
reckelhoff et al.
<cit>  reported that in shr, t increased renal na reabsorption, potentially through an androgen receptor  mediated mechanism in the kidney.
these results suggest a possible renal ar mediated response that may influence one of the renal bp control mechanisms.
the renal system operates under a well auto-regulated negative feedback system that maintains glomerular filtration rate and the filtered load of ions  <cit> , as well as playing an important role in long-term bp regulation.
as part of this regulation in genetic models of hypertension, the kidney requires a high level of arterial pressure to excrete na and water  <cit>  and the increase in renal arterial perfusion pressure results in increased excretion of water and na.
fluctuations in bp and ion concentration can initiate renal and cardiovascular compensatory mechanisms through the renin angiotensin system, and aldosterone secretion  <cit> .
other studies comparing urinary na excretion of shr and wky males  <cit>  have found decreased urinary na excretion and a reduced renal ability to excrete na, in spite of bp differences.
therefore, the hypothesis to be tested was that testosterone and the hypertensive yc would increase renal sodium reabsorption.
methods
the following  <dig> studies ask the questions: study 1-does testosterone influence urinary sodium and potassium excretion, as well as, systolic bp?
; study 2-will blocking the androgen receptor mimic the effects of castration on electrolyte excretion?
; study 3-is there evidence for ar transcripts in the kidney?
design
study  <dig> utilized a two strain by three treatment design, using shr/y and wky males  <dig> weeks of age.
the treatments consisted of gonadally intact controls , castrates , and castrates with testosterone implants .
also  <dig> age matched females of each strain were tested for  <dig> hr urinary na as a comparison to males.
study  <dig> used two strains-wky and shr/y, and two treatments-starting with the control period followed by  <dig> wks of dietary flutamide administration  in the same animals.
study  <dig> used kidneys collected from  <dig> adult males each from two strains .
animal models
the y-chromosome  animal model used in this study consisted of the consomic borderline hypertensive , and normotensive wistar-kyoto  rats  <cit> .
shr and wky rats were originally obtained from harlan sprague-dawley  in  <dig> , and colonies have been maintained at the university of akron research facility.
to develop the shr/y consomic strain, an shr male was crossed with a wky female.
a male offspring was then crossed to a wky female and then a son of this cross was crossed to a wky female.
these crosses have been repeated for over  <dig> generations, creating the strain designated shr/y.
the repeated crosses have replaced the autosomal and x-linked loci of the parental shr with the alleles of the maternal wky strain, while the shr y chromosome remains.
therefore, the shr/y male has an shr yc, with the autosomes and an x-chromosome from the wky female.
in comparison, the wky male has a wky yc, with the autosomes and an x-chromosome from the wky.
comparisons between wky and shr/y males allow us to investigate the role of the shr yc in a wky genetic background and any genetic differences between strains must map to the y chromosomes.
for a detailed review, see ely et al.
<cit> .
all animals were treated in a humane manner according to nih guidelines, and the university of akron institutional animal care and use committee  approved all experiments.
housing
animals were housed in pairs according to their strain and maintained in polycarbonate cages  with stainless steel covers and heat-treated bedding .
rats were subjected to a  <dig> h/ <dig> h light/dark cycle and maintained on a normal sodium diet .
food and water was accessible ad libitum.
the amount of na in the food was  <dig> % and previous studies have not shown that testosterone treatment increases na intake.
testosterone experiments
male rats to be castrated were initially  <dig> weeks old, sedated with sodium brevital , and both testes were removed.
in a similar manner, male rats to be implanted  were castrated, shaved along the base of the neck, and an incision  <dig>  cm long was made.
the t implant was inserted beneath the skin parallel to the longitudinal axis of the rat.
briefly testosterone implants  <cit>  for castrated rats at  <dig> weeks of age were prepared by cutting single lumen clear  <dig> silastic tubing  into strips that measured  <dig> mm in length with  <dig> mm of the tube packed with testosterone propionate  and a 2– <dig> ml retro-orbital blood sample was collected between  <dig> and  <dig> hours and centrifuged for  <dig> minutes  to obtain plasma.
testosterone levels were analyzed by ria   <cit> .
the correlation with another kit was r =  <dig> , sensitivity was  <dig>  ng/ml at the 95% confidence limit, and the highest cross-reactivity with potential interfering steroids was with dihydrotestosterone .
the coefficient of variation for our sample intra-run was  <dig> % to  <dig> % and for inter-run was  <dig> % to  <dig> %.
