Preparation and study of fragments of single rabbit nephrons

Burg, M. B.; Grantham, J. J.; Abramow, Maurice; Orloff, Jack

doi:10.1152/ajplegacy.1966.210.6.1293

Cited by 760 publications

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“…We hand dissected kidneys from ''pathogenfree'' female New Zealand White rabbits (1.4-2.0 kg) to yield individual segments of midcortical S2 PTs in accordance with an approved animal protocol. We used two assemblies of concentric glass pipettes to perfuse lumens of single isolated PTs at 37°C, as described by Burg and coworkers (18) and modified by Quigley and Baum (19). We used a calibrated collection pipette (volume Х 55 nl) to collect samples of fluid that had flowed down the PT lumen; the luminal collection rate was 12.6 Ϯ 0.3 nl͞min (n ϭ 50 J V ͞J HCO 3 measurements).…”

Section: Methodsmentioning

confidence: 99%

Evidence from renal proximal tubules that \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation}{\mathrm{HCO}}_{3}^{-}\end{equation}\end{document} and solute reabsorption are acutely regulated not by pH but by basolateral \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackag

Zhou

Zhao

Bouyer

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Respiratory acidosis, a decrease in blood pH caused by a rise in [CO 2], rapidly triggers a compensatory response in which the kidney markedly increases its secretion of H ؉ from blood to urine. However, in this and other acid-base disturbances, the equilibrium CO 2 kidney ͉ out-of-equilibrium solution ͉ acid-base disturbances ͉ volume reabsorption A major task of the kidney is to secrete H ϩ into the urine.Inadequate renal H ϩ secretion caused, for example, by mutations to acid-base transporters (1-4) or carbonic anhydrases (5), or by renal failure (6, 7), can lead to a life-threatening decrease in blood pH. Moreover, to maintain a stable blood pH, the kidney must appropriately increase H ϩ secretion in response to metabolic acidosis (a decrease in blood pH caused by a decrease in [HCO 3 Ϫ ] at a fixed CO 2 ) or to respiratory acidosis. However, a half century after the classical observation that respiratory acidosis rapidly stimulates renal H ϩ secretion (8, 9), we still have little insight into how the kidney senses acute acid-base disturbances.The renal proximal tubule (PT) reabsorbs (from lumen to blood) a liquid that contains Ϸ80% of the HCO 3 Ϫ filtered by the glomerulus. The PT cell does this reabsorbtion by secreting H ϩ into the PT lumen and using this H ϩ to titrate luminal HCO 3 Ϫ to CO 2 and H 2 O. After entering the cell across the apical membrane, the CO 2 and H 2 O recombine to produce H ϩ and HCO 3 Ϫ . The cell extrudes the H ϩ into the lumen across the apical membrane through Na-H exchangers (10-12) and H ϩ pumps (13) and moves the HCO 3 Ϫ out across the basolateral membrane via the electrogenic Na͞HCO 3 cotransporter NBCe1-A (14, 15). Carbonic anhydrases catalyze the interconversions between CO 2 and H 2 O on the one hand and HCO 3 Ϫ and H ϩ on the other (5). Because blood pH is the parameter regulated by these acid-base transport processes, blood pH or, more likely, intracellular pH (pH i ), has been thought to be the parameter sensed by renal cells. However, because of the interconversion CO 2 ϩ H 2 O^HCO 3 In the present study, we perfused single, isolated S2 segments of rabbit PTs and collected the fluid that had passed along the PT lumen. Analysis of this collected fluid allowed us to compute volume reabsorption (J V ); HCO 3 Ϫ reabsorption (J HCO 3 ), which is virtually the same as the H ϩ -secretion rate under the conditions of our experiments; and the reabsorption of solutes other than NaHCO 3 (J Other ). We made the surprising observation that, at least in the short term, J HCO 3 and J Other do not respond to changes in basolateral or intracellular pH. The most straightforward hypothesis is that PT cells have sensors for basolateral HCO 3 Ϫ and a parameter related to CO 2 . Materials and MethodsExcept for the compositions of most of the OOE CO 2 ͞HCO 3 Ϫ solutions, our methods are described in detail in ref. 17.Tubule Perfusion. We hand dissected kidneys from ''pathogenfree'' female New Zealand White rabbits (1.4-2.0 kg) to yield individual segments of midcortical S2 PTs in accorda...

