Medullary Thick Ascending Limb Of Henle Research Articles

Angiotensin II inhibits NaCl absorption in the rat medullary thick ascending limb.

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 (AT(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(Cl)) as well as transepithelial voltage (V(te)) were measured. Luminal or peritubular 10(-11) and 10(-10) M angiotensin II had no effect on J(Cl) or V(te). However, 10(-8) M luminal or peritubular angiotensin II reversibly decreased both J(Cl) and V(te). The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10(-6) M). By contrast, PD-23319, an AT(2)-receptor antagonist, did not alter the inhibitory effect of 10(-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 AT(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.

Basolateral membrane Cl(-)-, Na(+)-, and K(+)-coupled base transport mechanisms in rat MTALH.

Mechanisms involved in basolateral HCO 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(-) 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 CO(2)/HCO. The alkalinization elicited by removing peritubular Cl(-) persisted in the bilateral absence of Na(+), together with a voltage clamp. When studied in Cl(-)-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO(2)/HCO. Removing peritubular Na(+) elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na(+)-HCO cotransport and inhibition of a basolateral Na(+)/H(+) exchange. Increasing bath K(+) induced an intracellular alkalinization that was inhibited in the absence of external CO(2)/HCO. At 2 mM, peritubular Ba(2+), which inhibits the K(+)-Cl(-) cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K(+)-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba(2+)-sensitive K(+)-Cl(-) cotransport that may operate as a K(+)-HCO 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 HCO transport mechanisms are functional under physiological conditions: electroneutral Cl(-)/HCO exchange, DIDS- and EIPA-insensitive Na(+)-HCO cotransport, and Ba(2+)-sensitive electroneutral K(+)-Cl(-)(HCO) cotransport.

Cell Shrinkage Triggers the Activation of Mitogen-activated Protein Kinases by Hypertonicity in the Rat Kidney Medullary Thick Ascending Limb of the Henle's Loop: REQUIREMENT OF p38 KINASE FOR THE REGULATORY VOLUME INCREASE RESPONSE

The kidney medulla is exposed to very high interstitial osmolarity leading to the activation of mitogen-activated protein kinases (MAPK). However, the respective roles of increased intracellular osmolality and of cell shrinkage in MAPK activation are not known. Similarly, the participation of MAPK in the regulatory volume increase (RVI) following cell shrinkage remains to be investigated. In the rat medullary thick ascending limb of Henle (MTAL), extracellular hypertonicity produced by addition of NaCl or sucrose increased the phosphorylation level of extracellular signal-regulated kinase (ERK) and p38 kinase and to a lesser extent c-Jun NH(2)-terminal kinase with sucrose only. Both hypertonic solutions decreased the MTAL cellular volume in a dose- and time-dependent manner. In contrast, hypertonic urea had no effect. The extent of MAPK activation was correlated with the extent of MTAL cellular volume decrease. Increasing intracellular osmolality without modifying cellular volume did not activate MAPK, whereas cell shrinkage without variation in osmolality activated both ERK and p38. In the presence of 600 mosmol/liter NaCl, the maximal cell shrinkage was observed after 10 min at 37 degrees C and the MTAL cellular volume was reduced to 70% of its initial value. Then, RVI occurred and the cellular volume progressively recovered to reach about 90% of its initial value after 30 min. SB203580, a specific inhibitor of p38, almost completely inhibited the cellular volume recovery, whereas inhibition of ERK did not alter RVI. In conclusion, in rat MTAL: 1) cell shrinkage, but not intracellular hyperosmolality, triggers the activation of both ERK and p38 kinase in response to extracellular hypertonicity; and 2) RVI is dependent on p38 kinase activation.

Nitric oxide inhibits transcription of the Na+-K+-ATPase alpha1-subunit gene in an MTAL cell line.

Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-L-arginine methyl ester (L-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-D-arginine methyl ester (D-NAME), or L-NAME for 16 h, after which 86Rb+ uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle or D-NAME) exhibited lower rates of ouabain-sensitive 86Rb+ uptake ( approximately 65%), lower levels of Na+-K+-ATPase alpha1-subunit mRNA ( approximately 60%), and reduced rates of de novo Na+-K+-ATPase alpha1-subunit transcription compared with L-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase alpha1-subunit gene.

Adaptation of NHE-3 in the rat thick ascending limb: effects of high sodium intake and metabolic alkalosis.

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+ exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 microM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3 administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (-27% and -25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.

Osmotic regulation of the Na+/myo-inositol cotransporter and postinduction normalization

In renal cells, hyperosmolarity has been shown to induce the accumulation of myo-inositol, via the Na+/myo-inositol cotransporter (SMIT). Previously we showed that SMIT mRNA in the kidney is localized in the medullary thick ascending limb of Henle (TALH). Here we used renal cells derived from the rabbit outer medullary TALH to examine the regulation of myo-inositol transport by hyperosmolarity. In addition, using both cultured renal and endothelial cells, we examined the normalization of SMIT activity and mRNA levels following induction by hyperosmolarity. TALH cells were exposed to isotonic or hyperosmotic medium, and then SMIT mRNA levels and myo-inositol accumulation were determined. To examine postinduction normalization, cultured endothelial and renal cells were first exposed to hyperosmotic medium and then to isotonic medium containing actinomycin D or cycloheximide. Afterwards, SMIT mRNA levels and myo-inositol accumulation were determined. Hyperosmolarity increased SMIT mRNA levels and myo-inositol accumulation in TALH cells. The hyperosmolarity-induced increase in myo-inositol uptake by TALH cells was characterized by an increase in the Vmax for the high-affinity myo-inositol transport system, with no change in the Km. This increase was blocked by actinomycin D or cycloheximide. Examination of postinduction normalization showed that returning hyperosmotic-treated cells to isotonic medium caused a rapid reversion of SMIT mRNA levels, followed by a return of myo-inositol accumulation to basal values. However, the addition of cycloheximide or actinomycin D partially to totally prevented the reversal in SMIT mRNA levels and activity. These results suggest that RNA and protein synthesis is required for the hyperosmotic induction of SMIT mRNA levels and myo-inositol accumulation by TALH cells. Furthermore, normalization of SMIT mRNA levels and myo-inositol accumulation following hyperosmotic induction requires RNA transcription and protein synthesis.

Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle

Transport processes in the medullary thick ascending limb of Henle (MTAL) have previously been investigated predominantly using tubule suspensions, perfusion of the loop of Henle in vivo, or individual perfused tubules [1, 2]. One important approach to investigate mechanisms of transport and luminal-basolateral sidedness of ion movement directly has been to isolate highly purified vesicle preparations of both types of plasma membranes of epithelia, such as the renal proximal tubule and the small intestine [reviewed in 3—5]. Regarding the MTAL, however, isolation of luminal (LMV) and basolateral (BLMV) plasma membrane vesicles has posed considerable difficulties, and has generally yielded mixed plasma membrane fractions consisting of both LMV and BLMV [5]. It is also important to remember that most vesicle studies aimed at characterizing transport processes in the MTAL have been performed on plasma membrane vesicles derived either from the entire outer medulla or from outer medullary homogenates [5], and the results then extrapolated to the MTAL. Clearly, for direct investigation into the basic mechanisms of transtubular solute transport in the MTAL, it would be valuable to isolate both types of plasma membranes free of contamination with intracellular as well as with plasma membranes from other nephron segments. In the present paper, we describe a new and simple approach that allowed the simultaneous isolation of LMV and BLMV with high yields from a single homogenate sample of purified isolated rat MTALs. Because this is the first report of the isolation of both types of MTAL plasma membrane vesicles, we wanted to examine whether these membrane preparations may serve as a useful model to characterize the transport properties of the two surfaces of this nephron segment. This report describes our evidence for the expression of different Na/H antiporters on the LMV and BLMV isolated from rat MTAL.