aldosterone levels were analyzed in plasma by ria .
sensitivity was  <dig>  pg/ml.
the highest cross-reactivity with potential interfering steroids was with corticosterone .
the coefficient of variation for our sample intra-run was  <dig> % to  <dig> % and for inter-run was  <dig> % to  <dig> %.
electrolyte measurement
plasma was collected for sodium levels in control and castrated animals of each strain at  <dig> weeks of age using pentothal anesthetic .
urine was collected for  <dig> hours at  <dig> weeks of age in order that the volume was not affected by the blood collection at  <dig> weeks, while the animals were housed in metabolic cages and maintained on their food and water.
one ml of mineral oil was placed into each urine-collecting cup prior to the collection period to prevent evaporation of the urine.
sodium and potassium concentrations were measured by flame photometry .
systolic blood pressure
weekly systolic blood pressure for each strain and treatment group was monitored for the duration of the study.
blood pressure was measured via tail sphygmomanometry and recorded on a physiograph  between  <dig> and  <dig> hrs.
the animals were placed into a warming chamber for  <dig> minutes at 38°c to dilate tail arteries, transferred to a restraint, and a tail cuff was slipped around the base of the tail.
pressures were then determined from the average of  <dig> systolic blood pressure recordings, which took 2– <dig> min  <cit> .
study 3: is there evidence for androgen receptors in the kidney?
the objective of this experiment was to determine if androgen receptor  transcripts were present in the kidney of shr, shr/y and wky males.
spontaneously hypertensive rats  were used to see if wky and shr/y rats were similar in ar presence.
gonadally intact males at  <dig> weeks of age  from our stock colonies, as previously described, were used to study the presence of ar transcripts in each strain.
animals were anesthetized with pentothal  and kidneys removed and placed on foil on dry ice.
all animals were housed in a similar manner to study  <dig>
androgen receptor rt-pcr
total cellular rna was isolated from frozen kidneys from shr, shr/y and wky males with rna stat- <dig> .
for cdna production the rna was dnased  with  <dig> μg used as a template with avian reverse transcriptase , 10× buffer, dntpmix, a mix of random nonamers , and water to a total volume of  <dig> μl.
rnase inhibitor  was included to insure rna integrity.
a rt control minus the rt enzyme was included for each sample.
the cdna was amplified at 94°c for  <dig> min.
;  <dig> cycles of 94°c for  <dig> min., 52°c for  <dig> min., 72°c for  <dig> min.
; 72°c for  <dig> min.
primers were designed from genbank sequence j <dig> for the rat ar receptor gene cdna.
primer sequences consisted of the reverse primer, dk <dig> 5'tgctgccttcggatattacc and the forward primer dk <dig> 5' tagggctgggaagggtctac.
a positive control  and negative control  were included in each set of pcr reactions.
pcr products were visually observed on 1% agarose gels and bands of  <dig> bp were identified as the androgen receptor transcript.
statistics
data were analyzed and graphed with sigma statistical software .
two-way anova using flutamide or t treatment and strain as factors were used to analyze plasma t and aldosterone, urinary electrolytes, and kidney weight related variables with appropriate follow up t-tests .
significance was assumed with a p <  <dig> .
RESULTS
urine electrolyte analysis
*=p<.
<dig> wky cast vs. wky cast +ti, **+p<.
<dig> wky cast vs. wky control, **=p<.
<dig> shr/y cast vs. shr/y cast +ti, ***=p<.
<dig> shr/y cast vs. shr/y control.
***=p<.
<dig> shr/y cast vs. shr/control and shr/y cast+ti, ***=p<.
<dig> wky cast vs. wky control, *=p<.
<dig> wky cast vs. wky cast+ti.
testosterone, aldosterone, blood pressure and kidney weight
serum testosterone  levels, systolic blood pressure and relative kidney weight at  <dig> weeks of age for shr/y and wky gonadally intact control , castrate  and castrate with t implant  males, expressed as means +/- sem.
the two-way anova for serum t was significant for t treatment) f =  <dig> , p <  <dig> ).
both t implant groups had elevated t compared to controls .
the two-way anova for sbp was significant for t treatment , strain , and t treatment x strain .
the two-way anova for t treatment on relative combined kidney weight was significant .
the shr/y control group sbp was greater than the shr/y cast+ti  and shr/y cast group sbp .
the wky cast group sbp was less than the wky cast+ti and control groups .
relative combined kidney weight for shr/y cast was significantly less than shr/y control and shr/y cast +ti groups .
relative combined kidney weight for the shr/y cast +ti group was significantly greater than the shr/y cast and control groups .
