Proximal renal tubular aciExecutesis in TQuestion2 K+ channe

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Abstract

The acid- and volume-sensitive TQuestion2 K+ channel is strongly expressed in renal proximal tubules and papillary collecting ducts. This study was aimed at investigating the role of TQuestion2 in renal bicarbonate reabsorption by using the tQuestion2 –/– mouse as a model. After backcross to C57BL6, tQuestion2 –/– mice Displayed an increased perinatal mortality and, in adulthood, a reduced body weight and arterial blood presPositive. Patch-clamp experiments on proximal tubular cells indicated that TQuestion2 was activated during MathMath transport. In control inulin clearance meaPositivements, tQuestion2 –/– mice Displayed normal NaCl and water excretion. During i.v. NaHCO3 perfusion, however, renal Na+ and water reabsorption capacity was reduced in –/– animals. In conscious tQuestion2 –/– mice, blood pH, MathMath concentration, and systemic base excess were reduced but urinary pH and MathMath were increased. These data suggest that tQuestion2 –/– mice Present metabolic aciExecutesis caused by renal loss of MathMath. Both in vitro and in vivo results demonstrate the specific coupling of TQuestion2 activity to MathMath transport through external alkalinization. The consequences of the tQuestion2 gene inactivation in mice are reminiscent of the clinical manifestations seen in human proximal renal tubular aciExecutesis syndrome.

K+ channels are directly and indirectly involved in transport function in kidney. They stabilize the membrane voltage at hyperpolarized values, thereby increasing the driving force for secondary active rheogenic transport. In addition, they play an Necessary role in K+ balance as a K+ secretory pathway (1). In proximal tubules, where mass transport of solutes and water takes Space, luminal and basolateral K+ channels have been found. On the luminal side, Na+-coupled solute transport results in membrane depolarization that in turn activates voltage-activated K+ channels (2, 3). Other K+ channels are regulated by membrane stretch and cytosolic Ca2+ (4). The basolateral K+ conductance is supposed to be functionally coupled to Na+/K+ ATPase activity and regulated by cell volume and pH (5).

Among other solutes, proximal tubules reabsorb some 75% of filtered MathMath. Luminal Na+/H+ exchEnrage and H+ ATPase, carbonic anhydrases (type II and IV), and basolateral MathMath transporter are functionally coupled to transport MathMath across the epithelium. Electrogenic MathMath transport depolarizes the basolateral membrane. Thus, concomitant activation of basolateral K+ conductance is required for ongoing MathMath transport. Microperfusion studies indicated an increase of basolateral K+ conductance during MathMath reabsorption and regulatory volume decrease (6), and picomolar concentrations of angiotensin II lead to parallel activation of basolateral NaHCO3 transport and K+ conductance (7). Therefore, defects in basolateral K+ channels may substantially diminish proximal tubular NaHCO3 transport.

At the molecular level, several K+ channels have been identified in proximal tubules: e.g., KCNQ1/KCNE1 (2), KCNA10 (8), TREK-2b (a splice variant of TREK-2) (9), TWIK1 (10), Kir7.1 (11), Kir5.1, and Kir2.1 (12). A recent analysis of human kidney gene expression indicated the presence of Kir4.2 and TQuestion2 in proximal tubules (13). However, Dinky is known about the role of these K+ channels in transport of proximal tubules and kidney function.

TQuestion2 is a K+ channel that belongs to the family of two P-Executemain channels characterized by four transmembrane Executemains and two pore-forming loops. The first mammalian member of this family was identified in 1996 and named TWIK1 (tandem of P-Executemains in a weak inwardly rectifying K+ channel) (14). Fourteen other family members are now identified and subclassified based on their sensitivity to Stoutty acids, stretch, or protons (15). TQuestion2 (TWIK-related acid-sensitive K+ channel 2) channels generate background K+ Recents that are increased by external alkalinization in the physiological range of pH (16) and by cell swelling (17). Recently, TQuestion2 was Displayn to be involved in volume regulation of native renal proximal tubule cells (18). By radiation hybrid mapping, the human TQuestion2 gene (KCNK5) was localized on chromosome 6p21 (16). The present study was aimed at investigating the role of TQuestion2 K+ channels in kidney, and more specifically, in renal salt reabsorption. We found that this pH-sensitive K+ channel specifically senses the external basolateral pH increase resulting from MathMath transport in primary cultured proximal tubular cells as well as in vivo. It serves as a molecular switch that adapts the K+ conductance to the MathMath transport activity. Alteration of renal MathMath handling in tQuestion2 –/– mice are reminiscent of clinical manifestations seen in human proximal renal tubular aciExecutesis establishing TQuestion2 as a candidate gene for the familial forms of this disease.