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Section: Methodsmentioning

confidence: 99%

Evidence from renal proximal tubules that \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation}{\mathrm{HCO}}_{3}^{-}\end{equation}\end{document} and solute reabsorption are acutely regulated not by pH but by basolateral \documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackag

Zhou

Zhao

Bouyer

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The mTALs were microdissected with fine forceps under a stereoscopic microscope. A fragment of the mTAL was transferred to a chamber on the stage of an inverted microscope (IX-71, Olympus Co. Ltd., Tokyo) and was microperfused in vitro using Burg's method (Burg et al 1966), with some modifications as previously described (Kondo and Fromter 1987). Our system allowed the complete exchange of either luminal or basolateral solution in one second.…”

Section: Methodsmentioning

confidence: 99%

Chloride-Dependent Intracellular pH Regulation via Extracellular Calcium-Sensing Receptor in the Medullary Thick Ascending Limb of the Mouse Kidney

Aslanova

Morimoto

Farajov

et al. 2006

Tohoku J. Exp. Med.

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The extracellular calcium-sensing receptor (CaSR) located in either luminal or basolateral cell membranes of various types of renal tubules including proximal tubules, Henle's loop and collecting ducts has been thought to play a fundamental role in electrolyte metabolism. To further identify the physiological roles of the CaSR, we examined the effects of Ca 2+ and calcimimetics neomycin (Neo), gentamicin and gadolinium chloride (Gd 3+ ) on the intracellular pH (pHi) of in vitro microperfused mouse medullary thick ascending limb (mTAL) cells of Henle's loop, by loading the cells with fluorescent pH indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and measuring the ratio of fluorescence emission at 530 nm after exciting the dye at 490 and 440 nm. In a steady-state condition in Hepes-buffered solution, the pHi in the mTALs was 7.29 ± 0.04 (n = 9). A concentration of 200 μ mol/l Neo in the basolateral side decreased the pHi after 1 min by −0.13 ± 0.02 (n = 34, p < 0.0001). The other calcimimetics showed similar effects on pHi, whereas none of these calcimimetics in the lumen affected pHi. Na + removal or the inhibition of Na + and proton transport with amiloride, bumetanide, or bafilomycin did not eliminate the effect of Neo on pHi. On the other hand, Cl − removal clearly eliminated the Neo-induced pHi decrease (−0.06 ± 0.01 vs −0.00 ± 0.05 in Cl − removal, n = 4, p < 0.003). Thus, we have demonstrated for the first time that the CaSR is involved in the regulation of the pHi in the mTAL and requires Cl − to exert its effect. intracellular pH; renal medulla; chloride; amiloride; BCECF

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“…We perfused the PTs using methods that were similar to those originally described by Burg et al (10) and later modified by Baum et al (2) and also by our laboratory (31,41). Briefly, a rabbit weighing 1.4 -2.0 kg was euthanized by a single overdose of 3 ml (ϳ20 mg) of intravenous pentobarbital sodium.…”

Section: Biological Preparationmentioning

confidence: 99%

Effects of angiotensin II on the CO₂dependence of HCO₃⁻reabsorption by the rabbit S2 renal proximal tubule

