In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the ␣1 and ␤1 subunits. The ␥ variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

In the rat, the Na,K-ATPase alpha4 isoform exhibits unique enzymatic characteristics and is important for sperm motility. In this work, we studied expression, localization and function of alpha4 in human spermatozoa. We show two catalytically active Na,K-ATPase alpha polypeptides with different ouabain affinity and identified expression of alpha1, alpha4, beta1 and beta3 isoforms in the gametes. In addition, human sperm presented two Na,K-ATPases composed of alpha4, alpha4beta1 and alpha4beta3. Kinetic analysis of these isozymes produced in insect cells showed that, compared with human alpha1beta1, alpha4beta1 and alpha4beta3 exhibit higher Na(+) and lower K(+) affinity and higher sensitivity to ouabain. These particular enzymatic properties suggested a role for alpha4 in sperm function. Using computer-assisted sperm analysis (CASA), we found that ouabain inhibition of alpha4 significantly decreased percentage sperm motility. In contrast, ouabain did not affect linearity of forward progression, amplitude of lateral head displacement, beat cross frequency and sperm straight-line, curvilinear or average path velocities. This suggests a primary role of alpha4 in flagellar motility. Accordingly, we found alpha4 in the sperm tail, predominating in the mid-piece of the flagellum. Therefore, similar to the rat ortholog, human Na,K-ATPase alpha4 isoform has a distinct activity that is essential for sperm function.

In autosomal-dominant polycystic kidney disease (ADPKD), renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion. We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the EGF receptor (EGFR)-Src-MEK/ERK signaling pathway. We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers, ADPKD cell microcysts cultured in a three-dimensional collagen matrix, and metanephric organ cultures from Pkd1(m1Bei) mice. Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts. In contrast, in the presence of forskolin or 8-bromo-cAMP, ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion. Ouabain exerted this effect by enhancing cAMP-dependent Cl(-) secretion via the CFTR. Similarly, ouabain accelerated cAMP-dependent cyst enlargement in Pkd1(m1Bei) mice metanephroi, with a more prominent response in homozygous than heterozygous mice. Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys. The effects of ouabain in ADPKD cells and Pkd1(m1Bei) metanephroi were prevented by inhibitors of EGFR (AG1478), Src (PP2), and MEK (U0126). Together, our results show that ouabain, used in physiological concentrations, has synergistic effects on cAMP-mediated fluid secretion and cyst growth, via activation of the EGFR-Src-MEK pathway. These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression.

The Na,K-ATPase alpha4 (ATP1A4) isoform is specifically expressed in male germ cells and is highly prevalent in spermatozoa. Although selective inhibition of alpha4 activity with ouabain has been shown to affect sperm motility, a more direct analysis of the role of this isoform in sperm movement has not yet been demonstrated. To establish this, we engineered transgenic mice that express the rat alpha4 isoform fused to green fluorescent protein in male germ cells, under the control of the mouse protamine 1 promoter. We showed that the rat Atp1a4 transgene is expressed in mouse spermatozoa and that it is localized to the sperm flagellum. In agreement with increased expression of the alpha4 isoform, sperm from transgenic mice displayed higher alpha4-specific Na,K-ATPase activity and binding of fluorescently labeled ouabain than wild-type mice. In contrast, expression and activity of ATP1A1 (alpha1), the other Na,K-ATPase alpha isoform present in sperm, remained unchanged. Similar to wild-type mice, mice expressing the alpha4 transgene exhibited normal testis and sperm morphology and no differences in fertility. However, compared to wild-type mice, sperm from transgenic mice displayed plasma membrane hyperpolarization and higher total and progressive motility. Other parameters of motility also increased, including straight-line, curvilinear, and average path velocities and amplitude of lateral head displacement. In addition, sperm from the transgenic mice showed enhanced sperm hyperactive motility, but no changes in progesterone-induced acrosome reaction. Altogether, these results provide new genetic evidence for the role of the ATP1A4 isoform in sperm motility, under both noncapacitating and capacitating conditions.