The Forgotten Antiproteinuric Properties of Diuretics.

BACKGROUND: Although diuretics are one of the most widely used drugs by nephrologists, their antiproteinuric properties are not generally taken into consideration. SUMMARY: Thiazide diuretics have been shown to reduce proteinuria by >35% in several prospective controlled studies, and these values are markedly increased when combined with a low-salt diet. Thiazide-like diuretics (indapamide and chlorthalidone) have shown similar effectiveness. The antiproteinuric effect of mineralocorticoid receptor antagonists (spironolactone, eplerenone, and finerenone) has been clearly established through prospective and controlled studies, and treatment with finerenone reduces the risk of chronic kidney disease progression in type-2 diabetic patients. The efficacy of other diuretics such as amiloride, triamterene, acetazolamide, or loop diuretics has been less explored, but different investigations suggest that they might share the same antiproteinuric properties of other diuretics that should be evaluated through controlled studies. Although the inclusion of sodium-glucose cotransporter-2 inhibitors (SGLT2i) among diuretics is a controversial issue, their renoprotective and cardioprotective properties, confirmed in various landmark trials, constitute a true revolution in the treatment of patients with kidney disease. Recent subanalyses of these trials have shown that the early antiproteinuric effect induced by SGLT2i predicts long-term preservation of kidney function. Key Message: Whether the early reduction in proteinuria induced by diuretics other than finerenone and SGLT2i, as summarized in this review, also translates into long-term renoprotection requires further prospective and observational studies. In any case, it is important for the clinician to be aware of the antiproteinuric properties of drugs so often used in daily clinical practice.

Kir4.1/Kir5.1 Activity Is Essential for Dietary Sodium Intake-Induced Modulation of Na-Cl Cotransporter.

BACKGROUND: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. METHODS: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. RESULTS: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K+ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K+ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K+ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K+ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. CONCLUSIONS: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Glucocorticoid-induced leucine zipper protein regulates sodium and potassium balance in the distal nephron.

Glucocorticoid induced leucine zipper protein (GILZ) is an aldosterone-regulated protein that controls sodium transport in cultured kidney epithelial cells. Mice lacking GILZ have been reported previously to have electrolyte abnormalities. However, the mechanistic basis has not been explored. Here we provide evidence supporting a role for GILZ in modulating the balance of renal sodium and potassium excretion by regulating the sodium-chloride cotransporter (NCC) activity in the distal nephron. Gilz-/- mice have a higher plasma potassium concentration and lower fractional excretion of potassium than wild type mice. Furthermore, knockout mice are more sensitive to NCC inhibition by thiazides than are the wild type mice, and their phosphorylated NCC expression is higher. Despite increased NCC activity, knockout mice do not have higher blood pressure than wild type mice. However, during sodium deprivation, knockout mice come into sodium balance more quickly, than do the wild type, without a significant increase in plasma renin activity. Upon prolonged sodium restriction, knockout mice develop frank hyperkalemia. Finally, in HEK293T cells, exogenous GILZ inhibits NCC activity at least in part by inhibiting SPAK phosphorylation. Thus, GILZ promotes potassium secretion by inhibiting NCC and enhancing distal sodium delivery to the epithelial sodium channel. Additionally, Gilz-/- mice have features resembling familial hyperkalemic hypertension, a human disorder that manifests with hyperkalemia associated variably with hypertension.

Potassium Supplementation Prevents Sodium Chloride Cotransporter Stimulation During Angiotensin II Hypertension.

Angiotensin II (AngII) hypertension increases distal tubule Na-Cl cotransporter (NCC) abundance and phosphorylation (NCCp), as well as epithelial Na(+) channel abundance and activating cleavage. Acutely raising plasma [K(+)] by infusion or ingestion provokes a rapid decrease in NCCp that drives a compensatory kaliuresis. The first aim tested whether acutely raising plasma [K(+)] with a single 3-hour 2% potassium meal would lower NCCp in Sprague-Dawley rats after 14 days of AngII (400 ng/kg per minute). The potassium-rich meal neither decreased NCCp nor increased K(+) excretion. AngII-infused rats exhibited lower plasma [K(+)] versus controls (3.6±0.2 versus 4.5±0.1 mmol/L; P<0.05), suggesting that AngII-mediated epithelial Na(+) channel activation provokes K(+) depletion. The second aim tested whether doubling dietary potassium intake from 1% (A1K) to 2% (A2K) would prevent K(+) depletion during AngII infusion and, thus, prevent NCC accumulation. A2K-fed rats exhibited normal plasma [K(+)] and 2-fold higher K(+) excretion and plasma [aldosterone] versus A1K. In A1K rats, NCC, NCCpS71, and NCCpT53 abundance increased 1.5- to 3-fold versus controls (P<0.05). The rise in NCC and NCCp abundance was prevented in the A2K rats, yet blood pressure did not significantly decrease. Epithelial Na(+) channel subunit abundance and cleavage increased 1.5- to 3-fold in both A1K and A2K; ROMK (renal outer medulla K(+) channel abundance) abundance was unaffected by AngII or dietary K(+) In summary, the accumulation and phosphorylation of NCC seen during chronic AngII infusion hypertension is likely secondary to potassium deficiency driven by epithelial Na(+) channel stimulation.