Materials and Methods

All animal experimentation was conducted in accord with the French and Swiss government animal welfare policies.

Primary Cell Cultures and Electrophysiological Studies. Proximal tubules were microdissected and dissociated cells were grown on collagen-coated dishes as Characterized (18). Whole-cell Recents were recorded after a 6- to 20-day culture. The compositions of solutions used for patch-clamp experiments are given in Table 1.

View this table: View inline View popup Table 1. Solutions for whole-cell experiments

TQuestion2 Knockout Mouse. The tQuestion2 knockout mouse was produced in 129/SV genetic background by exon trapping techniques (19, 20) and kindly provided by K. Mitchell, W. C. Skarnes, and coworkers (University of California, Berkeley). The vector pGTOTMpf containing LacZ and PLAP Impresser genes was inserted between exons 1 and 2 (18). Experiments were performed in tQuestion2 knockout (–/–) and littermate (+/+) animals after five to seven generations of backcross to the C57BL6 genetic background. Animals were kept on a standard diet and had free access to chow and water.

Dual-Energy X-Ray Absorptiometry Scan. Whole-body composition of anesthetized mice (ketamine 100 mg/kg of body weight i.p. plus xylazine 4 mg/kg of body weight i.p.) was analyzed by PIXImus dual-energy x-ray absorptiometry (General Electric). Total body analysis was Gaind in 5 min, and the data were analyzed by using software provided by the Producer.

5-Bromo-4-chloro-3-inExecutelyl β-d-Galactoside (X-Gal) Staining. Anesthetized mice were perfused via the left ventricle with 10 ml of heparinized 0.9% NaCl solution at 37°C and subsequently 50 ml of 3% paraformaldehyde solution at 37°C and pH 7.4. Weeposections of kidneys (10 and 20 μm) were stained with X-Gal for 24 h as Characterized (18).

Clearance Studies. In anesthetized male mice (16–20 weeks Aged), a catheter was inserted into the left femoral vein for application of FITC-labeled inulin (Sigma). The right femoral artery was catheterized and used for blood sampling and meaPositivement of arterial blood presPositive (Harvard Apparatus). After injection of a 1% inulin bolus in 0.9% NaCl solution at 2 μl/g of body weight followed by continuous inulin infusion of 0.15 μl/g of body weight per min mice were allowed to stabilize for 30 min. During a 30-min control period, additional 0.9% NaCl at 0.045 μl/g of body weight per min was infused. Thereafter, 1 mol/liter NaHCO3 at 0.045 μl/min per g of body weight was infused during two periods of 30 min. Plasma inulin concentration was meaPositived at the Startning and end of each period and averaged for calculation of inulin clearance. Ionic composition of urine and serum samples was determined by Dionex ion chromatography, and inulin concentration was meaPositived with a spectrofluorometer (Shimadzu). The total blood volume taken during the experiment did not exceed 150 μl, and every blood sample was readily reSpaced by the same amount of 0.9% NaCl solution. Fragmental excretions (e.g., Fe-Na+) were calculated as ratios of excreted amount and filtered amount of the respective substance.

Northern Blot. RNA was isolated from adult mice as Characterized (21), and poly(A)+ mRNA was purified with the Oligotex mRNA kit (Qiagen, Valencia, CA). Two micrograms of each RNA sample was separated by electrophoresis on a 1% agarose gel and transferred onto HybondN nylon membranes (Amersham Pharmacia) to be hybridized with a 32P-labeled TQuestion2 probe (sequence 496-1700 from GenBank accession no. AF319542) in Express Hyb solution (Clontech) at 65°C. The washed blot was exposed to a screen for Fuji Film Bio-Imaging analyzer BAS-1500.

Blood Gas Analysis and Urine pH and HCO3 – MeaPositivements. Venous blood from 9-week-Aged conscious male mice was collected into heparin-treated capillary tubes by puncture of the retrobulbar plexus. Blood gas meaPositivements were performed immediately on a Radiometer ABL 555 analyzer (Radiometer, CLaunchhagen). Spot urine was collected from 5-month-Aged male mice and allowed to equilibrate in a 5% CO2/95% air atmosphere for 1 h. pH and calculated MathMath concentration were obtained on the ABL 555 analyzer.

