Reduced ENaC protein abundance contributes to the lower blood pressure observed in pendrin-null mice.

Pendrin (encoded by Pds, Slc26a4) is a Cl(-)/HCO(3)(-) exchanger expressed in the apical regions of type B and non-A, non-B intercalated cells of kidney and mediates renal Cl(-) absorption, particularly when upregulated. Aldosterone increases blood pressure by increasing absorption of both Na(+) and Cl(-) through increased protein abundance and function of Na(+) transporters, such as the epithelial Na(+) channel (ENaC) and the Na(+)-Cl(-) cotransporter (NCC), as well as Cl(-) transporters, such as pendrin. Because aldosterone analogs do not increase blood pressure in Slc26a4(-/-) mice, we asked whether Na(+) excretion and Na(+) transporter protein abundance are altered in kidneys from these mutant mice. Thus wild-type and Slc26a4-null mice were given a NaCl-replete, a NaCl-restricted, or NaCl-replete diet and aldosterone or aldosterone analogs. Abundance of the major renal Na(+) transporters was examined with immunoblots and immunohistochemistry. Slc26a4-null mice showed an impaired ability to conserve Na(+) during dietary NaCl restriction. Under treatment conditions in which circulating aldosterone is increased, alpha-, beta-, and 85-kDa gamma-ENaC subunit protein abundances were reduced 15-35%, whereas abundance of the 70-kDa fragment of gamma-ENaC was reduced approximately 70% in Slc26a4-null relative to wild-type mice. Moreover, ENaC-dependent changes in transepithelial voltage were much lower in cortical collecting ducts from Slc26a4-null than from wild-type mice. Thus, in kidney, ENaC protein abundance and function are modulated by pendrin or through a pendrin-dependent downstream event. The reduced ENaC protein abundance and function observed in Slc26a4-null mice contribute to their lower blood pressure and reduced ability to conserve Na(+) during NaCl restriction.

Dietary Cl(-) restriction upregulates pendrin expression within the apical plasma membrane of type B intercalated cells.

Pendrin, encoded by Slc26a4, is a Cl(-)/HCO(3)(-) exchanger expressed in the apical region of type B and non-A, non-B intercalated cells, which regulates renal NaCl excretion. Dietary Cl(-) restriction upregulates total pendrin protein expression. Whether the subcellular expression of pendrin and whether the apparent vascular volume contraction observed in Slc26a4 null mice are Cl(-) dependent, but Na(+) independent, is unknown. Thus the subcellular distribution of pendrin and its role in acid-base and fluid balance were explored using immunogold cytochemistry and balance studies of mice ingesting a NaCl-replete or a Na(+)-replete, Cl(-)-restricted diet, achieved through substitution of NaCl with NaHCO(3). Boundary length and apical plasma membrane pendrin label density each increased by approximately 60-70% in type B intercalated cells, but not in non-A, non-B cells, whereas cytoplasmic pendrin immunolabel increased approximately 60% in non-A, non-B intercalated cells, but not in type B cells. Following either NaCl restriction or Cl(-) restriction alone, Slc26a4 null mice excreted more Cl(-) and had a higher arterial pH than pair-fed wild-type mice. In conclusion, 1) following dietary Cl(-) restriction, apical plasma membrane pendrin immunolabel increases in type B intercalated cells, but not in non-A, non-B intercalated cells; and 2) pendrin participates in the regulation of renal Cl(-) excretion and arterial pH during dietary Cl(-) restriction.

Hypotension in NKCC1 null mice: role of the kidneys.

NKCC1 null mice are hypotensive, in part, from the absence of NKCC1-mediated vasoconstriction. Whether these mice have renal defects in NaCl and water handling which contribute to the hypotension is unexplored. Therefore, we asked 1) whether NKCC1 (-/-) mice have a defect in the regulation of NaCl and water balance, which might contribute to the observed hypotension and 2) whether the hypotension observed in these mice is accompanied by endocrine abnormalities and/or downregulation of renal Na+ transporter expression. Thus we performed balance studies, semiquantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (-/-) mice which consumed either a high (2.8% NaCl)- or a low-NaCl (0.01% NaCl) diet for 7 days. Blood pressure was lower in NKCC1 (-/-) than NKCC1 (+/+) mice following either high or low dietary NaCl intake. Relative to wild-type mice, NKCC1 null mice had a lower plasma ANP concentration, a higher plasma renin and a higher serum K+ concentration with inappropriately low urinary K+ excretion, although serum aldosterone was either the same or only slightly increased in the mutant mice. Expression of NHE3, the alpha-subunit of the Na-K-ATPase, NCC, and NKCC2 were higher in NKCC1 null than in wild-type mice, although differences were generally greater during NaCl restriction. NKCC1 null mice had a reduced capacity to excrete free water than wild-type mice, which resulted in hypochloremia following the NaCl-deficient diet. Hypochloremia did not occur from increased aquaporin-1 (AQP1) or 2 protein expression or from redistribution of AQP2 to the apical regions of principal cells. Instead, NKCC1 null mice had a blunted increase in urinary osmolality following vasopressin administration, which should increase free water excretion and attenuate the hypochloremia. In conclusion, aldosterone release is inappropriately low in NKCC1 null mice. Moreover, the action of aldosterone and vasopressin is altered within kidneys of NKCC1 null mice, which likely contributes to their hypotension. Increased Na+ transporter expression, increased plasma renin, and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thus minimizing hypotension.

Intercalated cell H+/OH- transporter expression is reduced in Slc26a4 null mice.

Slc26a4 (Pds) encodes pendrin, a Cl(-)/HCO(3)(-) exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH(-) equivalents. Thus genetic disruption of Slc26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in Slc26a4 (-/-) mice under basal conditions? 2) In the absence of pendrin-mediated OH(-) secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell H(+)/OH(-) transporter expression? To answer these questions, net acid excretion and H(+)/OH(-) transporter expression were examined in Slc26a4 (-/-) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and Pco(2) were much lower in Slc26a4 null relative to wild-type mice due to reduced urinary buffering of secreted H(+) by HCO(3)(-). Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of Slc26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced H(+)-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell H(+)/OH(-) transporter expression is observed in Slc26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4.