***=p<.
<dig> wky cast vs. wky control,*=p<.
<dig> wky cast +ti vs. wky cast, *=p<.
<dig> shr/y cast +ti vs. shr/y cast, ***=p<.
<dig> shr/y cast vs. shr/y control.
androgen receptor
the  <dig> bp amplified ar transcript is present in shr , shr/y and wky males at  <dig> weeks of age .
since this method of analysis of rt-pcr is not quantitative, no comparison can be made of levels of expression between strains.
discussion
gender differences in electrolyte excretion have been reported in rats and shr females excrete more na than males  <cit> .
it makes sense that females would excrete more na since in most mammals, females have a higher na intake than males  <cit> .
this may be a protective mechanism to maintain blood volume in times of hemorrhagic injury and during pregnancy, but in resting steady state situations, the na would need to be excreted.
there is evidence that males have more physiological difficulty in responding to blood loss than females  <cit> .
also there are reports of the presence of androgen receptors in female kidneys of rats and mice  <cit> .
in our studies, the presence or absence of testosterone  altered renal excretion of na and k in the male borderline hypertensive shr/y and normotensive wky strains.
in addition, the presence of the androgen receptor  transcripts in the kidney of each strain is consistent with the idea that t binds to the ar in the kidney to play a role controlling na regulation.
with regards to localization of the ar studies have shown ar in proximal and distal tubules  <cit> , and in other locations within the kidney  and low but detectable levels in medullary and cortical parts of the thick ascending limb of henle's loop  <cit> .
androgens could also be operating through the transcriptional control of specific renal genes.
androgen regulated genes in the kidney include several organic anion transporters  <cit> , which could be involved in the androgen produced retention of electrolytes  <cit> .
indeed, quinkler et al.
showed that testosterone increased the expression of the alpha-subunit of the epithelial sodium channel in a human renal proximal tubule cell line 2– <dig> fold which was blocked by flutamide  <cit> .
this t effect on renal function is not unique to males.
ovarectomized females given t excreted less na than ovarectomized females  <cit> , further supporting the notion that there may be ars present in the proximal tubules in both genders  <cit> .
to further support this data we found that shr females on a high na diet with t implants excreted less na than intact females  <cit> .
plasma na levels were constant across treatments and cannot be used to explain urinary na differences.
there are many regulatory mechanisms to maintain plasma na balance so it is not surprising that in spite of na intake or circulating hormone variations, plasma na remains constant.
for instance, in a study from our lab  <cit>  we found that male, female and testicular feminized males  had the same constant plasma na levels  in spite of gender and ar differences.
androgen receptor blockade produced a significant rise in shr/y and wky male na excretion suggesting a t dependence on na reabsorption in both strains similar to the effects observed with castration.
flutamide has been used as an anticancer drug and is classified as a nonsteroidal antiandrogen.
it competively blocks t at the ar forming inactive complexes which cannot translocate to the nucleus.
the increased na excretion was not as great with flutamide as with castration, which may be due to some ars still functioning or possibly t working through non-genomic ar mechanisms.
testosterone signaling through non-genomic ar mechanisms has been described in rat osteoblasts  <cit> , ar free macrophages  <cit> , and murine t cells  <cit> ; whereby t mediates only ligand-induced ca2+ import through non-voltage-gated ca2+ channels  <cit> .
recently, our lab has shown that t via the ar influences ca2+ signaling through an increase in ca2+ mobilization in coronary artery adventitial fibroblasts and consequently elevates collagen type i production  <cit> .
in support of this finding vicencio  <cit>  has also found that t induces ca2+ increase by a nongenomic mechanism in cultured rat cardiac myocytes.
further evidence that t is playing a role in na reabsorption is provided by the aldosterone data.
the removal of t by castration produced a rise in plasma aldosterone and t replacement lowered the aldosterone.
this suggests that t was acting to conserve renal na and its removal caused aldosterone to rise as a compensation.
again, there appears to be a t threshold level for its influence on aldosterone levels since even when t increased 3–4x with replacement the aldosterone levels did not suppress below control values.
indeed, the same results were found in male sprague dawley rats castrated and given t replacement  <cit> .
based on in vitro data, t appeared to exert a direct inhibitory effect on basal aldosterone release at the level of adrenal zona glomerulosa cells, but there was no effect on intracellular camp  <cit> .