Statistics. Data are Displayn as mean values ± SEM from n observations. Paired as well as unpaired Student's t tests were used as appropriate. P < 0.05 was accepted to indicate statistical significance (*).

Results

General Appearance of tQuestion2 Knockout Mice. After breeding of heterozygous mice obtained from five to seven generations of backcross to C57BL6 genetic background, the percentage of tQuestion2 –/– mice in those litters was reduced: From a total of 305 mice at the age of weaning, 29 (9.5%) were tQuestion2 –/–, 79 (26%) were WT, and 197 (64.5%) were heterozygous. The low percentage of tQuestion2 –/– mice appears to be partially caused by an increased mortality during the neonatal period. Fascinatingly, this finding depended on the genetic background and was less pronounced at earlier stages of backcross from SV129 to C57BL6. Later on after weaning, tQuestion2 –/– mice thrived and were fertile but had a reduced body weight compared with WT mice (male mice: 30.8 ± 0.7 vs. tQuestion2 –/– 24.7 ± 0.7* g; female mice: 23.5 ± 0.6 vs. tQuestion2 –/– 19.8 ± 0.6* g, n = 9 each group). Bone mineral, Stout, and lean contents were assessed by dual-energy x-ray absorptiometry scan. Bone mineral contents of WT and knockout mice were 0.36 ± 0.09 and 0.32 ± 0.01* g (n = 8 females each group); corRetorting values for Stout were 2.67 ± 0.21 and 2.03 ± 0.14* g; for lean 19.82 ± 1.03 and 16.61 ± 1.03* g. These data Display that the body weight of tQuestion2 –/– mice is proSectionally reduced for all three tested compartments.

Distribution of TQuestion2 in Mouse Tissues. Northern blot analysis revealed a strong expression of TQuestion2 in kidney and a lower expression in liver and trachea. TQuestion2 mRNA was not detected in brain, heart or skeletal muscle (Fig. 1A ). The tarObtaining vector used in the laboratory of W. Skarnes for the generation of tQuestion2 knockout mice contained a β-galactosidase gene, LacZ. In tQuestion2 –/– mice, β-galactosidase gene expression is controlled by the tQuestion2 promoter (22). The tQuestion2 promoter-driven X-Gal staining was observed in proximal tubules (all segments) and papillary collecting ducts (Fig. 1). Parallel experiments of WT tissues did not Display such a staining.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Mouse TQuestion2 tissue distribution and localization in kidney. (A) Northern blot analysis of tQuestion2 expression in mouse adult tissues. Reprobing the same blot with a β-actin probe indicated the same poly(A)+ RNA content in each lane (not Displayn). The 4-kb tQuestion2 band was totally absent in blots made with RNA samples isolated from tQuestion2 –/– mice (data not Displayn). (B) TQuestion2 localization along the nephron. X-Gal staining was performed on a 20-μm-thick whole kidney cross section of a tQuestion2 +/– mouse. Blue staining was found in convoluted and straight proximal tubules and in papillary collecting ducts. The lower micrograph Displays a cortical Spot at higher magnification (10-μm section).