Urinary extracellular vesicles as markers to assess kidney sodium transport.

PURPOSE OF REVIEW: This article summarizes studies that have analyzed sodium transporters in urinary extracellular vesicles (uEVs) in relation to hypertension. RECENT FINDINGS: The majority of kidney sodium transporters are detectable in uEVs. Patients with loss or gain of function mutations in sodium transporter genes have concomitant changes in the abundances of their corresponding proteins in uEVs. The effects of aldosterone on kidney sodium transport, including activation of the sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC), are transferred to uEVs as increases in phosphorylated NCC and the γ-subunit of ENaC. Specific forms of hypertension, including aldosteronism and pseudohypoaldosteronism, are characterized by higher abundances of total or phosphorylated NCC in uEVs. The proteolytic processing of ENaC by urinary proteases is detectable in uEVs as cleaved γ-ENaC, as demonstrated in hypertensive patients with diabetic nephropathy. Analysis of uEVs from patients with essential or salt-sensitive hypertension identified potential candidates for uEV markers of hypertension, including retinoic acid-induced gene 2 protein and hsa-miR-4516. SUMMARY: Analysis of sodium transporters in uEVs is a promising approach to study renal epithelial transport processes noninvasively in human hypertension. VIDEO ABSTRACT: http://links.lww.com/CONH/A16.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway.

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

A novel target for diuretic therapy.

The reabsorption of salt in the distal nephron is predominantly mediated via the thiazide-sensitive sodium chroride cotransporter, NCC (SLC12A3), and the chloride-bicarbonate exchanger pendrin (SLC26A4, PDS), with pendrin working in tandem with the epithelial sodium channel and NCC working by itself. Single deletion of NCC or pendrin in genetically engineered mouse models does not cause salt wasting or excessive diuresis under basal conditions. Both pendrin knockout and NCC knockout mice, however, show signs of volume depletion or develop hypotension during salt restriction. These findings have led investigators to conclude that pendrin and NCC are predominantly active during salt depletion and their contribution to salt reabsorption at baseline conditions is small. We hypothesized that pendrin may compensate for loss of NCC under basal conditions, thereby masking the role that each transporter plays in salt reabsorption. To test this hypothesis, double knockout of pendrin and sodium chloride cotransporter was generated by crossing animals with single deletion for NCC and pendrin. The double-knockout mice show significant salt and fluid wasting, along with severe volume depletion, metabolic alkalosis and prerenal failure under baseline conditions. Volume depletion, metabolic alkalosis and prerenal failure were significantly corrected with salt repletion. We conclude that pendrin plays an essential role in the distal tubule salt reabsorption in the setting of sodium-chloride cotransporter inactivation. We propose that pendrin could be a novel target for a new diuretic that in conjunction with thiazide can be an effective regimen for patients with fluid overload.

The phosphorylated sodium chloride cotransporter in urinary exosomes is superior to prostasin as a marker for aldosteronism.

Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain several disease-associated proteins and are thus useful targets for identifying biomarkers of disease. Here, we hypothesized that the phosphorylated (active) form of the sodium chloride cotransporter (pNCC) or prostasin could serve as biomarkers for aldosteronism. We tested this in 2 animal models of aldosteronism (aldosterone infusion or low-sodium diet) and in patients with primary aldosteronism. Urinary exosomes were isolated from 24-hour urine or spot urine using ultracentrifugation. In rats, a normal or a high dose of aldosterone for 2, 3, or 8 days increased pNCC 3-fold in urinary exosomes (P<0.05 for all). A low-sodium diet also increased pNCC in urinary exosomes approximately 1.5-fold after 4 and after 8 days of treatment. The effects of these maneuvers on prostasin in urinary exosomes were less clear, showing a significant 1.5-fold increase only after 2 and 3 days of high-aldosterone infusion. In urinary exosomes of patients with primary aldosteronism, pNCC was 2.6-fold higher (P<0.05) while prostasin was 1.5-fold higher (P=0.07) than in patients with essential hypertension. Urinary exosomal pNCC and, to a lesser extent, prostasin are promising markers for aldosteronism in experimental animals and patients. These markers may be used to assess the biological activity of aldosterone and, potentially, as clinical biomarkers for primary aldosteronism.

Pathogenesis of calcineurin inhibitor-induced hypertension.

This article reviews the current understanding of the mechanisms of calcineurin inhibitor-induced hypertension. Already early after the introduction of cyclosporine in the 1980s, vasoconstriction, sympathetic excitation and sodium retention by the kidney had been shown to play a role in this form of hypertension. The vasoconstrictive effects of calcineurin inhibitors are related to interference with the balance of vasoactive substances, including endothelin and nitric oxide. Until recently, the renal site of the sodium-retaining effect of calcineurin inhibitors was unknown. We and others have shown that calcineurin inhibitors increase the activity of the thiazide-sensitive sodium chloride cotransporter through an effect on the kinases WNK and SPAK. Here, we review the pertinent literature on the hypertensinogenic effects of calcineurin inhibitors, including neural, vascular and renal effects, and we propose an integrated model of calcineurin inhibitor-induced hypertension.

AT2 receptor non-peptide agonist C21 promotes natriuresis in obese Zucker rats.

Previously, we demonstrated that angiotensin II type 2 (AT(2)) receptors have a role in natriuresis in obese Zucker rats (OZR). In the present study, we investigated the role of a novel, non-peptide agonist, C21, in natriuresis via AT(2) receptor activation in OZR. Infusion of C21 (1 and 5 µg kg(-1) min(-1)) into rats under anesthesia caused a dose-dependent increase in urine flow (UF) and urinary Na volume (U(Na)V). These effects of C21 were blocked by pre-infusion of the AT(2) receptor antagonist, PD123319, (50 µg kg(-1) min(-1)), suggesting involvement of the AT(2) receptor. Infusion of C21 (5 µg kg(-1) min(-1)) significantly increased the fractional excretion of sodium without changing the glomerular filtration rate or blood pressure, suggesting a tubular effect. Similarly, C21 infusion increased the fractional excretion of lithium, suggesting a proximal tubular effect. Furthermore, we tested the effect of C21 on natriuresis after blocking two main, distal-nephron Na transporters, the epithelial Na channels (ENaC), with amiloride (AM, 3 mg kg(-1) body wt), and the NaCl cotransporters (NCC), with bendroflumethiazide (BFTZ, 7 mg kg(-1) body wt). Infusion of AM + BFTZ caused significant increases in both diuresis and natriuresis, which were further increased by infusion of C21 (5 µg kg(-1) min(-1)). Natriuresis in response to C21 was associated with increases in urinary NO and cGMP levels. The data indicate that the AT(2) receptor agonist, C21, promotes natriuresis via AT(2) receptor activation and that this effect is potentially based in the proximal tubules and linked to the nitric oxide/cyclic guanosine monophosphate pathway. The natriuretic response to C21 may have therapeutic significance by improving kidney function in obesity.

Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects.

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 µg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of ßENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 µg/day) together with aldosterone (12 µg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.

Identification of a novel target of thiazide diuretics.

Thiazide diuretics are the older but still one of the most effective therapies for human hypertension. They are believed to act exclusively by blocking renal sodium absorption by the NaCl cotransporter NCC. We recently identified, however, a novel NaCl transport system that is expressed in intercalated cells of the collecting duct. This novel mechanism of NaCl transport operates by the combined action of 2 chloride/bicarbonate exchangers, 1 sodium-independent and 1 sodium-dependent. We propose that part of the action of thiazide occurs through blockade of this novel system.

Novel mechanisms for NaCl reabsorption in the collecting duct.

PURPOSE OF REVIEW: There is consensus that the abnormal retention of sodium by the kidney is the most important pathophysiological event in hypertension. The present review summarizes our current understanding of sodium reabsorption in the distal nephron. RECENT FINDINGS: The antihypertensive effect of thiazides is thought to be mediated by inhibiting Na+ uptake via the NaCl cotransporter NCC in the distal convoluted tubule. Although it was known that thiazide-sensitive Na⁺ reabsorption in isolated cortical collecting ducts can occur independently of the epithelial Na⁺ channel ENaC, its molecular correlate was unresolved. It was absent in isolated cortical collecting ducts of mice with a targeted disruption of the Na⁺-driven chloride/bicarbonate exchanger NDCBE suggesting that this pathway involves apical Na⁺ uptake into intercalated cells via the Na⁺-driven anion-exchanger NDCBE (SLC4A8). SUMMARY: The finding that SLC4A8-dependent thiazide-sensitive Na⁺ reabsorption occurs in the cortical collecting duct challenges our current model of how thiazides mediate their antihypertensive action and identifies a potentially new target for antihypertensive strategies.

Angiotensin II induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter independent of aldosterone.

We studied here the independent roles of angiotensin II and aldosterone in regulating the sodium chloride cotransporter (NCC) of the distal convoluted tubule. We adrenalectomized three experimental and one control group of rats. Following surgery, the experimental groups were treated with either a high physiological dose of aldosterone, a non-pressor, or a pressor dose of angiotensin II for 8 days. Aldosterone and both doses of angiotensin II lowered sodium excretion and significantly increased the abundance of NCC in the plasma membrane compared with the control. Only the pressor dose of angiotensin II caused hypertension. Thiazides inhibited the sodium retention induced by the angiotensin II non-pressor dose. Both aldosterone and the non-pressor dose of angiotensin II significantly increased phosphorylation of NCC at threonine-53 and also increased the intracellular abundance of STE20/SPS1-related, proline alanine-rich kinase (SPAK). No differences were found in other modulators of NCC activity such as oxidative stress responsive protein type 1 or with-no-lysine kinase 4. Thus, our in vivo study shows that aldosterone and angiotensin II independently increase the abundance and phosphorylation of NCC in the setting of adrenalectomy; effects are likely mediated by SPAK. These results may explain, in part, the hormonal control of renal sodium excretion and the pathophysiology of several forms of hypertension.

Effects of dietary K on cell-surface expression of renal ion channels and transporters.

Changes in apical surface expression of ion channels and transporters in the superficial rat renal cortex were assessed using biotinylation and immunoblotting during alterations in dietary K intake. A high-K diet increased, and a low-K diet decreased, both the overall and surface abundance of the ß- and γ-subunits of the epithelial Na channel (ENaC). In the case of γ-ENaC, the effect was specific for the 65-kDa cleaved form of the protein. The overall amount of α-ENAC was also increased with increasing K intake. The total expression of the secretory K(+) channels (ROMK) increased with a high-K diet and decreased with a low-K diet. The surface expression of ROMK increased with high K intake but was not significantly altered by a low-K diet. In contrast, the amounts of total and surface protein representing the thiazide-sensitive NaCl cotransporter (NCC) decreased with increasing K intake. We conclude that modulation of K(+) secretion in response to changes in dietary K intake involves changes in apical K(+) permeability through regulation of K(+) channels and in driving force subsequent to alterations in both Na delivery to the distal nephron and Na(+) uptake across the apical membrane of the K(+) secretory cells.

Expression of the sodium potassium chloride cotransporter (NKCC1) and sodium chloride cotransporter (NCC) and their effects on rat lens transparency.

PURPOSE: To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. RESULTS: Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. CONCLUSIONS: We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.

Upregulation of renal sodium transporters in D5 dopamine receptor-deficient mice.

D(5) dopamine receptor (D(5)R)-deficient (D(5)(-/-)) mice have hypertension that is aggravated by an increase in sodium intake. The present experiments were designed to test the hypothesis that a dysregulation of renal sodium transporters is related to the salt sensitivity in D(5)(-/-) mice. D(5)R was expressed in the renal proximal tubule, thick ascending limb, distal convoluted tubule, and cortical and outer medullary collecting ducts in D(5)(+/+) mice. On a control Na(+) diet, renal protein expressions of NKCC2 (sodium-potassium-2 chloride cotransporter), sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel were greater in D(5)(-/-) than in D(5)(+/+) mice. Renal renin abundance and urine aldosterone levels were similar but renal angiotensin II type 1 receptor (AT(1)R) protein expression was increased in D(5)(-/-) mice. An elevated Na(+) diet increased further the elevated blood pressure of D(5)(-/-) mice but did not affect the normal blood pressure of D(5)(+/+) mice. The increased levels of NKCC2, sodium chloride cotransporter, and alpha and gamma subunits of the epithelial sodium channel persisted with the elevated Na(+) diet and unaffected by chronic AT(1)R blockade (losartan) in D(5)(-/-) mice. The expressions of proximal sodium transporters NHE3 (sodium hydrogen exchanger type 3) and NaPi2 (sodium phosphate cotransporter type 2) were increased by the elevated Na(+) diet in D(5)(-/-) mice; the increased expression of NHE3 but not NaPi2 was abolished by AT(1)R blockade. Our findings suggest that the increased protein expression of sodium transporters/channels in distal nephron segments may be the direct consequence of the disruption of D(5)R, independent of the renin-angiotensin aldosterone system.