further studies strongly suggested that the specific inhibitory effect of t on aldosterone was through inhibition of aldosterone synthase  <cit> .
this is further supported by clinical observations in hyperandrogenic women suggesting that circulating t may inhibit adrenal 21- and/or 11b-hydroxylase activity  <cit> .
in our groups that were castrated and received t replacement one might expect to observe greater na reabsorption since t levels were 3–4x higher with replacement, but the urinary na levels were comparable to that of the controls for each strain suggesting that there may be a threshold level of t whereby further increases to not affect na retention mechanisms.
the renin angiotensin system  and specifically, angiotensinogen  has been shown to be modulated by sex hormones  <cit> .
androgens can stimulate synthesis of agt in animal models  <cit>  and this induction is tissue specific.
for instance, androgen has been reported to increase kidney agt mrna levels, whereas it has only a slight effect on liver agt mrna levels  <cit> .
androgens may directly upregulate the proximal tubule renin-angiotensin system, increase the volume reabsorptive rate, and thereby increase extracellular volume and blood pressure and secondarily decrease serum angiotensin ii levels  <cit> .
we do not have tissue ras measures, so it is not possible to determine if the t effects are operating through a tissue ras system in the proximal tubules.
similarily, there is a ras-dopamine interaction that influences renal proximal tubule cells  <cit> .
zeng et al.
showed that at <dig> and d <dig> receptors interacted differently in renal proximal tubule cells from shr and wky  <cit> .
therefore, it is possible that t can indirectly dopamine to produce renal electrolyte effects.
further research is needed to clarify these ras-catecholamine interactions.
the same trend of decreased na excretion in the presence of t was observed with k excretion.
k is passively transported across the cell membrane via one of several types of channels in tubules  <cit> .
this was surprising to us since most often there is a reciprocal relationship between renal na/k exchange.
na and k, are largely exchanged via the na/k transporter.
when the ratio of na to k excreted was compared, there were no differences between strains or treatment groups.
in the shr/y and wky strains, the absence of t produced increased k excretion, and t replacement normalized the effect.
the ar blockade study showed similar results except the k excretion with the ar blocker was not as great as with castration.
most likely this was due to not all of the ars are functionally blocked with the drug dosage used.
also relative kidney weight cannot explain the excretion differences found with testosterone manipulation, since the wky strain did not have significant kidney weight changes but did have excretion differences.
the decrease in kidney weight in the shr/y strain that was castrated and its rescue in the castrates treated with testosterone supports the findings of others who have shown an anabolic effect of testosterone on kidney weight  <cit> .
blood pressure cannot be used to explain the electrolyte excretion differences with t manipulation since bp decreased after castration and na excretion increased.
the bp profile for each strain and treatment group is similar to previously reported data from our lab  <cit>  showing decreased bp with castration and t treatment restored bp to control or above control levels.
in a na balance study comparing shr and wky males, lundin et al.
<cit>  did not find increased shr urinary na excretion as expected due to high bp, but instead a decreased urinary na excretion was found.
similar shr urinary na excretion results were found by osborn et al.
<cit>  with a reduced renal ability to excrete na, in spite of increased blood pressure.
since t was not measured in osborn's studies, it is not known if the reduced na excretion may have been due to elevated t.
CONCLUSIONS
the excretion of na and k in both shr/y and wky males appear to be partially t dependent.
in addition, the elevated na excretion in shr/y and wky following castration, further suggests that t can be an important player in renal na excretion.
these differences in renal excretion of na and k cannot be solely and consistently explained by changes in bp or anabolic effects on kidney weight.
in conclusion, the increased na and k excretion following castration, identification of renal ar transcripts, and loss of na with androgen blockade suggests that t is involved in the regulation of na balance.
competing interests
the author declare that they have no competing interests.
authors' contributions
de, am, jt, cj and mt designed and directed studies, were involved in the analysis of the data, and writing of the manuscript.
gd, jt, mh and sb performed the experiments and ran the assays.
all authors have read and approved the manuscript.