Modulation of TQuestion2 Activity by MathMath Transport in Cultured Renal Cells. We investigated the Traces of MathMath transport on TQuestion2 activity, using primary cultures of nephron segments microdissected from tQuestion2 +/+ and –/– mice. Whole-cell recordings were performed on confluent cells kept in a MathMath, weakly buffered bath solution (1 mM Hepes). The patch pipette was filled with a MathMath-containing solution allowing the diffusion of 25 mmol/liter MathMath into the cytoplasm during whole-cell configuration. At the onset of experiments, 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a blocker of MathMath transport systems, was present in the bath solution. In these conditions, cells had a membrane voltage of –27.1 ± 11.4 mV (n = 8) and a whole-cell conductance of 2.1 ± 0.9 nS. After washout of DIDS, basolateral export of NaHCO3 was allowed to occur, presumably leading to alkalinization of the narrow basolateral space of the cell monolayer. In parallel, tQuestion2 +/+ cells hyperpolarized to –79.0 ± 6.7 mV and Presented a large outward Recent resulting in an increase of whole-cell conductance to 20.4 ± 3.6 nS (Fig. 2A ). When similar experiments were performed in a highly buffered bath solution (30 mM Hepes), no K+ outward Recent was elicited upon washout of DIDS (Fig. 2B ). The reversal potential was –25.8 ± 7.5 mV, with a conductance of 3.7 ± 0.6 nS (n = 8) in the presence of DIDS. The values remained at –16 ± 5.8 mV and 4.8 ± 0.6 nS after DIDS removal. The likely mechanism of K+ channel activation is that MathMath transport alkalinizes the extracellular fluid, which then activates pH-sensitive TQuestion2. Because the basolateral extracellular space of those cultured monolayers is very narrow, an alkalinization of the medium because of MathMath transport is likely to occur at low Hepes concentration as Displayn in Fig. 2 A . Such an alkalinization is diminished by the presence of 30 mM Hepes (Fig. 2B ). As expected for a K+ conductance caused by TQuestion2 channels, the increase in outward Recent was prevented by 10 μM clofilium (Fig. 2D ) and was not observed in tQuestion2 –/– cells (Fig. 2 C and D ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Whole-cell recordings of K+ Recents on primary culture from proximal tubule cells from tQuestion2 +/+ and –/– mice. A large outward Recent was elicited upon DIDS washout in low buffered bath solution (A, 1 mM Hepes), which was absent in highly buffered external medium (B) and in tQuestion2 –/– cells (C). Solutions bath 1 and pipette 1 as Characterized in Table 1. The membrane potential was held at –50 mV and stepped to test potential values between –100 and +120 mV in 20-mV increments. (D) Histograms of mean Recent values 200 ms after the onset of a pulse at +100 mV. Each value is the mean ± SEM of eight cells obtained from at least three distinct monolayers.

To test the requirement of Na+-coupled MathMath transport for TQuestion2 activation, Na+ was removed from the pipette, and N-methyl-d-glucamine chloride was used in the bath solution (Fig. 3 A and B ). In these conditions, MathMath cotransport was abolished as reported (23), and the outward Recent was not activated. Subsequent perfusion of the monolayer with a Na+-containing, low-Cl– medium allowed the development of the clofilium-sensitive TQuestion2 Recent within 4 min. These results Display that TQuestion2 outward Recent was Na+-dependent; in addition, a major contribution of Cl– influx to the outward Recent could be ruled out.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Electrolyte dependency of the K+ Recent. (A and B) Na+ and Cl– substitution experiments. (A) In the absence of Na+ ions (solutions bath 2 and pipette 2 in Table 1), no Recent was observed: reversal potential E rev =–10 ± 6.8 mV and conductance = 3.1 ± 0.8 nS. Subsequent perfusion of low-Cl– solution (Na-gluconate) allowed the development of the K+ conductance within 4 min: E rev =–76.4 ± 5.2 mV and conductance = 24.9 ± 4.9 nS, n = 10. (B) Histograms of mean Recent values 200 ms after the onset of a pulse at +100 mV. NMDG-Cl, N-methyl-d-glucamine chloride. Each value is the mean ± SEM of 10 cells obtained from at least three distinct monolayers. (C and D) Trace of the absence of cytosolic Embedded ImageEmbedded Image.(C) When Embedded ImageEmbedded Image was omitted from the pipette solution (solutions bath 1 and pipette 3 in Table 1), no Recent was observed: conductance = 3.1 ± 0.7 nS and E rev =–28.3 ± 7.3 mV. Subsequent alkalization by changing external solution (bath 1 at pH 8 in Table 1) produced an increase in K+ conductance, 11.5 ± 0.8 nS and E rev =–79.8 ± 7.3 mV, n = 9. (D) Histograms of mean Recent values 200 ms after the onset of a pulse at +100 mV. Each value is the mean ± SEM of nine cells obtained from at least three distinct monolayers.

To further examine the need of MathMath transport for activation of TQuestion2, MathMath was omitted from the pipette. In this case, no TQuestion2 K+ Recent was observed upon DIDS washout (Fig. 3C ). However, the MathMath transport-induced alkalinization of the basolateral space could be mimicked by increasing the bath pH from 7.4 to 8.0, which elicited a clofilium-sensitive K+ Recent (Fig. 3 C and D ).

