Rita D. Sigmund

Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity

Rats on a low-NaCl diet have a high Na+ channel activity in colon and kidney. To address the mechanism of this increased activity, we measured mRNA levels of three Na+channel subunits in epithelial tissue (rENaC) from rats having been fed either a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. The size of the mRNA for each of the rENaC subunits as determined by Northern blot was unaffected by diet. RNase protection assay showed heterogeneity of response by organs and subunit. In lung, there was no effect of diet on any of the three subunits. In descending colon, the low-NaCl diet increased β- and γ-rENaC mRNA, with no effect on α-rENaC mRNA. In the kidney, the response to dietary NaCl was dependent on the region. In cortex and outer medulla, diet had no effect on any of the subunits. Rats fed the low-NaCl diet had greater α-rENaC in inner medulla but not β- or γ-rENaC mRNA. We next asked whether acute administration of pure glucocorticoid (GC) or mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the effects of a low-NaCl diet. Six hours after administration of GC or MC, a somewhat different heterogeneity occurred. In lung, α-rENaC mRNA was increased but only in response to GC. In colon, either GC or MC increased β- or γ-rENaC, and there was no effect on α-rENaC. In kidney, either GC or MC increased α-rENaC, without an effect on β- or γ-rENaC. In contrast to the response to a low-NaCl diet, all three regions were similarly affected by acute steroids. These results demonstrate a striking heterogeneity in response to physiological stimuli that regulate ENaC function. The mRNA levels of each of the rENaC subunits can be determined by the type of steroid and by factors unique to the organ and even to the specific region of the kidney.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

rENaC Is the Predominant Na + Channel in the Apical Membrane of the Rat Renal Inner Medullary Collecting Duct

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

Amiloride-sensitive Na+channels in pelvic uroepithelium involved in renal sensory receptor activation

Stretching the renal pelvic wall increases ipsilateral afferent renal nerve activity (ARNA). This response is enhanced by inhibiting Na+-K+-ATPase with ouabain, suggesting a modulatory role for intracellular Na+ in the activation of mechanosensitive neurons. The messenger RNA for alpha-, beta-, and gamma-subunits of epithelial Na+ channels (ENaC) is found in collecting duct cells. Because ENaC subunits show homology with genes involved in mechanosensation, we examined whether ENaC mRNA could be found in the pelvic wall and whether the ARNA response to increased renal pelvic pressure was modulated by blockers of the Na+ channel. alpha-, beta-, and gamma-subunits are present in the pelvis. The messenger RNA for the beta- and gamma-subunits is readily detected by in situ hybridization throughout the uroepithelium. The ARNA response to increased renal pelvic pressure was reduced by 53 +/- 10% and 40 +/- 10% (P < 0.01) by renal pelvic perfusion with the inhibitors amiloride and benzamil, respectively. Amiloride inhibited the ouabain-induced enhancement of the ARNA response to increased renal pelvic pressure. The magnitude of this inhibition was inversely correlated with the magnitude of the amiloride-mediated blockade of the ARNA response to increased renal pelvic pressure (P < 0.001). Amiloride also reduced the ARNA response to renal pelvic administration of substance P, a mediator of the ARNA response to increased renal pelvic pressure. We conclude that the ENaC complex in the pelvic uroepithelium participates in the activation of renal pelvic mechanosensitive neurons.Stretching the renal pelvic wall increases ipsilateral afferent renal nerve activity (ARNA). This response is enhanced by inhibiting Na+-K+-ATPase with ouabain, suggesting a modulatory role for intracellular Na+ in the activation of mechanosensitive neurons. The messenger RNA for α-, β-, and γ-subunits of epithelial Na+channels (ENaC) is found in collecting duct cells. Because ENaC subunits show homology with genes involved in mechanosensation, we examined whether ENaC mRNA could be found in the pelvic wall and whether the ARNA response to increased renal pelvic pressure was modulated by blockers of the Na+channel. α-, β-, and γ-subunits are present in the pelvis. The messenger RNA for the β- and γ-subunits is readily detected by in situ hybridization throughout the uroepithelium. The ARNA response to increased renal pelvic pressure was reduced by 53 ± 10% and 40 ± 10% ( P < 0.01) by renal pelvic perfusion with the inhibitors amiloride and benzamil, respectively. Amiloride inhibited the ouabain-induced enhancement of the ARNA response to increased renal pelvic pressure. The magnitude of this inhibition was inversely correlated with the magnitude of the amiloride-mediated blockade of the ARNA response to increased renal pelvic pressure ( P < 0.001). Amiloride also reduced the ARNA response to renal pelvic administration of substance P, a mediator of the ARNA response to increased renal pelvic pressure. We conclude that the ENaC complex in the pelvic uroepithelium participates in the activation of renal pelvic mechanosensitive neurons.

Osmoregulation of ceroid neuronal lipofuscinosis type 3 in the renal medulla

Recessive inheritance of mutations in ceroid neuronal lipofuscinosis type 3 (CLN3) results in juvenile neuronal ceroid lipofuscinosis (JNCL), a childhood neurodegenerative disease with symptoms including loss of vision, seizures, and motor and mental decline. CLN3p is a transmembrane protein with undefined function. Using a Cln3 reporter mouse harboring a nuclear-localized bacterial beta-galactosidase (beta-Gal) gene driven by the native Cln3 promoter, we detected beta-Gal most prominently in epithelial cells of skin, colon, lung, and kidney. In the kidney, beta-Gal-positive nuclei were predominant in medullary collecting duct principal cells, with increased expression along the medullary osmotic gradient. Quantification of Cln3 transcript levels from kidneys of wild-type (Cln3(+/+)) mice corroborated this expression gradient. Reporter mouse-derived renal epithelial cultures demonstrated a tonicity-dependent increase in beta-Gal expression. RT-quantitative PCR determination of Cln3 transcript levels further supported osmoregulation at the Cln3 locus. In vivo, osmoresponsiveness of Cln3 was demonstrated by reduction of medullary Cln3 transcript abundance after furosemide administration. Primary cultures of epithelial cells of the inner medulla from Cln3(lacZ/lacZ) (CLN3p-null) mice showed no defect in osmolyte accumulation or taurine flux, arguing against a requirement for CLN3p in osmolyte import or synthesis. CLN3p-deficient mice with free access to water showed a mild urine-concentrating defect but, upon water deprivation, were able to concentrate their urine normally. Unexpectedly, we found that CLN3p-deficient mice were hyperkalemic and had a low fractional excretion of K(+). Together, these findings suggest an osmoregulated role for CLN3p in renal control of water and K(+) balance.

Oxygen regulation of the epithelial Na channel in the collecting duct

The PO(2) within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing PO(2) on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O(2) concentration from 20 to 8% decreased ENaC activity by 40%; increasing O(2) content to 40% increased ENaC activity by 50%. The O(2) effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O(2) medium. Corticosteroids increased ENaC activity at each O(2) concentration; there was no interaction. The pathways for O(2) and steroid regulation of ENaC are different since O(2) did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2-45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O(2) appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O(2) concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O(2) induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O(2); both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.