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Phosphoenolpyruvate carboxykinase 1 (PCK1 or PEPCK-C) is a cytosolic enzyme converting oxaloacetate to phosphoenolpyruvate, with a potential role in gluconeogenesis, ammoniagenesis, and cataplerosis in the liver. Kidney proximal tubule cells display high expression of this enzyme, whose importance is currently not well defined. We generated PCK1 kidney-specific knockout and knockin mice under the tubular cell-specific PAX8 promoter. We studied the effect of PCK1 deletion and overexpression at the renal level on tubular physiology under normal conditions and during metabolic acidosis and proteinuric renal disease. PCK1 deletion led to hyperchloremic metabolic acidosis characterized by reduced but not abolished ammoniagenesis. PCK1 deletion also resulted in glycosuria, lactaturia, and altered systemic glucose and lactate metabolism at baseline and during metabolic acidosis. Metabolic acidosis resulted in kidney injury in PCK1-deficient animals with decreased creatinine clearance and albuminuria. PCK1 further regulated energy production by the proximal tubule, and PCK1 deletion decreased ATP generation. In proteinuric chronic kidney disease, mitigation of PCK1 downregulation led to better renal function preservation. PCK1 is essential for kidney tubular cell acid-base control, mitochondrial function, and glucose/lactate homeostasis. Loss of PCK1 increases tubular injury during acidosis. Mitigating kidney tubular PCK1 downregulation during proteinuric renal disease improves renal function. [ABSTRACT FROM AUTHOR]

Tight regulation of calcium levels is required for many critical biological functions. The [Ca.sup.2+]-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism., Introduction Calcium is the most abundant mineral in the human body, and its tight regulation is required for many critical biological functions, such as bone mineralization, muscle contraction, nerve conduction, [...]

Ammonia absorption by the medullary thick ascending limb of Henle's loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral [Na.sup.+]/[H.sup.+] exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: [Na.sup.+]/[H.sup.+] exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that [NHE4.sup.[-/-]] mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, [NHE4.sup.[-/-]] mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of [NH.sub.3]/[NH.sub.4.sup.+] needed to excrete an acid load, contributing to systemic metabolic acidosis., Introduction Ammonium is the main component of urinary [H.sup.+] excretion. In addition, its renal production and excretion rise in response to an acid load, thereby allowing the kidney to appropriately [...]

The kidney has an important role in the regulation of acid-base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. [...]

Macula densa (MD) cells express the [Na.sup.+]/[H.sup.+] exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE[2.sup.+/+]) and NHE2 knockout (NHE[2.sup.-/-]) mice. Immunofluorescence studies indicated that NHE[2.sup.-/-] mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of reninexpressing cells in the afferent arteriole in NHE[2.sup.-/-] mice was increased ~2.5-fold using renin immunohistochemistry. Western blotting confirmed ~20% higher renal cortical renin content in NHE[2.sup.-/-] mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE[2.sup.+/+] mice, but the response was blunted in NHE[2.sup.-/-] mice. Renal tissue renin activity and plasma renin concentration were elevated three-and two-fold, respectively, in NHE[2.sup.-/-] mice compared with wild type. NHE[2.sup.-/-] mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE[2.sup.-/-] kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE2-renin release pathway. macula densa; cell volume; renin release; mitogen-activated protein kinases; cyclooxygenase-2

Growth hormone (GH) has antidiuretic and antinatriuretic effects in rats and humans, but the molecular mechanisms responsible for these effects are unknown. The aim of this study was to investigate the mechanisms behind the acute renal effects of GH in rats. Female rats received rat (r)GH (2.8 mg/kg sc) or saline and were placed in metabolic cages for 5 h. Urinary excretion of electrolytes and urinary volume were reduced after rGH injection, while urine osmolality was increased. Creatinine and lithium clearance remained unchanged, suggesting that rGH increases reabsorption in segments distal to the proximal tubule. Total plasma insulin-like growth factor I (IGF-I) levels did not change, while cortical IGF-I mRNA abundance was increased. The relative abundance of total and [Ser.sup.256]-phosphorylated aquaporin 2 was found to be unchanged by immunoblotting, whereas a significant increase of [Thr.sup.96] and [Thr.sup.101]-phosphorylated NKCC2 (renal [Na.sup.+], [K.sup.+], 2[Cl.sup.-] cotransporter) was found in the inner stripe of outer medulla thick ascending limbs (mTAL). Additionally, an increased NKCC2 expression was observed in the cortical region. Immunohistochemistry confirmed these findings. The density of NKCC2 molecules in the apical membrane of mTAL cells appeared to be unchanged after rGH injection evaluated by immunoelectron microscopy. Basolateral addition of rGH or IGF-I to microperfused rat mTAL segments did not change transepithelial voltage. In conclusion, GH appears to exert its acute antinatriuretic and antidiuretic effects through indirect activation of NKCC2 in the mTAL. thick ascending limb; sodium; insulin-like growth factor I; GH; kidney