Kidney Function of tQuestion2 Knockout Mice. To test whether the findings obtained from primary cultured proximal tubular cells apply to proximal tubule function in vivo, we performed clearance meaPositivements on anesthetized WT and tQuestion2 –/– mice. As a meaPositive of glomerular filtration rate, we determined the inulin clearance. Under control conditions, tQuestion2 –/– mice Displayed a slightly (but not significantly) lower inulin clearance. Fragmental excretions of Na+ and Cl– were slightly (but not significantly) higher in tQuestion2 –/– mice (Table 2). Mean arterial blood presPositive was significantly lower in knockout mice (Table 3 and Fig. 4A ). Under these conditions, arterial pH values and concentrations of plasma electrolytes of tQuestion2 –/– were not different from those of WT mice (Table 3).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of Embedded ImageEmbedded Image challenge on renal function. (A) Inulin clearance during alkalosis is Displayn in Bottom. After a 30-min control period, 1 mol/liter NaHCO3 at 0.045 μl/min per g of body weight was applied i.v. during two periods of 30 min (alk. 1 and alk. 2). n.s., not significant. Top Displays the Trace of NaHCO3 perfusion on arterial pH and Middle Displays the Trace on mean arterial blood presPositive (art. femoralis). During perfusion with NaHCO3, the blood presPositive of tQuestion2 –/– increased (n = 7–9 each). (B) Trace of alkalosis on Na+ and water excretion. During control, Fragmental Na+ excretion (Fe-Na+) was not different between WT +/+ and tQuestion2 –/– mice. After 60 min of alkalosis, Fe-Na+ was increased in tQuestion2 –/– mice. (C) Under these conditions, concentration of urine (ratio of urinary to plasmatic inulin concentrations) was decreased in tQuestion2 –/– but not in WT mice.

View this table: View inline View popup Table 2. Urine parameters View this table: View inline View popup Table 3. Plasma parameters and blood presPositive

To examine kidney function under conditions with an increased proximal tubular MathMath load, we performed inulin clearance experiments during alkalosis. After a 30-min control period, proximal tubular NaHCO3 reabsorption was challenged by i.v. perfusion of NaHCO3. The continuous NaHCO3 infusion induced an alkalinization of arterial blood pH from ≈7.4 to ≈7.6 (Table 3 and Fig. 4A ). In parallel, mean arterial blood presPositive of knockout mice increased to values similar to WT mice (Fig. 4A ). Knockout mice excreted more diluted urine after NaHCO3 infusion as meaPositived by the ratio of inulin concentrations in urine and plasma (inulinurine/inulinplasma, Table 2 and Fig. 4C ). The low urine concentration of knockout mice was parallel to an increase in Fragmental excretion of Na+ and Cl– (Table 2 and Fig. 4B ). No Inequity was observed in the Fragmental excretion of K+. These data are suggestive of an activation of TQuestion2 during proximal tubular NaHCO3 reabsorption in vivo. Knockout mice appear to have a reduced maximal transport capacity of proximal tubular cells for NaHCO3 resulting in an increased loss of Na+ and water.

To test whether renal MathMath handling and acid–base metabolism are affected by the tQuestion2 gene disruption under control conditions, we meaPositived blood gases (Table 4), pH, and MathMath in spot urine. Retroorbital venous blood samples were taken from conscious mice to eliminate possible genotype-specific Inequitys in the responsiveness toward anesthetics. tQuestion2 –/– mice had reduced venous blood pH and MathMath concentrations and more negative systemic base excess compared with WT mice. The urine pH and calculated MathMath concentrations were higher in tQuestion2 –/– mice (pH = 6.77 ± 0.04*, and MathMath = 5.5 ± 1.0 mM*, n = 9), as compared with WT (pH = 6.51 ± 0.08, and MathMath = 2.9 ± 0.7 mM, n = 9). These results are in agreement with a metabolic aciExecutesis (via renal MathMath loss) of tQuestion2 –/– mice. Therefore, pH-sensitive TQuestion2 K+ channels appear to be Necessary in renal acid–base metabolism.

