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Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]

Supplementary Data from Development of a Small-Molecule Serum- and Glucocorticoid-Regulated Kinase-1 Antagonist and Its Evaluation as a Prostate Cancer Therapeutic

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]

The inhibition of nitric oxide production has been shown to reduce RBF. The effects of the nitric oxide synthase inhibitors, N omega-nitro-L-arginine and NG-monomethyl-L-arginine, on afferent and efferent arterioles isolated from rabbit kidneys were examined. Under basal conditions, N omega-nitro-L-arginine (10(-7) to 10(-3) M) had no effect on efferent arteriole lumen diameter but caused a 40% decrease in the lumen diameter of afferent arterioles. In afferent and efferent arterioles precontracted with norepinephrine, N omega-nitro-L-arginine and NG-monomethyl-L-arginine (3 x 10(-4) M) markedly attenuated the vasorelaxant effects of the endothelium-dependent vasodilator acetylcholine. In both arterioles, the inhibitory effect of N omega-nitro-L-arginine on acetylcholine-induced relaxation could be reversed by L- but not D-arginine (10(-3) M). However, N omega-nitro-L-arginine had no effect on the relaxation produced by the endothelium-independent vasodilators prostaglandin E2 (afferent) and dopamine (efferent). These observations demonstrate that under the in vitro conditions used in this study, afferent arterioles but not efferent arterioles synthesize and release nitric oxide in the basal state. However, both arterioles release nitric oxide in response to an endothelium-dependent vasodilator. The results of this study provide further evidence for an important role of nitric oxide in the regulation of renal hemodynamics.

Effects of vasopressin on the isolated perfused human collecting tubule. Cortical collecting tubules (CCT) were dissected from the surviving normal tissue of human kidneys removed at operation for either carcinoma or calculus. These CCT's were perfused in vitro shortly after the nephrectomy was performed. Transtubular potential differences in different tubules varied from + 3.2 to -2.0 mV and were reduced towards zero by lowering the temperature or by adding ouabain to the bath. In the absence of vasopressin, tubules were essentially impermeable to water with extremely low net water fluxes even in the presence of a transtubular osmotic gradient. Addition of vasopressin to the bath caused the transtubular osmotic water permeability coefficient to increase to values of 125, 175, and 155 × 10 -4 cm/sec in three tubules thus studied. These results demonstrate close similarities between the human CCT and the more extensively studied rabbit CCT. Effet de la vasopressine sur le tube collecteur humain isole et perfuse. Des tubes collecteurs corticaux (CCT) ont ete disseques a partir du tissu normal de rein humain obtenu dans des pieces operatoires de carcinome ou de lithiase. Ces CCT ont ete perfuses in vitro peu de temps apres le prelevement. La difference de potentiel trans-tubulaire dans les differents tubules a varie de +3,2 a -2,0 mV et elle a ete ramenee vers zero par l'abaissement de la temperature ou l'addition d'ouabaine au bain. En l'absence de vasopressine les tubes etaient impermeables a l'eau avec des flux nets d'eau extremement faibles meme en presence d'un gradient osmotique trans-tubulaire. L'addition de vasopressine au bain entraine une augmentation du coefficient de permeabilite osmotique a l'eau jusqu'a des valeurs de 125, 175, et 155 × 10 -4 cm/sec dans trois tubules ainsi etudies. Ces resultats demontrent de grandes ressemblances entre le CCT humain et celui, plus etudie, du lapin.