Zhou¹,

Bouyer²,

Boron³

2006

American Journal of Physiology-Renal Physiology

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M tended to reduce stimulation by CO2, and at 10 Ϫ9 M, produced an even greater reduction. In conclusion, the mutual effects of 1) ANG II on the JHCO 3 response to basolateral CO2 and 2) basolateral CO2 on the JHCO 3 responses to ANG II suggest that the signal-transduction pathways for ANG II and basolateral CO2 intersect or merge.kidney; out-of-equilibrium solutions; acid-base; volume reabsorption ONE OF THE MAJOR tasks of the kidneys is to participate, along with the lungs, in the regulation of the acid-base balance of the extracellular fluid. For example, it has long been appreciated that acute respiratory acidosis (i.e., a rise in PCO 2 that causes a fall in pH) rapidly stimulates renal H ϩ secretion (8, 16). The proximal tubule (PT) plays a key role in this acid secretion. In addition to reabsorbing a near-isosmotic fluid that represents about two-thirds of the fluid filtered by the glomerulus, the PT reabsorbs ϳ80% of the filtered HCO 3 Ϫ as follows. The PT cell actively secretes H ϩ into the tubule lumen (1, 6, 35) and uses this H ϩ to titrate filtered HCO 3 Ϫ in the lumen to CO 2 and H 2 O, catalyzed by apical carbonic anhydrase IV (9, 36, 37). The newly formed CO 2 and H 2 O then diffuse into the PT cells, where carbonic anhydrase II (36, 37) catalyzes the regeneration of H ϩ and HCO 3 Ϫ . The cell extrudes the H ϩ across the apical membrane via Na-H exchangers (4, 5, 30) and H ϩ pumps (20), while exporting the HCO 3 Ϫ across the basolateral membrane, mainly via the electrogenic Na/HCO 3 cotransporter (7,15,32,33 (12) on isolated, perfused rabbit PTs. They noted that adding ϳ2 ϫ 10 Ϫ6 M ANG II to the basolateral solution had no detectable effect on the rate of fluid absorption (J V ). In a 1974 rat micropuncture study, Steven (38) reported that ϳ2 ϫ 10 Ϫ5 M basolateral ANG II lowered J V , whereas 1 ϫ 10 Ϫ7 M had no effect. Harris and Young (23) later showed that basolateral ANG II has a biphasic effect on Na ϩ reabsorption in the rat PT, stimulating at low doses (10 Ϫ12 -10 Ϫ10 M) and inhibiting at higher doses (3 ϫ 10 Ϫ7 -3 ϫ 10 Ϫ6 M). Shuster and colleagues (34) in 1984 demonstrated a similar biphasic effect of basolateral ANG II on J V in isolated, perfused rabbit PTs, ruling out a role of sympathetic innervation on the J V response. In 1991, working in isolated, perfused rat proximal straight tubules (PSTs), Garvin (19) found that 10 Ϫ10 M basolateral ANG II increases both J HCO 3 and J V . At about the same time, Chatsudthipong and Chan (14) found that high levels of basolateral ANG II reduce J HCO 3 in rats and that these effects are blocked by saralasin, an antagonist of ANG II receptors.As far as the effects of luminal ANG II are concerned, in a 1988 micropuncture study, Liu and Cogan (26) showed that low-dose luminal ANG II (10 Ϫ12 -10 Ϫ11 M) increases HCO 3 Ϫ reabsorption (J HCO 3 ) even in a denervated rat kidney. The 1990 study on the rat PT by Wang and Chan (39) extended the earlier observations by showing that increasing levels of luminal ANG II have biphasic effects on J HCO 3 as well...

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Preparation and study of fragments of single rabbit nephrons

Cited by 760 publications

References 0 publications

Chloride-Dependent Intracellular pH Regulation via Extracellular Calcium-Sensing Receptor in the Medullary Thick Ascending Limb of the Mouse Kidney

Effects of angiotensin II on the CO₂dependence of HCO₃⁻reabsorption by the rabbit S2 renal proximal tubule

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Preparation and study of fragments of single rabbit nephrons

Cited by 760 publications

References 0 publications

Chloride-Dependent Intracellular pH Regulation via Extracellular Calcium-Sensing Receptor in the Medullary Thick Ascending Limb of the Mouse Kidney

Effects of angiotensin II on the CO2dependence of HCO3−reabsorption by the rabbit S2 renal proximal tubule

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Effects of angiotensin II on the CO₂dependence of HCO₃⁻reabsorption by the rabbit S2 renal proximal tubule