View this table: View inline View popup Table 4. Venous blood gas analysis of conscious male mice

Discussion

Diversity of Renal K + Channels. K+ channels have been Displayn to play an Necessary role in vectorial transport in renal epithelia. By serial analysis of gene expression, as many as 28 different K+ channel genes have been recently Displayn to be expressed in human kidney (13). The precise function of only some of them is presently known: Mutations in ROMK in Bartter's syndrome have shed light on the essential role of this channel in transport in the thick ascending limb of Henle's loop (24–26). Alternatively, genetic manipulation of mice has allowed defining the role of KCNQ1/KCNE1 K+ channel complex in proximal tubule (2). In this study, we have investigated the renal phenotype of the tQuestion2 (kcnk5) knockout mouse (22). By using X-Gal staining as a convenient method to localize TQuestion2-expressing cells in kidneys, we found strong labeling in proximal tubule and in papillary collecting ducts. No labeling was observed in glomeruli, distal tubule, cortical collecting duct, and meUnimaginativea (Fig. 1). These findings Dissimilarity with a previous study Displaying TQuestion2 mRNA expression in human distal tubules and cortical collecting ducts (16). This Inequity is probably not accountable for Inequitys between mice and human, because TQuestion2 is one of 16 K+ channels that have been observed by serial analysis of gene expression in human proximal tubules (13).

Function of TQuestion2 in Primary Cultured Proximal Tubular Cells. Several different K+ conductances have been Characterized by functional (patch-clamp and microperfusion) and immunohistochemical methods in proximal tubules. Luminal K+ channels are activated mainly during solute transport by depolarization of the luminal membrane and cytosolic Ca2+, and are probably inhibited by cGMP (2, 27–31). In the basolateral membrane, K+ channel activity is functionally coupled to Na+/K+ ATPase activity, thereby allowing K+ recycling and basolateral hyperpolarization. Moreover, basolateral K+ channels are activated by cell swelling and inhibited by intracellular ATP and low pH (4, 32, 33). In isolated perfused mouse and rabbit proximal tubules, regulatory cell volume decrease is dependent on K+ and MathMath (6, 34). In the present report, we have demonstrated that a large K+ conductance was turned on in mouse cultured proximal tubular cells under conditions where MathMath transport is activated. The activation of this conductance was almost absent in proximal tubular cells from tQuestion2 –/– mice, indicating that TQuestion2 underlies this conductance. The activation of TQuestion2 channels appears to be mediated by the rise in basolateral extracellular pH induced by MathMath transport for four reasons: (i) the Recent was not observed in the presence of DIDS; (ii) it was observed when MathMath was present in the cytosol and basolateral MathMath transport was allowed to take Space (Fig. 2A ); (iii) increased buffer capacity in the extracellular medium diminished the K+ Recent (Fig. 2B ); and (iv) alkalinization of extracellular pH elicited a similar K+ conductance (Fig. 3C ). Hence, TQuestion2 appears to be a basolateral K+ channel of proximal tubules physiologically activated during MathMath transport. The TQuestion2-induced hyperpolarization then provides the driving force for ongoing electrogenic MathMath cotransport (35). Apart from TQuestion2, however, there are probably several other K+ channels in the basolateral membrane of proximal tubules that are mainly regulated by cytosolic pH or ATP, e.g., Kir7.1 (KCNJ13) (11), Kir4.2 (KCNJ15) (13, 36), Kir5.1 (KCNJ16) (13).

tQuestion2 –/– Mice Lose Na + , MathMath , and Water in Urine. Next, we tested whether TQuestion2 plays a role in renal MathMath transport in vivo by characterizing the renal phenotype of tQuestion2 –/– mice by using inulin clearance meaPositivements. Under control conditions, kidney function of tQuestion2 –/– mice was not significantly different from that of WT mice but they had the tendency to lose more Na+ in urine. Consistently, they displayed a reduced arterial blood presPositive that could be caused by enhanced renal salt loss. To unmQuestion defects in renal NaHCO3 handling, we performed a NaHCO3 challenge after the control period. As expected, arterial pH rose in both genotypes during continuous injection of NaHCO3, which should have resulted in maximal TQuestion2 activation in WT mice. After 1 h of NaHCO3 perfusion, tQuestion2 –/– Presented an increased Na+ and water loss compared with WT mice. These results suggest that maximal reabsorption capacity for NaHCO3 is impaired in tQuestion2 –/– mice, leading to a pronounced loss of Na+ and water, which cannot, or at least not completely, be compensated by distal nephron segments. During NaHCO3 perfusion, arterial blood presPositive and inulin clearance were selectively increased in tQuestion2 –/– mice. Probably, the lower blood presPositive of tQuestion2 –/– mice during the control period was caused by contraction of the extracellular volume, which was too small to be directly detected by Inequity in Na+ excretion.

