I. Jeanette Lynch

The circadian clock protein Period 1 regulates expression of the renal epithelial sodium channel in mice

The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the alpha subunit of the epithelial sodium channel (alphaENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding alphaENaC in the presence or absence of aldosterone. Furthermore, expression of alphaENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal alphaENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance.

The renal H+-K+-ATPases: physiology, regulation, and structure

The H(+)-K(+)-ATPases are ion pumps that use the energy of ATP hydrolysis to transport protons (H(+)) in exchange for potassium ions (K(+)). These enzymes consist of a catalytic alpha-subunit and a regulatory beta-subunit. There are two catalytic subunits present in the kidney, the gastric or HKalpha(1) isoform and the colonic or HKalpha(2) isoform. In this review we discuss new information on the physiological function, regulation, and structure of the renal H(+)-K(+)-ATPases. Evaluation of enzymatic functions along the nephron and collecting duct and studies in HKalpha(1) and HKalpha(2) knockout mice suggest that the H(+)-K(+)-ATPases may function to transport ions other than protons and potassium. These reports and recent studies in mice lacking both HKalpha(1) and HKalpha(2) suggest important roles for the renal H(+)-K(+)-ATPases in acid/base balance as well as potassium and sodium homeostasis. Molecular modeling studies based on the crystal structure of a related enzyme have made it possible to evaluate the structures of HKalpha(1) and HKalpha(2) and provide a means to study the specific cation transport properties of H(+)-K(+)-ATPases. Studies to characterize the cation specificity of these enzymes under different physiological conditions are necessary to fully understand the role of the H(+)-K(+) ATPases in renal physiology.

The Circadian Protein Period 1 Contributes to Blood Pressure Control and Coordinately Regulates Renal Sodium Transport Genes

The circadian clock protein period 1 (Per1) contributes to the regulation of expression of the &agr; subunit of the renal epithelial sodium channel at the basal level and in response to the mineralocorticoid hormone aldosterone. The goals of the present study were to define the role of Per1 in the regulation of additional renal sodium handling genes in cortical collecting duct cells and to evaluate blood pressure (BP) in mice lacking functional Per1. To determine whether Per1 regulates additional genes important in renal sodium handling, a candidate gene approach was used. Immortalized collecting duct cells were transfected with a nontarget small interfering RNA or a Per1-specific small interfering RNA. Expression of the genes for &agr;-epithelial sodium channel and Fxyd5, a positive regulator of Na, K-ATPase activity, decreased in response to Per1 knockdown. Conversely, mRNA expression of caveolin 1, Ube2e3, and ET-1, all negative effectors of epithelial sodium channel, was induced after Per1 knockdown. These results led us to evaluate BP in Per1 KO mice. Mice lacking Per1 exhibit significantly reduced BP and elevated renal ET-1 levels compared with wild-type animals. Given the established role of renal ET-1 in epithelial sodium channel inhibition and BP control, elevated renal ET-1 is one possible explanation for the lower BP observed in Per1 KO mice. These data support a role for the circadian clock protein Per1 in the coordinate regulation of genes involved in renal sodium reabsorption. Importantly, the lower BP observed in Per1 KO mice compared with wild-type mice suggests a role for Per1 in BP control as well.

Regulation of αENaC expression by the circadian clock protein Period 1 in mpkCCDc14 cells