As a renal function declines in patients and experimental animals with chronic renal disease, potassium homeostasis is maintained by a progressive increase in potassium secretion by the surviving nephrons, a phenomenon known as potassium adaptation. To determine the nephron site and the underlying mechanisms responsible for this phenomenon, studies were performed on normal and 75% nephrectomized rabbits maintained on normal or high-potassium diets. Cortical collecting tubules (CCT) were dissected from the normal and remnant kidneys and perfused in vitro in an artificial solution. In normal CCT mean (+/- SE) net K secretion, JK, (peq/cm per s) was 1.26 +/- 0.43 (normal diet) and 3.27 +/- 0.66 (high-K diet). In uremic CCT, JK was 3.55 +/- 0.60 (normal diet) and 6.83 +/- 0.58 (high-K diet). By reducing the dietary intake of potassium in proportion to the reduction of renal mass in these uremic animals, the adaptation in K secretion was prevented (JK: 1.22 +/- 0.40). Transepithelial potential difference was similar in CCT from normal and uremic animals on a normal diet despite the fact that JK was significantly greater in the latter group. However, in both normal and uremic CCT, the increase in JK caused by potassium loading was associated with an increase in luminal negativity. Uremic CCT underwent significant compensatory hypertrophy regardless of the dietary intake or potassium secretory rates. Plasma aldosterone levels were elevated only in the uremic-high potassium rabbits suggesting that a mineralocorticoid effect on the CCT may be exaggerated when potassium loading is superimposed upon decreased excretory capacity. The activity of Na-K ATPase was comparable in normal and uremic CCT from rabbits on either normal or high-K diets indicating that potassium adaptation may occur independently of changes in the activity of this enzyme. Intracellular potassium content measured chemically and by 42K exchange, was not significantly altered in either normal or uremic CCT when dietary potassium intake was increased, despite the fact the JK was increased under these circumstances. These data indicate that the CCT is an important site of potassium adaptation in the surviving nephrons of animals with reduced renal mass. This adaptation is an intrinsic property of the CCT and is expressed in the absence of a uremic milieu. Potassium adaptation by the uremic CCT is not fixed according to the degree of compensatory hypertrophy but varies according to the excretory requirements of the animal. Transepithelial potential difference and circulating aldosterone levels contribute to the adaptation but neither factor can entirely account for the phenomenon. Potassium adaptation by the CCT occurs in the absence of changes in Na-K ATPase activity and intracellular potassium content.

The present studies examined whether vasopressin increases prostaglandin biosynthesis in isolated rabbit cortical collecting tubules (CCT) and whether endogenous prostaglandin biosynthesis plays a role in modulating the response of this nephron segment to vasopressin. Three groups of studies were performed. In the first group, CCT and proximal straight tubules (PST) were incubated with [3H]arachidonic acid, and metabolites were separated and identified using silica gel thin-layer chromatography. CCT were capable of producing all of the major prostaglandins (PG) (PGE2 > thromboxane B2[TxB2] > PGF2α > PGI2). PST produced significantly lesser quantities of these lipids. In the second group, radiolabeled arachidonic acid was incorporated into the phospholipid pool of both CCT and PST, vasopressin was added to the incubation medium, and metabolities were separated and identified as above. Vasopressin stimulated the release of all of the major prostaglandins in CCT but had no effect on PST. PGE release into the incubation medium, as assessed by a radioreceptor assay, increased 108%, and a vasopressin analogue, 1-desamino-8-d-arginine vasopressin, had a quantitatively similar effect. In the third group, a submaximal dose of vasopressin was administered to isolated, perfused CCT studied in the presence and absence of indomethacin to assess whether endogenous prostaglandins play a role in modulating the antidiuretic response to vasopressin. Studies were performed in rabbits on a normal diet and in desoxycorticosterone acetate (DOCA)- or KCl-loaded animals. In the state of mineralocorticoid excess, basal prostaglandin synthesis was 63% lower, and vasopressin-stimulated prostaglandin synthesis 76% lower, than the synthesis observed in rabbits on a normal diet. Cyclooxygenase inhibition exposed a significant hydroosmotic response to a submaximal dose of vasopressin in CCT from DOCA- or KCl-loaded animals. With arachidonic acid in the bath, the same dose of vasopressin failed to elicit a hydroosmotic response in CCT from rabbits on a normal diet even in the presence of a cyclooxygenase inhibitor. However, removal of exogenous arachidonic acid, with a consequently lower rate of prostaglandin synthesis, allowed the cyclooxygenase inhibitor to enhance the hydroosmotic response to vasopressin in these tubules. We conclude from these studies that the rabbit CCT has the capacity to synthesize all of the major prostaglandins and that the rate of synthesis of these lipids is enhanced by vasopessin. Prostaglandin synthesis by the CCT is postulated to modulate the antidiuretic action of vasopressin via a closed feedback loop. The effectiveness of this feedback regulation is dependent upon the mineralocorticoid status of the animal, which determines the level of basal and vasopressin-stimulated prostaglandin synthesis by the CCT.

