Aldosterone, but not Increased Na⁺Influx or NF-κB Activation, Increases Kidney-specific WNK1 Gene Expression in Renal Collecting Duct Cells

Introduction

Kidneys play a chief role in the homeostasis of mammalian body fluid compartments. The fine-tuning of Na⁺balance occurs at the level of the renal collecting duct and is tightly controlled by hormonal factors. The collecting duct may reabsorb between 0 and 5% of the filtered sodium and may or may not reabsorb water. One of the major hormonal factors that positively controls Na⁺reabsorption in the collecting duct is aldosterone, which acts physiologically by intracellular binding of mineralocorticoid and glucocorticoid receptors and by positive and coordinated regulation of the activities of the apical Na⁺entry site (ENaC) and of basolateral Na,K-ATPase [1]. Recent studies shed light on an intriguing and previously unappreciated connection in the collecting duct between aldosterone-dependent Na⁺transport and the NF-κB transcriptional pathway, classically involved in the cellular signaling of inflammatory cytokines [2]: it was shown that aldosterone [3], or increased Na⁺influx [4], triggers NF-κB activation and, vice versa, that NF-κB-activating inflammatory agents stimulate Na⁺ transport in collecting duct cells [4].

WNKs (with-no-lysine [K]) are a recently discovered family of serine–threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of renal sodium and potassium transport was originally discovered by the findings that mutations of two members, WNK1 and WNK4, cause a rare form of salt-sensitive hypertension called pseudohypoaldosteronism type II [5]. A plethora of subsequent studies, using cultured cells and animal models, unraveled the molecular crosstalk that WNK1 and WNK4 display in the regulation of blood pressure by participating in a complex signaling cascade that affects the expression, localization, and activity of several ion transporters and co-transporters in the kidney epithelia [6]. Moreover, an association of polymorphisms of WNK genes with essential hypertension in the general population has been described [6]. Two alternative spliced isoforms of WNK1 exist: a full-length ubiquitous one (L-WNK1) and a kidney-specific shorter isoform lacking the kinase domain (KS-WNK1), which functionally antagonize each other [6]. Interestingly, the expression of KS-WNK1 is upregulated by aldosterone, and overexpressed KS-WNK1 increases Na⁺transport in collecting duct cells [7]. WNK4 also augments Na⁺transport in the collecting duct by increasing the activity of ENaC [6]. Furthermore, an additional molecular link between aldosterone and WNK1/WNK4 is provided by the aldosterone-induced protein serum– and glucocorticoid-regulated kinase (SGK1): activated L-WNK1 directly phosphorylates SGK1, which in turn phosphorylates WNK4, translating functionally into increased Na⁺reabsorption [6]. Given that accumulating evidence shows a link between aldosterone signaling/Na⁺transport and the inflammatory NF-κB pathway [3] [4], we designed this study to determine whether and how the gene expression of these signaling effectors of the WNK family (L-WNK1, KS-WNK1, and WNK4) is modulated by aldosterone, intracellular Na⁺ influx, and/or NF-κB-activating agents (tumor necrosis factor α [TNFα], lipopolysaccharide [LPS]) in the kidney collecting duct. To this aim, we used two well-established immortalized mouse kidney collecting duct cell lines, mpkCCD_c14 and mCCD_cl1. These cell lines differ in that the mpkCCD_c14 cells were derived from micro-dissected collecting ducts from mice expressing the large T antigen under the control of a truncated pyruvate kinase promoter fused to an early SV40 enhancer, while mCCD_cl1 cells were derived using the same micro-dissection procedure starting from wild-type mice [4] [8]. Functionally, however, both lines represent valuable collecting duct cell models, as they similarly retain transepithelial and aldosterone-regulated Na⁺transport [4] [8].

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