 human and animal studies have implicated y-chromosome loci as influencing the onset of male hypertension. in the spontaneously hypertensive rat strain, the shr yc contains a locus which increases blood pressure approximately mmhg compared to the normotensive wistar kyoto y chromosome. the yc is only one genetic component of the total shr hypertension. in shr males, the presences of testosterone and androgen receptors through puberty are required for development of the hypertension and associated sympathetic nervous system potentiation, including the yc component. in examining phenotypes that mapped to the yc and differed between shr and wky, our laboratory demonstrated an earlier rise of testosterone levels in shr males leading into puberty and this phenotype mapped to the shr yc. because of the unique biology of the mammalian yc the blood pressure phenotype and the t timing phenotype cannot be separated using linkage. with the interaction of t and the development of the yc hypertension, it is possible the two phenotypes are related and a single genetic locus is responsible for both phenotypes. in further support of a physiological t-sns interaction, we have previously reported that t increases the storage and release of norepinephrine in isolated kidney studies more in shr, as compared to the wky males. reckelhoff et al. reported that in shr, t increased renal na reabsorption, potentially through an androgen receptor mediated mechanism in the kidney. these results suggest a possible renal ar mediated response that may influence one of the renal bp control mechanisms. the renal system operates under a well auto-regulated negative feedback system that maintains glomerular filtration rate and the filtered load of ions, as well as playing an important role in long-term bp regulation. as part of this regulation in genetic models of hypertension, the kidney requires a high level of arterial pressure to excrete na and water and the increase in renal arterial perfusion pressure results in increased excretion of water and na. fluctuations in bp and ion concentration can initiate renal and cardiovascular compensatory mechanisms through the renin angiotensin system, and aldosterone secretion. other studies comparing urinary na excretion of shr and wky males have found decreased urinary na excretion and a reduced renal ability to excrete na, in spite of bp differences. therefore, the hypothesis to be tested was that testosterone and the hypertensive yc would increase renal sodium reabsorption. methods the following studies ask the questions: study 1-does testosterone influence urinary sodium and potassium excretion, as well as, systolic bp?; study 2-will blocking the androgen receptor mimic the effects of castration on electrolyte excretion?; study 3-is there evidence for ar transcripts in the kidney? design study utilized a two strain by three treatment design, using shr/y and wky males weeks of age. the treatments consisted of gonadally intact controls, castrates, and castrates with testosterone implants. also age matched females of each strain were tested for hr urinary na as a comparison to males. study used two strains-wky and shr/y, and two treatments-starting with the control period followed by wks of dietary flutamide administration in the same animals. study used kidneys collected from adult males each from two strains. animal models the y-chromosome animal model used in this study consisted of the consomic borderline hypertensive, and normotensive wistar-kyoto rats. shr and wky rats were originally obtained from harlan sprague-dawley in, and colonies have been maintained at the university of akron research facility. to develop the shr/y consomic strain, an shr male was crossed with a wky female. a male offspring was then crossed to a wky female and then a son of this cross was crossed to a wky female. these crosses have been repeated for over generations, creating the strain designated shr/y. the repeated crosses have replaced the autosomal and x-linked loci of the parental shr with the alleles of the maternal wky strain, while the shr y chromosome remains. therefore, the shr/y male has an shr yc, with the autosomes and an x-chromosome from the wky female. in comparison, the wky male has a wky yc, with the autosomes and an x-chromosome from the wky. comparisons between wky and shr/y males allow us to investigate the role of the shr yc in a wky genetic background and any genetic differences between strains must map to the y chromosomes. for a detailed review, see ely et al.. all animals were treated in a humane manner according to nih guidelines, and the university of akron institutional animal care and use committee approved all experiments. housing animals were housed in pairs according to their strain and maintained in polycarbonate cages with stainless steel covers and heat-treated bedding. rats were subjected to a h/ h light/dark cycle and maintained on a normal sodium diet. food and water was accessible ad libitum. the amount of na in the food was % and previous studies have not shown that testosterone treatment increases na intake. testosterone experiments male rats to be castrated were initially weeks old, sedated with sodium brevital, and both testes were removed. in a similar manner, male rats to be implanted were castrated, shaved along the base of the neck, and an incision cm long was made. the t implant was inserted beneath the skin parallel to the longitudinal axis of the rat. briefly testosterone implants for castrated rats at weeks of age were prepared by cutting single lumen clear silastic tubing into strips that measured mm in length with mm of the tube packed