Functional profile of the isolated uremic nephron: Intrinsic adaptation of phosphate transport in the rabbit proximal tubule. The maintenance of phosphate homeostasis in uremia appears to be governed both by parathyroid hormone (PTH) and by PTH-independent adaptations in renal tubular function. The relative contributions of these two mechanisms that control phosphate excretion by the diseased kidney have been difficult to define in intact animals. The present study was designed to examine the nature of the adaptation of phosphate handling by the proximal tubule of subtotally nephrectomized, uremic rabbits in vitro. Euparathyroid and hyperparathyroid uremic rabbits were studied. Tubular sensitivity to PTH was examined in vitro. The dose-response relationship between bath PTH concentration and inhibition of lumen-to-bath phosphate flux (Jp-lb) in isolated perfused proximal straight tubules (PSTs) revealed that PTH sensitivity was increased in the euparathyroid uremic rabbits and was decreased in hyperparathyroid uremic rabbits. The dose-response to dibutyryl cAMP was the same as normal in both uremic groups. These data strongly suggest the existence of a receptor-mediated adaptation in the effects of PTH on the uremic proximal tubule. In addition to an altered PTH-sensitivity the uremic PST also exhibited an alteration in the basal rate of phosphate transport studied in the presence of normal rabbit serum. Although net volume flux across the PST increased in both uremic groups as a possible consequence of hypertrophy, net phosphate flux per unit length was unchanged. Considering the increase of luminal area in these tubules, net phosphate flux per unit reabsorptive surface area was actually decreased. This dissociation is supportive of the existence of an intrinsic tubular adaptation which is independent of the size of the tubule per se. These studies indicate that there is an intrinsic adaptation of the basal rate of phosphate transport by the uremic rabbit proximal tubule and that the sensitivity of the tubule to PTH is altered. The data are strongly suggestive of an increase in the number of PTH receptors in the proximal tubule of the euparathyroid uremic rabbit and suggest that “down regulation” or persistent occupancy of these receptors occurs when hyperparathyroidism supervenes.Profil fonctionnel du néphron urémique isolé: Adaptation intrinsèque du transport de phosphates dans le tubule proximal de lapin. La conservation de l'homéostasie des phosphates dans l'urémie semble être gouvernée par l'hormone parathyroïdienne (PTH) et par une adaptation indépendante de la PTH de la fonction tubulaire rénale. La contribution relative de ces deux mécanismes de contrôle de l'excrétion de phosphates par le rein malade est difficile à définir chez des animaux intacts. Cette etude á été conçue pour examiner la nature de l'adaptation de la réabsorption de phosphates par le tubule proximal in vitro de lapins urémiques après néphrectomie sub-totale. Des lapins urémiques euparathyroïdiens et hyperparathyroïdiens ont été étudiés. La sensibilité tubulaire à la PTH a été examinée in vitro. La courbe dose-réponse entre la concentration de PTH du bain et l'inhibition du flux de phosphates de la lumière vers le bain (Jp-lb) dans des tubules proximales droites (PSTs) isolés perfusés a révélé que la sensibilité à la PTH était augmentée chez les lapins urémiques euparathyroïdiens et était diminuée chez les lapins urémiques hyperparathyroïdiens. La courbe dose-réponse au dibutyryl cAMP était la même dans les deux groupes urémiques. Ces données suggèrent fortement l'existence d'une adaptation médiée par des récepteurs des effets de la PTH dans le tubule proximal urémique. En plus d'une altération de la sensibilité à la PTH, le PST urémique avait également une altération de la vitesse basale de transport de phosphates étudiée en présence de sérum de lapin normal. Bien que le flux volumique net à travers le PST se soit élevé chez les deux groupes urémiques, représentant une conséquence possible de l'hypertrophie, le flux de phosphates net par unité de longueur était inchangé. En prenant en compte l'augmentation de la surface luminale de ces tubules, le flux net de phosphate par unité de surface de réabsorption était en fait diminué. Cette dissociation est en faveur de l'existence d'une adaptation tubulaire intrinsèque indépendante de la taille du tubule. Ces études indiquent qu'il y a une adaptation intrinsèque de la vitesse de base du transport de phosphates par le tubule proximal du lapin urémique et que la sensibilité du tubule à la PTH est altérée. Ces données suggèrent fortement une augmentation du nombre de récepteurs de la PTH dans le tubule proximal du lapin urémique euparathyroïdien et suggèrent qu'une ”régulation basse“ ou une occupation persistante de ces récepteurs se produit lorsque l'hyperparathyroïdisme s'installe.