The epithelial sodium channel (ENaC) mediates the fine-tuned regulation of external sodium (Na) balance. The circadian clock protein Period 1 (Per1) is an aldosterone-induced gene that regulates mRNA expression of the rate-limiting alpha subunit of ENaC (αENaC). In the present study, we examined the effect of Per1 on αENaC in the cortex, the site of greatest ENaC activity in the collecting duct, and examined the mechanism of Per1 action on αENaC. Compared to wild type mice, Per1 knockout mice exhibited a 50% reduction of steady state αENaC mRNA levels in the cortex. Importantly, siRNA-mediated knockdown of Per1 decreased total αENaC protein levels in mpkCCD(c14) cells, a widely used model of the murine cortical collecting duct (CCD). Per1 regulated basal αENaC expression and participated in the aldosterone-mediated regulation of αENaC in mpkCCD(c14) cells. Because circadian clock proteins mediate their effects as part of multi-protein complexes at E-box response elements in the promoters of target genes, the ability of Per1 to interact with these sequences from the αENaC promoter was tested. For the first time, we show that Per1 and Clock are present at an E-box response element found in the αENaC promoter. Together these data support an important role for the circadian clock protein Per1 in the direct regulation of αENaC transcription and have important implications for understanding the role of the circadian clock in the regulation of renal function.

Aldosterone modulates steroid receptor binding to the endothelin-1 gene (Edn1)

Aldosterone and endothelin-1 (ET-1) act on collecting duct cells of the kidney and are important regulators of renal sodium transport and cardiovascular physiology. We recently identified the ET-1 gene (edn1) as a novel aldosterone-induced transcript. However, aldosterone action on edn1 has not been characterized at the present time. In this report, we show that aldosterone stimulated edn1 mRNA in acutely isolated rat inner medullary collecting duct cells ex vivo and ET-1 peptide in rat inner medulla in vivo. Aldosterone induction of edn1 mRNA occurred in cortical, outer medullary, and inner medullary collecting duct cells in vitro. Inspection of the edn1 promoter revealed two putative hormone response elements. Levels of heterogeneous nuclear RNA synthesis demonstrated that edn1 mRNA stimulation occurred at the level of transcription. RNA knockdowns corroborated pharmacological studies and demonstrated both mineralocorticoid receptor and glucocorticoid receptor participated in this response. Aldosterone resulted in dose-dependent nuclear translocation and binding of mineralocorticoid receptor and glucocorticoid receptor to the edn1 hormone response elements. Hormone receptors mediated the association of chromatin remodeling complexes, histone modification, and RNA polymerase II at the edn1 promoter. Direct interaction between aldosterone and ET-1 has important implications for renal and cardiovascular function.

Endothelin-1 inhibits sodium reabsorption by ETA and ETB receptors in the mouse cortical collecting duct

The collecting duct (CD) is a major renal site for the hormonal regulation of Na homeostasis and is critical for systemic arterial blood pressure control. Our previous studies demonstrated that the endothelin-1 gene (edn1) is an early response gene to the action of aldosterone. Because aldosterone and endothelin-1 (ET-1) have opposing actions on Na reabsorption (JNa) in the kidney, we postulated that stimulation of ET-1 by aldosterone acts as a negative feedback mechanism, acting locally within the CD. Aldosterone is known to increase JNa in the CD, in part, by stimulating the epithelial Na channel (ENaC). In contrast, ET-1 increases Na and water excretion through its binding to receptors in the CD. To date, direct measurement of the quantitative effect of ET-1 on transepithelial JNa in the isolated in vitro microperfused mouse CD has not been determined. We observed that the CD exhibits substantial JNa in male and female mice that is regulated, in part, by a benzamil-sensitive pathway, presumably ENaC. ENaC-mediated JNa is greater in the cortical CD (CCD) than in the outer medullary CD (OMCD); however, benzamil-insensitive JNa is present in the CCD and not in the OMCD. In the presence of ET-1, ENaC-mediated JNa is significantly inhibited. Blockade of either ETA or ETB receptor restored JNa to control rates; however, only ETA receptor blockade restored a benzamil-sensitive component of JNa. We conclude 1) Na reabsorption is mediated by ENaC in the CCD and OMCD and also by an ENaC-independent mechanism in the CCD; and 2) ET-1 inhibits JNa in the CCD through both ETA and ETB receptor-mediated pathways.

A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention

The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na(+) transport genes in the collecting duct, including the α-subunit of the renal epithelial Na(+) channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3β-dehydrogenase isomerase (3β-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na(+) retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal Na(+)-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na(+) compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na(+) retention.