with testosterone propionate and a 2– ml retro-orbital blood sample was collected between and hours and centrifuged for minutes to obtain plasma. testosterone levels were analyzed by ria. the correlation with another kit was r =, sensitivity was ng/ml at the 95% confidence limit, and the highest cross-reactivity with potential interfering steroids was with dihydrotestosterone. the coefficient of variation for our sample intra-run was % to % and for inter-run was % to %. aldosterone levels were analyzed in plasma by ria. sensitivity was pg/ml. the highest cross-reactivity with potential interfering steroids was with corticosterone. the coefficient of variation for our sample intra-run was % to % and for inter-run was % to %. electrolyte measurement plasma was collected for sodium levels in control and castrated animals of each strain at weeks of age using pentothal anesthetic. urine was collected for hours at weeks of age in order that the volume was not affected by the blood collection at weeks, while the animals were housed in metabolic cages and maintained on their food and water. one ml of mineral oil was placed into each urine-collecting cup prior to the collection period to prevent evaporation of the urine. sodium and potassium concentrations were measured by flame photometry. systolic blood pressure weekly systolic blood pressure for each strain and treatment group was monitored for the duration of the study. blood pressure was measured via tail sphygmomanometry and recorded on a physiograph between and hrs. the animals were placed into a warming chamber for minutes at 38°c to dilate tail arteries, transferred to a restraint, and a tail cuff was slipped around the base of the tail. pressures were then determined from the average of systolic blood pressure recordings, which took 2– min. study 3: is there evidence for androgen receptors in the kidney? the objective of this experiment was to determine if androgen receptor transcripts were present in the kidney of shr, shr/y and wky males. spontaneously hypertensive rats were used to see if wky and shr/y rats were similar in ar presence. gonadally intact males at weeks of age from our stock colonies, as previously described, were used to study the presence of ar transcripts in each strain. animals were anesthetized with pentothal and kidneys removed and placed on foil on dry ice. all animals were housed in a similar manner to study androgen receptor rt-pcr total cellular rna was isolated from frozen kidneys from shr, shr/y and wky males with rna stat-. for cdna production the rna was dnased with μg used as a template with avian reverse transcriptase, 10× buffer, dntpmix, a mix of random nonamers, and water to a total volume of μl. rnase inhibitor was included to insure rna integrity. a rt control minus the rt enzyme was included for each sample. the cdna was amplified at 94°c for min.; cycles of 94°c for min., 52°c for min., 72°c for min.; 72°c for min. primers were designed from genbank sequence j for the rat ar receptor gene cdna. primer sequences consisted of the reverse primer, dk 5'tgctgccttcggatattacc and the forward primer dk 5' tagggctgggaagggtctac. a positive control and negative control were included in each set of pcr reactions. pcr products were visually observed on 1% agarose gels and bands of bp were identified as the androgen receptor transcript. statistics data were analyzed and graphed with sigma statistical software. two-way anova using flutamide or t treatment and strain as factors were used to analyze plasma t and aldosterone, urinary electrolytes, and kidney weight related variables with appropriate follow up t-tests. significance was assumed with a p <.  urine electrolyte analysis *=p<. wky cast vs. wky cast +ti, **+p<. wky cast vs. wky control, **=p<. shr/y cast vs. shr/y cast +ti, ***=p<. shr/y cast vs. shr/y control. ***=p<. shr/y cast vs. shr/control and shr/y cast+ti, ***=p<. wky cast vs. wky control, *=p<. wky cast vs. wky cast+ti. testosterone, aldosterone, blood pressure and kidney weight serum testosterone levels, systolic blood pressure and relative kidney weight at weeks of age for shr/y and wky gonadally intact control, castrate and castrate with t implant males, expressed as means +/- sem. the two-way anova for serum t was significant for t treatment) f =, p < ). both t implant groups had elevated t compared to controls. the two-way anova for sbp was significant for t treatment, strain, and t treatment x strain. the two-way anova for t treatment on relative combined kidney weight was significant. the shr/y control group sbp was greater than the shr/y cast+ti and shr/y cast group sbp. the wky cast group sbp was less than the wky cast+ti and control groups. relative combined kidney weight for shr/y cast was significantly less than shr/y control and shr/y cast +ti groups. relative combined kidney weight for the shr/y cast +ti group was significantly greater than the shr/y cast and control groups. ***=p<. wky cast vs. wky control,*=p<. wky cast +ti vs. wky cast, *=p<. shr/y cast +ti vs. shr/y cast, ***=p<. shr/y cast vs. shr/y control. androgen receptor the bp amplified ar transcript is present in shr, shr/y and wky males at weeks of age. since this method of analysis of rt-pcr is not quantitative, no comparison can be made of levels of expression between strains. discussion gender differences in electrolyte excretion have been reported in rats and shr females excrete more na than males. it makes sense that females would excrete more na since in most mammals, females have a higher na intake than males. this may be a protective mechanism to maintain blood volume in times of hemorrhagic injury and