Novel role of ouabain as a cystogenic factor in autosomal dominant polycystic kidney disease

Abstract

The classic role of the Na-K-ATPase is that of a primary active transporter that utilizes cell energy to establish and maintain transmembrane Na(+) and K(+) gradients to preserve cell osmotic stability, support cell excitability, and drive secondary active transport. Recent studies have revealed that Na-K-ATPase located within cholesterol-containing lipid rafts serves as a receptor for cardiotonic steroids, including ouabain. Traditionally, ouabain was viewed as a toxin produced only in plants, and it was used in relatively high concentrations to experimentally block the pumping action of the Na-K-ATPase. However, the new and unexpected role of the Na-K-ATPase as a signal transducer revealed a novel facet for ouabain in the regulation of a myriad of cell functions, including cell proliferation, hypertrophy, apoptosis, mobility, and metabolism. The seminal discovery that ouabain is endogenously produced in mammals and circulates in plasma has fueled the interest in this endogenous molecule as a potentially important hormone in normal physiology and disease. In this article, we review the role of the Na-K-ATPase as an ion transporter in the kidney, the experimental evidence for ouabain as a circulating hormone, the function of the Na-K-ATPase as a signal transducer that mediates ouabain's effects, and novel results for ouabain-induced Na-K-ATPase signaling in cystogenesis of autosomal dominant polycystic kidney disease.

Keywords: Na-K-ATPase signalosome; cardiotonic steroids; polycystic kidney disease.

Fig. 1.

Diagram showing Na-K-ATPase control of cell function by ouabain via its ion transport and signaling functions. A: by inhibiting the Na-K-ATPase pumping mode, ouabain can directly modify intracellular Na⁺ levels and secondarily cytosolic Ca²⁺ via the Na/Ca exchanger (NCX). This represents the well-described mechanism of action of the cardiotonic steroids that lead to cardiac- and smooth muscle-enhanced contraction. B: by acting through a subpopulation of Na-K-ATPase in caveolae, ouabain activates the Na-K-ATPase signaling pathway, which involves a series of intracellular intermediates that ultimately modulate gene expression and cause a variety of cell type-specific effects, such as those listed in the figure. O, ouabain, (α and β) subunits of the Na-K-ATPase. See the text for additional definitions.

Fig. 2.

Evolution of autosomal dominant polycystic kidney disease (ADPKD) cysts from epithelial cells of the renal tubules. Polycystic kidney (left), result from the growth of multiple fluid-filled cysts that originate in the renal tubules (middle) and continue developing through different pathophysiological mechanisms (right). Cysts evolve from individual cells carrying a germ-line mutation in one of the alleles for the Pkd1 or Pkd2 genes (right). Every cell in the tubule carries a mutated allele, and a somatic mutation or haploinsufficiency of the second allele is believed to trigger the disease (cells marked with stars). Initially, cell proliferation leads to the development of blister-like structures. Several abnormalities, including cell cilia malfunction, alteration in planar cell polarity, and remodeling of the extracellular matrix are also implicated in ADPKD cystogenesis. Once the cysts separate from the nephron that originated them, they continue growing through cell proliferation and transepithelial fluid secretion into the expanding cyst cavity. In addition to the genetic abnormality, nongenetic factors, such as several circulating agents, aid to enhance cyst growth and determine the progression of the disease.

Fig. 3.

Ouabain stimulates proliferation of ADPKD cells. A: physiological concentrations of ouabain stimulate the growth of human renal epithelial ADPKD cells but has only a slight effect on normal human kidney epithelial cells (NHK). Values are expressed as percentage of untreated controls, without ouabain. Modified from Ref. . B: pathway by which ouabain enhances cell proliferation and fluid secretion in ADPKD cells. Levels of ouabain far below those needed to completely block Na-K-ATPase pumping, bind to the basolaterally located Na-K-ATPase of ADPKD cells and triggers the Na-K-ATPase signaling machinery in the cell caveolae. Ouabain binding induces association of the Na-K-ATPase to EGFR and the kinase Src. Activation of Src initiates the phosphorylation of the B-Raf-MEK and ERK cascade. This, by actions that may be direct or indirect, leads to downregulation of the cyclin kinase inhibitors p21 and p27, relieving inhibition of the cell cycle to favor the increase in cell growth. O, ouabain, (α and β) subunits of the Na-K-ATPase; Cav, caveolin.

Fig. 4.

Ouabain stimulates cAMP-induced fluid secretion in ADPKD monolayers. A: physiological levels of ouabain (3 nM) enhanced the movement of transepithelial basolateral to apical fluid in the presence of 5 μM forskolin but had no effect when used alone. Ouabain, either alone or in the presence of forskolin, did not affect fluid secretion of NHK cells. *Significant differences (P < 0.05). (Modified from reference 85). B: pathway involved in the activation of cAMP-dependent fluid secretion in ADPKD cells. Physiological amounts of ouabain, acting on the Na-K-ATPase signalosome, stimulates Src-dependent phosphorylation and the downstream activation of the B-Raf-MEK and ERK cascade. This, by actions that may be direct or indirect, produces activation of the CFTR, enhancing Cl⁻ movement across the apical membrane of ADPKD cells by a mechanism that has not yet been determined. Also, low physiological concentrations of ouabain partially inhibit Na-K-ATPase activity, which may slightly reduce Na⁺ and water reabsorption by the epithelium. Both of these actions of ouabain may shift the reabsorption/secretion balance of the epithelium to favor fluid secretion and help cystogenesis.

Fig. 5.

Ouabain stimulates ADPKD cystogenesis. A: ouabain alone (3 nM) had no effect but enhanced the increase in size of microcysts caused by forskolin in ADPKD cells grown in a 3-dimensional collagen matrix. In contrast, ouabain did not significantly influence the growth of NHK cells. Top: microphotographs of representative ADPKD microcysts in the absence and presence of ouabain with or without added forskolin. Bottom: average total surface area of the formed microcysts. B: ouabain induces tubular dilations in metanephric organ cultures from Pkd1^m1Bei mice (Pkd1^−/−), an established model of ADPKD. Ouabain (30 nM) enhanced the effect of the cell-permeable cAMP analog 8-Br-cAMP to increase cyst area (volume) in intact embryonic kidneys from Pkd1^m1Bei mice, but had little effect on metanephroi from wild-type mice. Ouabain by itself did not induce cystic dilations in either wild-type or Pkd1^m1Bei mice organ cultures. Top: representative images of metanephroi. Bottom: average fractional cyst area for the different experimental conditions. *Significant differences (P < 0.05). Modified from reference .