In experimental models of glomerular and nonglomerular renal disease, single nephron filtration rate and proximal tubular reabsorption of fluid decrease or increase in parallel in the same nephron. To assess whether intrinsic adaptations in proximal tubular function, i.e., changes that are independent of the peritubular or humoral milieu, contribute to this phenomenon, segments of rabbit late superficial proximal convoluted tubules (PCT) were studied by in vitro perfusion. PCT were obtained from normal kidneys, from remnant kidneys, and from kidneys embolized with microspheres. Single nephron filtration rates are increased in the remnant and decreased in the embolized kidneys. Whereas the embolized-kidney rabbits were nonazotemic (the contralateral kidney was left in situ), the remnant-kidney animals were uremic. In order to study a nonazotemic model of increased single nephron filtration rate, PCT were also obtained from uninephrectomized rabbits. Significant compensatory hypertrophy occurred in the PCT of the remnant kidney. Net fluid reabsorption (Jv) per unit length was increased by approximately 60%; Jv per unit luminal surface area was the same as in the normal PCT. Transepithelial potential difference (PD) was significantly greater than normal. This was associated with a reversal of the normal permselective properties (P(Cl) > P(Na)) of the late superficial PCT so that P(Na) exceeded P(Cl). The changes could not be ascribed to some undetermined effect of the uremic state in vivo, since increases in tubule size, Jv per unit length, and PD also occurred in PCT from nonazotemic uninephrectomized rabbits. In contrast, Jv, per unit length or per unit luminal surface area, was decreased by approximately 50% in PCT from embolized kidneys and PD was also reduced. In these tubules, the normal permselective properties were also reversed. Tubule size, however, was not significantly different from normal. The increases or decreases in Jv that occurred in the different disease models were not dependent on tubular fluid flow rate or the uremic milieu in vitro. These studies indicate that intrinsic proximal tubular function is modified by the disease state in vivo and that the "memory" of this adaptation is expressed in the in vitro situation. The changes in Jv observed in vitro parallel the increases or decreases in single nephron filtration rates that occur in vivo. Compensatory hypertrophy, with an attendant increase in luminal surface area, could explain the increased Jv per millimeter in the remnant kidneys, but the adaptation observed in the embolized kidneys cannot be ascribed to changes in tubule size.

A B S T RA C T Resistance of the chronically diseased kidney to vasopressin has been proposed as a possible explanation for the urinary concentrating defect of uremia. The present studies examined the water permeability and adenylate cyclase responsiveness of isolated cortical collecting tubules (CCT) from remnant kidneys of uremic rabbits to vasopressin. In the absence of vasopressin the CCTs of both normal and uremic rabbits were impermeable to water. At the same osmotic gradient, addition of a supramaximal concentration ofvasopressin to the peritubular bathing medium led to a significantly lower net water flux per unit length (and per unit luminal surface area) in uremic CCTs than in normal CCTs. Transepithelial osmotic water permeability coefficient, Pf, was 0.0232 +0.0043 cm/s in normal CCTs and 0.0059+0.001 cm/s in uremic CCTs (P < 0.001). The impaired vasopressin responsiveness of the uremic CCTs was observed whether normal or uremic serum was present in the

An in vitro approach to the study of single nephron function in uremia has been employed in evaluating the control of fluid reabsorption by the renal superficial proximal straight tubule (PST). Isolated segments of PSTs from the remnant kidneys of uremic rabbits (stage III) were perfused in vitro and their rate of fluid reabsorption compared with normal PSTs and with PSTs derived from the remnant kidneys of nonuremic rabbits (stage II). All segments were exposed to a peritubular bathing medium of both normal and uremic rabbit serum thereby permitting a differentiation to be made between adaptations in function which are intrinsic to the tubular epithelium and those which are dependent upon a uremic milieu.Compared with normal and stage II PSTs, there was significant hypertrophy of the stage III tubules as evidenced by an increase in length and internal diameter, and a twofold increase in the dry weight per unit length. Fluid reabsorption per unit length of tubule was 70% greater in stage III than in normal and stage II PSTs, and was closely correlated with the increase in dry weight. Substitutions between normal and uremic rabbit serum in the peritubular bathing medium did not affect fluid reabsorption significantly in any of the three groups of PSTs. Perfusion of the tubules with an ultrafiltrate of normal vs. uremic serum likewise failed to influence the rate of net fluid reabsorption. It has previously been observed that net fluid secretion may occur in nonperfused or stop-flow perfused normal rabbit PSTs exposed to human uremic serum. Additional studies were thus performed on normal and stage III PSTs to evaluate whether net secretion occurs in the presence of rabbit uremic serum. No evidence for net secretion was found. These studies demonstrate that fluid reabsorption is greatly increased in the superficial PST of the uremic remnant kidney and that this functional adaptation is closely correlated with compensatory hypertrophy of the segment. Humoral factors in the peritubular environment do not appear to be important mediators of the enhanced fluid reabsorption.