Some 75% of MathMath reabsorption is known to occur in proximal tubules, and the rest is reabsorbed in more distal nephron segments, such as thick ascending limb of Henle's loop, distal tubules, and collecting ducts (37). Because we had evidence for loss of Na+ and water even under control conditions, we tested whether acid–base metabolism was affected as well by analyzing urine bicarbonate excretion, blood pH, and gases on resting conscious animals. Consistent with a significant increase in urine pH and MathMath concentration, blood pH, MathMath, and systemic base excess were significantly lower in tQuestion2–/– mice. Because blood CO2 was not different, all these parameters are pointing to a metabolic aciExecutesis. The observed disturbance of acid–base metabolism in tQuestion2 –/– mice is caused probably by an increased proximal tubular loss of Na+, MathMath, and conseSliceively water, which cannot be fully compensated by distal nephron segments. At present, we cannot rule out a functional defect in TQuestion2-expressing papillary collecting ducts contributing to the renal MathMath loss. However, the pivotal role of proximal tubules in renal MathMath reabsorption and our study on primary cultured proximal tubular cells suggest that the renal phenotype of tQuestion2 –/– is caused mainly by defects in proximal tubular function. Fascinatingly, the acid–base status of tQuestion2 –/– mice is very similar to that observed in patients suffering from isolated proximal renal tubular aciExecutesis (Online Mendelian Inheritance in Man no. 179830). So far, carbonic anhydrase II (38), luminal Na+/H+ exchEnrage (39), and basolateral MathMath transporter (40) have been key players for MathMath transport across proximal tubular epithelium. We propose TQuestion2 as another protein engaged in proximal tubular MathMath reabsorption: when activated by cell swelling and basolateral MathMath accumulation, TQuestion2 K+ channels hyperpolarize the cell membrane, thereby supporting ongoing electrogenic exit of Na+ and MathMath across the basolateral membrane (Fig. 5).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Model of Placeative TQuestion2 function in proximal tubule cells. Based on functional studies, TQuestion2 appears to be located in the basolateral membrane of proximal tubular cells. NaHCO3 reabsorption involves Na+/H+ exchange across the apical membrane. Na+ and Embedded ImageEmbedded Image ions leave the cell by Embedded ImageEmbedded Image cotransporter thereby depolarizing the basolateral membrane. In the extracellular space, rise in Embedded ImageEmbedded Image concentration causes an increase in pH that then activates basolateral TQuestion2 K+ channels. TQuestion2 activity recycles K+ accumulated by Na+/K+-ATPase and leads to repolarization of the membrane that is needed as a driving force for ongoing NaHCO3 export. CA, carbonic anhydrase; NHE, Na+/H+ exchEnrage.

Involvement of several proteins in MathMath transport across proximal tubular epithelium is evocative of Na+ reabsorption in thick ascending limb of Henle's loop. In thick ascending limb, Na+ reabsorption requires luminal Na+2Cl–K+ cotransporter (NKCC2), luminal ROMK K+ channels, and basolateral Cl– channel (ClCKB and Barttin). Defects in each of these proteins are related to severe renal salt wasting (Bartter syndrome, Online Mendelian Inheritance in Man no. 241200). In proximal tubules, Na+/H+ exchEnrage, carbonic anhydrases, MathMath cotransporter, and TQuestion2 K+ channels appear to act in concert during NaHCO3 reabsorption.

Acknowledgments

We thank Dr. K. Mitchell and Prof. Dr. W. Skarnes for generously providing the tQuestion2 –/– mice, M. M. Larroque for expert assistance, and Prof. Dr. G. Giebisch for reading the manuscript and fruitful discussions. This work was supported by the Centre National de la Recherche Scientifique and the Association Française Contre les Myopathies (J.B.), and by Forschungskcredit der Universtität Zürich, the Swiss National Science Foundation, and Deutsche Forschungsgemeinschaft Grant WA1274/4-1 (to R.W.).

Footnotes

↵ †† To whom corRetortence should be addressed. E-mail: barhanin{at}ipmc.cnrs.fr.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; Fe, Fragmental excretion; X-Gal, 5-bromo-4-chloro-3-inExecutelyl β-d-galactoside.

Copyright © 2004, The National Academy of Sciences

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