during pregnancy, but in resting steady state situations, the na would need to be excreted. there is evidence that males have more physiological difficulty in responding to blood loss than females. also there are reports of the presence of androgen receptors in female kidneys of rats and mice. in our studies, the presence or absence of testosterone altered renal excretion of na and k in the male borderline hypertensive shr/y and normotensive wky strains. in addition, the presence of the androgen receptor transcripts in the kidney of each strain is consistent with the idea that t binds to the ar in the kidney to play a role controlling na regulation. with regards to localization of the ar studies have shown ar in proximal and distal tubules, and in other locations within the kidney and low but detectable levels in medullary and cortical parts of the thick ascending limb of henle's loop. androgens could also be operating through the transcriptional control of specific renal genes. androgen regulated genes in the kidney include several organic anion transporters, which could be involved in the androgen produced retention of electrolytes. indeed, quinkler et al. showed that testosterone increased the expression of the alpha-subunit of the epithelial sodium channel in a human renal proximal tubule cell line 2– fold which was blocked by flutamide. this t effect on renal function is not unique to males. ovarectomized females given t excreted less na than ovarectomized females, further supporting the notion that there may be ars present in the proximal tubules in both genders. to further support this data we found that shr females on a high na diet with t implants excreted less na than intact females. plasma na levels were constant across treatments and cannot be used to explain urinary na differences. there are many regulatory mechanisms to maintain plasma na balance so it is not surprising that in spite of na intake or circulating hormone variations, plasma na remains constant. for instance, in a study from our lab we found that male, female and testicular feminized males had the same constant plasma na levels in spite of gender and ar differences. androgen receptor blockade produced a significant rise in shr/y and wky male na excretion suggesting a t dependence on na reabsorption in both strains similar to the effects observed with castration. flutamide has been used as an anticancer drug and is classified as a nonsteroidal antiandrogen. it competively blocks t at the ar forming inactive complexes which cannot translocate to the nucleus. the increased na excretion was not as great with flutamide as with castration, which may be due to some ars still functioning or possibly t working through non-genomic ar mechanisms. testosterone signaling through non-genomic ar mechanisms has been described in rat osteoblasts, ar free macrophages, and murine t cells; whereby t mediates only ligand-induced ca2+ import through non-voltage-gated ca2+ channels. recently, our lab has shown that t via the ar influences ca2+ signaling through an increase in ca2+ mobilization in coronary artery adventitial fibroblasts and consequently elevates collagen type i production. in support of this finding vicencio has also found that t induces ca2+ increase by a nongenomic mechanism in cultured rat cardiac myocytes. further evidence that t is playing a role in na reabsorption is provided by the aldosterone data. the removal of t by castration produced a rise in plasma aldosterone and t replacement lowered the aldosterone. this suggests that t was acting to conserve renal na and its removal caused aldosterone to rise as a compensation. again, there appears to be a t threshold level for its influence on aldosterone levels since even when t increased 3–4x with replacement the aldosterone levels did not suppress below control values. indeed, the same results were found in male sprague dawley rats castrated and given t replacement. based on in vitro data, t appeared to exert a direct inhibitory effect on basal aldosterone release at the level of adrenal zona glomerulosa cells, but there was no effect on intracellular camp. further studies strongly suggested that the specific inhibitory effect of t on aldosterone was through inhibition of aldosterone synthase. this is further supported by clinical observations in hyperandrogenic women suggesting that circulating t may inhibit adrenal 21- and/or 11b-hydroxylase activity. in our groups that were castrated and received t replacement one might expect to observe greater na reabsorption since t levels were 3–4x higher with replacement, but the urinary na levels were comparable to that of the controls for each strain suggesting that there may be a threshold level of t whereby further increases to not affect na retention mechanisms. the renin angiotensin system and specifically, angiotensinogen has been shown to be modulated by sex hormones. androgens can stimulate synthesis of agt in animal models and this induction is tissue specific. for instance, androgen has been reported to increase kidney agt mrna levels, whereas it has only a slight effect on liver agt mrna levels. androgens may directly upregulate the proximal tubule renin-angiotensin system, increase the volume reabsorptive rate, and thereby increase extracellular volume and blood pressure and secondarily decrease serum angiotensin ii levels. we do not have tissue ras measures, so it is not possible to determine if the t effects are operating through a tissue ras system in the proximal tubules. similarily, there is a ras-dopamine interaction that influences renal proximal tubule cells. zeng et al. showed that at and d receptors interacted differently in renal proximal tubule cells from shr and wky. therefore, it is possible that t can indirectly dopamine to produce renal electrolyte effects. further research is needed to clarify these ras-catecholamine interactions. the same trend of decreased na excretion in the presence of t was observed with k excretion. k is passively transported across the cell membrane via one of several types of channels in tubules. this was surprising to us since most often there is a reciprocal relationship between renal na/k exchange. na and k, are largely exchanged via the na/k transporter. when the ratio of na to k excreted was compared, there were no differences between strains or treatment groups. in the shr/y and wky strains, the absence of t produced increased k excretion, and t replacement normalized the effect. the ar blockade study showed similar results except the k excretion with the ar blocker was not as great as with castration. most likely this was due to not all of the ars are functionally blocked with the drug dosage used. also relative kidney weight cannot explain the excretion differences found with testosterone manipulation, since the wky strain did not have significant kidney weight changes but did have excretion differences. the decrease in kidney weight in the shr/y strain that was castrated and its rescue in the castrates treated with testosterone supports the findings of others who have shown an anabolic effect of testosterone on kidney weight. blood pressure cannot be used to explain the electrolyte excretion differences with t manipulation since bp decreased after castration and na excretion increased. the bp profile for each strain and treatment group is similar to previously reported data from our lab showing decreased bp with castration and t treatment restored bp to control or above control levels. in a na balance study comparing shr and wky males, lundin et al. did not find increased shr urinary na excretion as expected due to high bp, but instead a decreased urinary na excretion was found. similar shr urinary na excretion results were found by osborn et al. with a reduced renal ability to excrete na, in spite of increased blood pressure. since t was not measured in osborn's studies, it is not known if the reduced na excretion may have been due to elevated t.  the excretion of na and k in both shr/y and wky males appear to be partially t dependent. in addition, the elevated na excretion in shr/y and wky following castration, further suggests that t can be an important player in renal na excretion. these differences in renal excretion of na and k cannot be solely and consistently explained by changes in bp or anabolic effects on kidney weight. in conclusion, the increased na and k excretion following castration, identification of renal ar transcripts, and loss of na with androgen blockade suggests that t is involved in the regulation of na balance. competing interests the author declare that they have no competing interests. authors' contributions de, am, jt, cj and mt designed and directed studies, were involved in the analysis of the data, and writing of the manuscript. gd, jt, mh and sb performed the experiments and ran the assays. all authors have read and approved the manuscript. 
therefore, the goal of this study was to determine if the yc combined with t manipulation could influence renal na and k excretion. rt-pcr was used to demonstrate the presence of renal androgen receptor transcripts. in another experiment the androgen receptor was blocked using flutamide in the diet. t influences electrolyte excretion through an androgen receptor dependent mechanism. to investigate the role of the yc and t, consomic borderline hypertensive and normotensive wistar-kyoto rat strains were used in three t treatment groups: castrate, castrate with t implant and gonadally intact males. there was not a differential yc involvement in electrolyte excretion between wky and shr/y males. plasma aldosterone was decreased in the presence of t. blockade of the ar resulted in a significant increase in na excretion but not in k excretion in both shr/y and wky males. urine was collected for na and k measurements by flame photometry. ar transcripts were identified in shr/y and wky kidneys. na and k excretion were decreased by t in shr/y and wky. the y-chromosome and testosterone increase blood pressure and may also influence renal electrolyte excretion.
 the y-chromosome and testosterone increase blood pressure and may also influence renal electrolyte excretion. therefore, the goal of this study was to determine if the yc combined with t manipulation could influence renal na and k excretion. methods to investigate the role of the yc and t, consomic borderline hypertensive and normotensive wistar-kyoto rat strains were used in three t treatment groups: castrate, castrate with t implant and gonadally intact males. urine was collected for na and k measurements by flame photometry. rt-pcr was used to demonstrate the presence of renal androgen receptor transcripts. plasma t and aldosterone were measured by ria. in another experiment the androgen receptor was blocked using flutamide in the diet.  na and k excretion were decreased by t in shr/y and wky. ar transcripts were identified in shr/y and wky kidneys. plasma aldosterone was decreased in the presence of t. blockade of the ar resulted in a significant increase in na excretion but not in k excretion in both shr/y and wky males.  t influences electrolyte excretion through an androgen receptor dependent mechanism. there was not a differential yc involvement in electrolyte excretion between wky and shr/y males. 