Attempts to establish a primary cell culture from a well-defined segment of the mammalian nephron have been fraught with difficulties, and at present no such cell line exists [1]. Handler et al have recently established and maintained a line of toad bladder epithelial cells in culture and have shown that these cells form a transporting epithelial monolayer that is responsive to specific hormonal stimulation [2]. Our initial efforts to achieve the same objective with mammalian tubular epithelial cells have not met with the same success, presumably due to inadequacies in the composition of the culture media used.

Obstructive uropathy is associated with changes in several renal functions. As some of these are mediated by cAMP, we examined whether changes in cAMP metabolism could contribute to the pathophysiology of unilateral ureteral ligation in rats. We found that basal adenylate cyclase in the cortex of the obstructed kidney doubled after 3-7 days, but the response to NaF and parathyroid hormone was decreased. Similarly, in vivo infusion of parathyroid hormone resulted in lower cAMP levels in the cortex of the obstructed kidney. Parathyroidectomy or pretreatment with beta-adrenergic blocker had no effect. Prostaglandin inhibition by indomethacin decreased, but did not abolish, the differences.On the other hand, basal adenylate cyclase in glomeruli from contralateral kidneys was higher. Pretreatment with indomethacin reduced the basal activity in contralateral glomeruli, and led to increased degrees of stimulation with parathyroid hormone and prostaglandins.Furthermore, we demonstrated that isolated glomeruli contain cAMP-dependent protein kinase that is found predominantly in the 30,000 g supernatant fraction and that by DEAE-cellulose chromatography has the characteristics of a type II kinase. In glomeruli from rats with unilateral ureteral ligation, the distribution and degree of activation of the cAMP-dependent protein kinase are affected differently on the ligated and contralateral sides, probably reflecting the change in glomerular cAMP generation.These results indicate that changes in cAMP metabolism may contribute to the altered cortical tubular function in unilateral ureteral ligation and may be partially related to enhanced prostaglandin synthesis in the obstructed kidney. On the other hand, the higher adenylate cyclase activity and the change in cAMP-dependent protein kinase activity in glomeruli from the contralateral kidney may in part reflect increased PGE2 production by these glomeruli. The results support the concept that similar to their in vitro action, locally generated prostaglandins may in vivo affect cortical tubular function by influencing the tubular cAMP system while those generated in the glomerulus alter glomerular cAMP metabolism.

Summary: Guanylate cyclase, cGMP phosphodiesterase and protein kinase activities were determined in kidneys of developing and adult rats. Guanylate cyclase activities of crude kidney homogenates, 100,000 × g supernatant and pellet of 7− and 21-day-old and adult rats were determined (Table I). In the kidneys of 7-day-old rats activity was 162% of adult controls in the homogenates (P < .001), 144% in the soluble (P < .005) and 308% in the particulate fraction (P < .001). In 3-week-old rats activity was still significantly higher at 144% in the homogenate (P < 0.02) and at 225% in the particulate (P < .001). Phosphodiesterase activity for cGMP was 7488 ± 831 pmol cGMP/mg protein · min in 1-week-old and 7674 ± 1120 in 3-week-old rats vs. 4042 ± 122 in the adults (P < .025) (Table II). Chromatography on Sephadex G-200 showed two peaks of cGMP-stimulatable protein kinase in both the adult and newborn kidney and in addition a minor peak of cGMP-stimulatable kinase in the newborn kidney only (Fig. 2). Speculation: These results provide further evidence for altered cGMP metabolism during kidney development. Whether cGMP is directly involved in organ growth remains open to speculation.