NHf transport by the distal nephron and N[H.sup.+.sub.4] detoxification by the liver are critical for achieving regulation of acid-base balance and to avoid hyperammonemic hepatic encephalopathy, respectively. Therefore, it has been proposed that rhesus type B glycoprotein (Rhbg), a member of the Mep/Amt/Rh N[H.sub.3] channel superfamily, may be involved in some forms of distal tubular acidosis and congenital hyperammonemia. We have tested this hypothesis by inactivating the RHbg gene in the mouse by insertional mutagenesis. Histochernical studies analyses confirmed that RHbg knockout (KO) mice did not express Rhbg protein. Under basal conditions, the KO mice did not exhibit encephalopathy and survived well. They did not exhibit hallmarks of distal tubular acidosis because neither acid-base status, serum potassium concentration, nor bone mineral density was altered by RHbg disruption. They did not have hyperammonemia or disturbed hepatic N[H.sub.3] metabolism. Moreover, the KO mice adapted to a chronic acidloading challenge by increasing urinary N[H.sup.+.sub.4] excretion as well as their wild-type controls. Finally, transepithelial N[H.sub.3] diffusive permeability, or N[H.sub.3] and N[H.sup.+.sub.4] entry across the basolateral membrane of cortical collecting duct cells, measured by in vitro microperfusion of collecting duct from KO and wild-type mice, was identical with no apparent effect of the absence of Rhbg protein. We conclude that Rhbg is not a critical determinant of N[H.sup.+] excretion by the kidney and of N[H.sup.+] detoxification by the liver in vivo. rhesus protein; acid-base; tubular acidosis; ammonia

NaCl reabsorption in the medullary thick ascending limb of Henle (MTALH) contributes to NaCl balance and is also responsible for the creation of medullary interstitial hypertonicity. Despite the presence of angiotensin II subtype 1 (A[T.sub.1]) receptors in both the luminal and the basolateral plasma membranes of MTALH cells, no information is available on the effect of angiotensin II on NaCl reabsorption in MTALH and, furthermore, on angiotensin II-dependent medullary interstitial osmolality. MTALHs from male Sprague-Dawley rats were isolated and microperfused in vitro; transepithelial net chloride absorption ([J.sub.Cl]) as well as transepithelial voltage ([V.sub.te]) were measured. Luminal or peritubular [10.sup.-11] and [10.sup.-10] M angiotensin II had no effect on [J.sub.Cl] or [V.sub.te]. However, [10.sup.-8] M luminal or peritubular angiotensin II reversibly decreased both [J.sub.Cl] and [V.sub.te]. The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan ([10.sup.-6] M). By contrast, PD-23319, an A[T.sub.2]-receptor antagonist, did not alter the inhibitory effect of [10.sup.-8] M angiotensin II. Finally, no additive effect of luminal and peritubular angiotensin II was observed. We conclude that both luminal and peritubular angiotensin II inhibit NaCl absorption in the MTALH via A[T.sub.1] receptors. Because of intrarenal angiotensin II synthesis, angiotensin II concentration in medullary tubular and interstitial fluids may be similar in vivo to the concentration that displays an inhibitory effect on NaCl reabsorption under the present experimental conditions. in vitro microperfusion; renal tubule; NaCl transport; angiotensin II subtype 1; losartan

Mechanisms involved in basolateral HC[O.sup.-.sub.3] transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular [Cl.sup.-] induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and blunted in the absence of external C[O.sub.2]/HC[O.sup.-.sub.3]. The alkalinization elicited by removing peritubular [Cl.sup.-] persisted in the bilateral absence of [Na.sup.+], together with a voltage clamp. When studied in [Cl.sup.-]-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external C[O.sub.2]/HC[O.sup.-.sub.3]. Removing peritubular [Na.sup.+] elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive [Na.sup.+]-HC[O.sup.-.sub.3] cotransport and inhibition of a basolateral [Na.sup.+]/[H.sup.+] exchange. Increasing bath [K.sup.+] induced an intracellular alkalinization that was inhibited in the absence of external C[O.sub.2]/ HC[O.sup.-.sub.3]. At 2 mM, peritubular [Ba.sup.2+], which inhibits the [K.sup.+]-[Cl.sup.-] cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited [K.sup.+]-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral [Ba.sup.2+]-sensitive [K.sup.+]-[Cl.sup.-] cotransport that may operate as a [K.sup.+]-HC[O.sup.-.sub.3] cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HC[O.sup.-.sub.3] transport mechanisms are functional under physiological conditions: electroneutral [Cl.sup.-]/HC[O.sup.-.sub.3] exchange, DIDS- and EIPA-insensitive [Na.sup.+]-HC[O.sup.-.sub.3] cotransport, and [Ba.sup.2+]-sensitive electroneutral [K.sup.+]-[Cl.sup.-](HC[O.sup.-.sub.3]) cotransport. medullary thick ascending limb; bicarbonate; in vitro microperfusion; intracellular pH