Human 12 and animal studies 3 have implicated Y-chromosome (Yc) loci as influencing the onset of male hypertension. In the Spontaneously Hypertensive Rat strain (SHR), the SHR Yc contains a locus which increases blood pressure (BP) approximately 20 mmHg compared to the normotensive Wistar Kyoto (WKY) 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 45. In examining phenotypes that mapped to the Yc and differed between SHR and WKY, our laboratory demonstrated an earlier rise of testosterone (T) levels in SHR males leading into puberty 4 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 6, 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 7. Reckelhoff et al. 8 reported that in SHR, T increased renal Na reabsorption, potentially through an androgen receptor (AR) 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 9, 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 10 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 11. Other studies comparing urinary Na excretion of SHR and WKY males 1213 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.

The following 3 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?

Gender differences in electrolyte excretion have been reported in rats and SHR females excrete more Na than males 1617. It makes sense that females would excrete more Na since in most mammals, females have a higher Na intake than males 18. 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 1920. Also there are reports of the presence of androgen receptors in female kidneys of rats and mice 2122. In our studies, the presence or absence of testosterone (T) 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 (AR) 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 23, and in other locations within the kidney (pars recta and cortical collecting duct (by real-time PCR) and low but detectable levels in medullary and cortical parts of the thick ascending limb of Henle's loop 24.

Androgens could also be operating through the transcriptional control of specific renal genes. Androgen regulated genes in the kidney include several organic anion transporters 23, which could be involved in the androgen produced retention of electrolytes 2425. 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 2–3 fold which was blocked by flutamide 26. This T effect on renal function is not unique to males. Ovarectomized females given T excreted less Na than ovarectomized females 27, further supporting the notion that there may be ARs present in the proximal tubules in both genders 28. To further support this data we found that SHR females on a high Na diet with T implants excreted less Na than intact females 27. 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 5 we found that male, female and testicular feminized males (mutant AR) had the same constant plasma Na levels (145, 146,144 meq/L, respectively) 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 29, AR free macrophages 30, and murine T cells 3132; whereby T mediates only ligand-induced Ca²⁺ import through non-voltage-gated Ca²⁺ channels 3132. 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 33. In support of this finding Vicencio 34 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 35. 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 35. Further studies strongly suggested that the specific inhibitory effect of T on aldosterone was through inhibition of aldosterone synthase 35. This is further supported by clinical observations in hyperandrogenic women suggesting that circulating T may inhibit adrenal 21- and/or 11B-hydroxylase activity 36. 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 (RAS) and specifically, angiotensinogen (AGT) has been shown to be modulated by sex hormones 37. Androgens can stimulate synthesis of AGT in animal models 38 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 39. 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 40. 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 41. Zeng et al. showed that AT1 and D1 receptors interacted differently in renal proximal tubule cells from SHR and WKY 42. 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 43. 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 444546.

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 4547 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. 12 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. 13 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.

The author(s) declare that they have no competing interests.

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.