Mineralocorticoids Stimulate the Activity and Expression of Renal H+,K+-ATPases

In the renal collecting duct, mineralocorticoids drive Na(+) reabsorption, K(+) secretion, and H(+) secretion through coordinated actions on apical and basolateral transporters. Whether mineralocorticoids act through H(+),K(+)-ATPases to maintain K(+) and acid-base homeostasis is unknown. Here, treatment of mice with the mineralocorticoid desoxycorticosterone pivalate (DOCP) resulted in weight gain, a decrease in blood [K(+)] and [Cl(-)], and an increase in blood [Na(+)] and [HCO(3)(-)]. DOCP treatment increased the rate of H(+),K(+)-ATPase-mediated H(+) secretion in intercalated cells of the inner cortical collecting duct. mRNA expression of the catalytic subunit HKα(1) did not significantly change, whereas HKα(2) mRNA expression dramatically increased in the outer and inner medulla of DOCP-treated mice. A high-K(+) diet abrogated this increase in renal HKα(2) expression, showing that DOCP-mediated stimulation of HKα(2) expression depends on dietary K(+) intake. DOCP treatment of mice lacking HKα(1) (HKα(1)(-/-)) resulted in greater urinary Na(+) retention than observed in either wild-type mice or mice lacking both HKα(1) and HKα(2) (HKα(1,2)(-/-)). DOCP-treated HKα(1,2)(-/-) mice exhibited a lower blood [HCO(3)(-)] and less Na(+) and K(+) retention than either wild-type or HKα(1)(-/-) mice. Taken together, these results indicate that H(+),K(+)-ATPases-especially the HKα(2)-containing H(+),K(+)-ATPases-play an important role in the effects of mineralocorticoids on K(+), acid-base, and Na(+) balance.

Heterogeneity of H-K-ATPase-mediated acid secretion along the mouse collecting duct

In the collecting duct (CD), H-K-ATPases function in cation reabsorption and H secretion. This study evaluated H-K-ATPase-mediated H secretion along the mouse CD, measured as EIPA- and luminal bafilomycin A(1)-insensitive intracellular pH (pH(i)) recovery from acute H loading (NH(4)) using BCECF. pH(i) recovery was measured in 1) microperfused cortical, outer medullary, and inner medullary CDs (CCD, OMCD, and IMCD) from C57BL/6J mice fed a normal diet and 2) common murine CD cell lines. H-K-ATPase activity along the native, microperfused CD was greatest in the CCD, less in the OMCD, and least in the IMCD (0.10 +/- 0.02, 0.04 +/- 0.01, and 0.01 +/- 0.002 U/min, respectively). H-K-ATPase activity was 0.30 +/- 0.03 and 0.26 +/- 0.03 in A- and B-type ICs, respectively, and was sensitive to Sch-28080 or ouabain. pH(i) recovery was greatest in the OMCD(1) cell line (0.25 +/- 0.01) and less in mpkCCD(c14) (0.17 +/- 0.01), mIMCD-K2 (0.12 +/- 0.01), and mIMCD-3 (0.05 +/- 0.01) cells. EIPA inhibited the majority of pH(i) recovery in these cells (100%, 64%, 75%, and 80% in mpkCCD(c14), OMCD(1), mIMCD-K2, and mIMCD-3, respectively). In OMCD(1) cells, where EIPA-insensitive pH(i) recovery was greatest, H-K-ATPase activity was 0.10 +/- 0.01 and was significantly inhibited (80%) by Sch-28080. We conclude that 1) H-K-ATPase-mediated H secretion in the native mouse CD is greatest in the ICs of the CCD, 2) A- and B-type ICs possess HKalpha(1) and HKalpha(2) H-K-ATPase activity, and 3) the OMCD(1) cell line best exhibits H-K-ATPase.

Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1

Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids.