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The present study directly tested the hypothesis that the NHE3 (Na+/H+ exchanger 3) in the proximal tubules of the kidney is required for the development of Ang II (angiotensin II)-induced hypertension using PT-Nhe3-/- (proximal tubule-specific NHE3 knockout) mice. Specifically, PT-Nhe3-/- mice were generated using the SGLT2-Cre/Nhe3loxlox approach, whereas Ang II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT-Nhe3-/- mice. Under basal conditions, systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were significantly lower in male and female PT-Nhe3-/- than WT mice (P<0.01). A high pressor, 1.5 mg/kg per day, intraperitoneal or a slow pressor dose of Ang II, 0.5 mg/kg per day, intraperitoneal for 2 weeks significantly increased systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure in male and female WT mice (P<0.01), but the hypertensive response to Ang II was markedly attenuated in male and female PT-Nhe3-/- mice (P<0.01). Ang II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in Ang II-infused PT-Nhe3-/- mice (P<0.01). AT1 receptor blocker losartan completely blocked Ang II-induced hypertension in both WT and PT-Nhe3-/- mice (P<0.01). However, inhibition of nitric oxide synthase with L-NG-Nitroarginine methyl ester had no effect on Ang II-induced hypertension in WT or PT-Nhe3-/- mice (not significant). Furthermore, Ang II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney may be a therapeutical target in hypertension induced by Ang II or with increased NHE3 expression in the proximal tubules. [ABSTRACT FROM AUTHOR]

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites. [ABSTRACT FROM AUTHOR]

The present study directly tested the hypothesis that deletion of the NHE3 (Na+/H+ exchanger 3) selectively in the proximal tubules of the kidney lowers basal blood pressure by increasing the pressure-natriuresis response in mice. Adult male and female, age-matched wild-type (WT) littermates and proximal tubule-specific NHE3 knockout mice (PT- Nhe3-/-; n=6-16 per group) were studied for (1) basal phenotypes of electrolytes and pH, blood pressure, and kidney function; (2) the pressure-natriuresis response using the mesenteric, celiac, and abdominal arterial occlusion technique; and (3) the natriuretic responses to acute saline expansion (0.9% NaCl, 10% body weight, intraperitoneal) or 2-week of 2% NaCl diet. Under basal conditions, PT- Nhe3-/- mice showed significantly lower systolic, diastolic, and mean arterial blood pressure ( P<0.01) than WT mice ( P<0.01). PT- Nhe3-/- mice also exhibited significantly greater diuretic ( P<0.01) and natriuretic responses than WT mice ( P<0.01), without altering 24-hour fecal Na+ excretion, plasma pH, Na+, and bicarbonate levels. In response to increased renal perfusion pressure by 30 mm Hg, the pressure-natriuresis response increased 5-fold in WT mice ( P<0.01), but it increased 8-fold in PT- Nhe3-/- mice ( P<0.01). In response to 10% acute saline expansion or 2-week 2% NaCl diet, more pronounced natriuretic responses were demonstrated in PT- Nhe3-/- than WT mice ( P<0.01). Our results support the scientific premise and physiological relevance that NHE3 in the proximal tubules plays an essential role in maintaining basal blood pressure homeostasis, and genetic deletion of NHE3 selectively in the proximal tubules of the kidney lowers blood pressure by increasing the pressure natriuretic response. [ABSTRACT FROM AUTHOR]

To study the role of [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal [Na.sup.+] conservation, mice with Nhe2 ([Nhe2.sup.-/-]) and/or Nhe3 ([Nhe3.sup.-/-]) null mutations were fed a [Na.sup.+]-restricted diet, and urinary [Na.sup.+] excretion, blood pressure, systemic acid-base and electrolyte status, and renal function were analyzed. [Na.sup.+]-restricted [Nhe2.sup.-/-] mice, on either a wild-type or Nhe3 heterozygous mutant ([Nhe3.sup.+/-]) background, did not exhibit excess urinary [Na.sup.+] excretion. After 15 days of [Na.sup.+] restriction, blood pressure, fractional excretion of [Na.sup.+], and the glomerular filtration rate (GFR) of [Nhe2.sup.-/-][Nhe3.sup.+/-] mice were similar to those of [Nhe2.sup.+/+] and [Nhe3.sup.+/-] mice, and no metabolic disturbances were observed. [Nhe3.sup.-/-] mice maintained on a [Na.sup.+]-restricted diet for 3 days exhibited hyperkalemia, urinary salt wasting, acidosis, sharply reduced blood pressure and GFR, and evidence of hypovolemic shock. These results negate the hypothesis that NHE2 plays an important renal function in sodium-fluid volume homeostasis; however, they demonstrate that NHE3 is critical for systemic electrolyte, acid-base, and fluid volume homeostasis during dietary [Na.sup.+] restriction and that its absence leads to renal salt wasting. sodium absorption; sodium/hydrogen exchanger; slc9a2; slc9a3

Amlal, Hassane, Qian Chen, Khalid Habo, Zhaohui Wang, and Manoocher Soleimani. Fasting downregulates renal water channel AQP2 and causes polyuria. Am J Physiol Renal Physiol 280: F513-F523, 2001.--Starvation causes impairment in the urinary concentrating ability. The mechanism of this defect, however, remains unknown. We tested the possibility that food deprivation might affect the expression and activity of aquaporins (AQP1, 2), thereby impairing renal water reabsorption in the kidney. Rats fasted for 24 h exhibited severe polyuria (urine volume increased from 11 before fasting to 29 ml/24 h after fasting, P [is less than] 0.0001) along with failure to concentrate their urine (urine osmolality decreased from 1,485 before fasting to 495 mosmol/kg[H.sub.2]O after fasting, P [is less than] 0.0001). Refeeding for 24 h returned the urinary concentrating ability back to normal. Northern hybridization and immunoblot analysis demonstrated that fasting was associated with a decrease in AQP2 protein (-80%, P [is less than or equal to] 0.002) and mRNA levels (-69%, P [is less than or equal to] 0.003) in the outer medulla. In the cortex, fasting decreased AQP2 protein abundance by 60% (P [is less than or equal to] 0.004) but did not alter its mRNA expression. During the recovery phase, AQP2 expression returned to normal level in both tissues. In the inner medulla, the expression of AQP2 was not altered in fasting, but was increased significantly at both protein ([+ or -] 92%) and mRNA ([+ or -] 43%) levels during the recovery from fasting. The proximal nephron water channel (AQP1) was not affected in response to fasting or recovery from fasting. We conclude that 1) fasting impairs the urinary concentrating ability in rats, and 2) the renal water-handling defect in fasting results specifically from the downregulation of AQP2 in the cortical and outer medullary collecting duct. urinary concentrating mechanism; aquaporin 2; hypoglycemia

Pendrin: an apical [Cl.sup.-]/[OH.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356-F364, 2001.--The identities of the apical [Cl.sup.-]/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brushborder membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the [Cl.sup.-]/[OH.sup.-], [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], and [Cl.sup.-]/formate exchange modes. The conclusion is that pendrin is an apical [Cl.sup.-]/base exchanger in the kidney proximal tubule and CCD and mediates [Cl.sup.-]/[OH.sup.-], [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], and [Cl.sup.-]/formate exchange. proximal tubule; cortical collecting duct; anion exchanger; apical membrane

Amlal, Hassane, Carissa M. Krane, Qian Chen, and Manoocher Soleimani. Early polyuria and urinary concentrating defect in potassium deprivation. Am J Physiol Renal Physiol 279: F655-F663, 2000.--The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in [K.sup.+] deprivation (KD) remains unknown. Rats were fed a [K.sup.+]-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum [K.sup.+] concentration was decreased only after, but not before, 3 days of a [K.sup.+]-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 [+ or -] 26 vs. 1,487 [+ or -] 102 mosmol/kg[H.sub.2]0 in control, P [is less than] 0.01). It decreased further at 24 h (to 858 [+ or -] 162 mosmol/ kg[H.sub.2]0 in KD, P [is less than] 0.004) and remained low at 21 days of KD (436 [+ or -] 58 mosmol/kg[H.sub.2]0, P [is less than] 0.0001 compared with baseline). Water intake decreased at 12 h (P [is less than] 0.002) but increased at 24 h (P [is less than] 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 [+ or -] 2 to 15 [+ or -] 2 ml/24 h, P [is less than] 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h (P [is greater than] 0.05), decreased at 24 h (~44%, P [is less than] 0.001), and remained suppressed (~52%, P [is less than] 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h (~47%, P [is less than] 0.01) and remained suppressed at 24 h (~77%, P [is less than] 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex (P [is less than] 0.02) and outer medulla (P [is less than] 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2. antidiuretic hormone; aquaporin-2; kidney

Potassium deprivation upregulates expression of renal basolateral [Na.sup.+]-[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] cotransporter (NBC-1). Am J Physiol Renal Physiol 279: F532-F543, 2000.--The purpose of the present experiments was to examine the effect of potassium deprivation on the expression of the renal basolateral [Na.sup.+]-[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] cotransporter (NBC-1). Rats were placed on a [K.sup.+]-free diet for various time intervals and examined. NBC-1 mRNA levels increased by about threefold in the cortex (P [is less than] 0.04,) at 72 h of [K.sup.+] deprivation and remained elevated at 21 days. NBC activity increased by ~110% in proximal tubule suspensions, with the activity increasing from 0.091 in control to 0.205 pH/min in the [K.sup.+]-deprived group (P [is less than] 0.005). The inner stripe of outer medulla and cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in [K.sup.+]-deprived rats, with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the [K.sup.+]-deprived group (P [is less than] 0.004). [K.sup.+] deprivation also increased NBC-1 mRNA levels in the renal papilla (P [is less than] 0.02). We conclude that 1) [K.sup.+] deprivation increases NBC-1 expression and activity in proximal tubule and 2) [K.sup.+] deprivation causes induction of NBC-1 expression and activity in mTAL tubule and inner medulla. We propose that NBC-1 likely mediates enhanced [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reabsorption in proximal tubule, mTAL, and inner medullary collecting duct in [K.sup.+] deprivation and contributes to the maintenance of metabolic alkalosis in this condition. acid-base transporters; proximal tubule; medullary thick ascending limb

CFTR induces the expression of DRA along with [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity in tracheal epithelial cells. Am J Physiol Cell Physiol 279: C62-C71, 2000.--Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of [Cl.sup.-] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] across the tracheal epithelium. The mechanism of the defect in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion is ill defined; however, a lack of apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange may exist in CF. To test this hypothesis, we examined the expression of [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells (P [is less than] 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 [Cl.sup.-]/ [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855-22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of 'downregulated in adenoma' (DRA) and, as a result, upregulates the apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger activity in tracheal cells. We propose that the tracheal [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion defect in patients with CF is partly due to the downregulation of the apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity mediated by DRA. cystic fibrosis transmembrane conductance regulator; cystic fibrosis; trachea; bicarbonate secretion; downregulated in adenoma; chloride ion/bicarbonate exchange

Shumaker, Holli, and Manoocher Soleimani. CFTR upregulates the expression of the basolateral [Na.sup.+][-K.sup.+] [-2Cl.sup.-] cotransporter in cultured pancreatic duct cells. Am. J. Physiol. 277 (Cell Physiol. 46): C1100-C1110, 1999.--The purpose of the current experiments was 1) to assess basolateral [Na.sup.+][-K.sup.+] [-2Cl.sup.-] cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypica] pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-beating CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells (P [is less than] 0.01, n = 3). [Na.sup.+][-K.sup.+][-2Cl.sup.-] cotransporter activity, assayed as the bumetanide-sensitive, [Na.sup.+] - and [Cl.sup.-]-dependent [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] entry into the cell (with [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] acting as a substitute for [K.sup.+]), increased by ~115% in CFPAC-WT cells compared with CFPAC-1 cells (P [is less than] 0.01, n = 6). Reducing the intracellular [Cl.sup.-] by incubating the cells in a [Cl.sup.-]-free medium increased [Na.sup.+][-K.sup.+][-2Cl.sup.-] cotransporter activity by twofold (P [is less than] 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of [Cl.sup.-]. NKCC1 activity is enhanced in the presence of an inward [Cl.sup.-] gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a [Cl.sup.-]-dependent manner. cystic fibrosis transmembrane conductance regulator; [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion; cystic fibrosis

Background: Probenecid is a uricosuric agent that in addition to exerting a positive ionotropic effect in the heart, blocks the ATP transporter Pannexin 1 and inhibits the Cl-/HCO3- exchanger, pendrin. In the kidney, pendrin blunts the loss of salt wasting secondary to the inhibition of the thiazide-sensitive Na+-Cl- co-transporter (NCC/SLC12A3). Hypothesis: Pre-treatment with probenecid down-regulates pendrin; therefore, leaving NCC as the main salt absorbing transporter in the distal nephron, and hence enhances the hydrochlorothiazide (HCTZ)-induced diuresis. Methods: Daily balance studies, blood and urine chemical analysis, immunofluorescence, as well as western and northern blot analyses were utilized to examine the effects of probenecid alone (at 250 mg/kg/day) or in combination with HCTZ (at 40 mg/kg/day) on kidney function and on salt and water transporters in the collecting duct. Results: Male Sprague Dawley rats were subjected to three different protocols: (1) HCTZ for 4 days, (2) probenecid for 10 days, and (3) primed with probenecid for 6 days followed by probenecid and HCTZ for 4 additional days. Treatment protocol 1 (HCTZ for 4 days) only mildly increased the urine volume (U Vol) from a baseline of 9.8–13.4 ml/day. In response to treatment protocol 2 (probenecid for 10 days), U Vol increased to 15.9 ml/24 h. Treatment protocol 3 (probenecid for 6 days followed by probenecid and HCTZ for 4 additional days) increased the U Vol to 42.9 ml/day on day 4 of co-treatment with HCTZ and probenecid (compared to probenecid p = 0.003, n = 5 or HCTZ alone p = 0.001, n = 5). Probenecid treatment at 250 mg/kg/day downregulated the expression of pendrin and led to a decrease in AQP2 expression. Enhanced diuresis by probenecid plus HCTZ was not associated with volume depletion. Conclusion: Probenecid pre-treatment downregulates pendrin and robustly enhances diuresis by HCTZ-mediated NCC inhibition in kidney. [ABSTRACT FROM AUTHOR]

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients. [ABSTRACT FROM AUTHOR]