Coordinate development of oxidative enzymes and Na-K-ATPase in thick ascending limb: role of corticosteroids.

The postnatal development of mitochondrial ATP-producing pathways and Na-K-adenosinetriphosphatase (ATPase) in the rat medullary thick ascending limb of Henle (MTAL) was studied by measuring the activities of 3-ketoacid-CoA transferase, fumarase, citrate synthase, and Na-K-ATPase in microdissected MTAL of 16, 21, and 30-day-old pups and in adults. The role of adrenal steroids in the development of these four markers was also investigated by studying 21-day-old rats adrenalectomized on day 16 and given dexamethasone or aldosterone or NaCl injections from day 16 to day 21. There were large and correlated increases in the activities of the oxidative enzymes in the MTAL of control rat kidneys between 16 and 30 days after birth; Na-K-ATPase activity in the MTAL also greatly increased during the same period. Adrenalectomy completely prevented the developmental increases in MTAL oxidative enzymes and Na-K-ATPase; dexamethasone restored the development of all four enzymes, whereas aldosterone had no effect. We conclude that the postnatal maturation of Na+ reabsorption functions in MTAL cells involves coordinated increases in the capacity to produce ATP by oxidative metabolism and in Na-K-ATPase activity. This maturation process is probably triggered by the rise in circulating glucocorticoids that occurs during the weaning period.

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH.

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.

In situ hybridization of prepro-epidermal growth factor mRNA in the mouse kidney.

The presence of prepro-epidermal growth factor (prepro-EGF) mRNA was studied in the mouse kidney by in situ hybridization using [3H]prepro-EGF cDNA and 35S-labeled prepro-EGF cRNA probes. In addition, anti-EGF serum was utilized to immunolocalize the peptide by the avidin-biotin complex immunoperoxidase method. Both EGF immunoreactivity and prepro-EGF mRNA hybridization were localized to the thick ascending limb of Henle (TAL) and the distal convoluted tubule (DCT), whereas the macula densa was negative. The glomerulus, the proximal portion of the nephron, and the collecting system were negative. Computer-assisted image analysis of the optical density of the autoradiographic and immunocytochemical signals were performed. The medullary TAL expressed significantly less prepro-EGF than the cortical TAL and the DCT.

Effects of various transport inhibitors on oscillating TGF pressure responses in the rat.

The present study examines the effect in free flow microperfusion experiments of various diuretics with different sites of action on an oscillating tubular pressure response to changes in Henle loop flow rate. Amiloride (1 mM) and bendroflumethiazide (0.1 mM) had no effect outside the stimulation caused by the solvent (Ringer solution). Acetazolamide (0.5 mM) stimulated a slow (30 mHz) oscillation and often activated a fast (130-190 mHz) rhythm. Furosemide (FUR) (0.1-2.0 mM) abolished the slow oscillation and caused the intratubular pressure to rise by 2-3 mm Hg. FUR (0.05 mM) caused partial inhibition of the slow rhythm, which usually became irregular. Bumetanide (BUM) (0.05 mM) elicited a biphasic response. Initially the pressure decreased, while the slow rhythm was amplified; then the fast oscillation was activated; after 4-6 min the oscillations disappeared, while the pressure increased. In the recovery period the oscillations often became irregular. The results confirm that the oscillating pressure response is mediated by a transport function of the macula densa (MD); and, thus, has the character of a tubulo-glomerular feedback (TGF) response. The data suggest 1. that BUM inhibition of NaCl co-transport in the cortical thick ascending limb of Henle (TAL) is delayed as compared to the medullary TAL; and 2. that FUR action at the MD interferes with more than one carrier mechanism. Partial inhibition gives rise to irregular oscillations.

Distribution of prostaglandin E 2-sensitive adenylate cyclase along the rat nephron

To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E 2 (PGE 2) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE 2 and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE 2. PGE 2 stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10 −5 to 2.8 × 10 −5 M. PGE 2 was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE 2, at both high (2.8 × 10 −5 M) and low (2.8 × 10 −8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE 2-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE 2 on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.