Laboratory of Renal and Body Fluid Physiology, Medical Research Centre of the Polish Academy of Sciences, PL 02-106 Warsaw, Poland
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ABSTRACT |
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The relationship of renal medullary tissue ion concentration and
medullary blood flow (MBF) has never been closely evaluated because of
limitations of available measuring methods. In an attempt to overcome
this difficulty, an integrated probe was developed for simultaneous
recording in rat renal medulla of tissue electrical admittance
(Y), an index of interstitial ion
concentration, and tissue perfusion with blood (laser-Doppler method).
During spontaneous-selective MBF variations tissue
Y showed inverse changes
(r = 0.77,
P < 0.001). The inverse correlation
of the two variables was also seen after MBF has been reduced
(
43%) by indomethacin, 5 mg/kg body wt iv
(r =
0.77,
P < 0.01). A modest selective MBF
reduction (15%) induced by glibenclamide, an inhibitor of
ATP-dependent K channels, did not alter medullary tissue admittance.
The data support experimentally the concept that the rate of medullary tissue perfusion with blood is one determinant of interstitial solute
concentration; however, changes in the latter were demonstrable only
with major alterations of the MBF.
kidney medulla; medullary sodium concentration; medullary blood flow
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INTRODUCTION |
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THE VASA RECTA SYSTEM of the renal medulla enables countercurrent exchange of solutes in this zone and preservation of the corticopapillary NaCl and urea gradient, a basis for efficient urine concentration. Thurau et al. (18) were first to show that urine concentration (U/Posm) was inversely correlated to medullary blood flow (MBF). In accordance with these early data, a current model of renal microcirculation predicts that increased blood flow should diminish the efficiency of countercurrent exchange (10).
In studies of the effect of MBF changes on the rate of solute exchange in the medulla, it would be more appropriate to correlate flow changes with those in tissue solute concentration, rather than with U/Posm, which reflects the final outcome of the complex urine concentration process. Concurrent measurement of the two variables has never been made for methodological reasons. Because analysis of tissue slices and most of earlier methods for MBF measurement could be applied only once per kidney, it was difficult to design protocols for study of the real time relationship between MBF and tissue solute concentration.
Application of tissue electrical admittance (reciprocal impedance) recording in the in situ kidney enabled us to continuously estimate of tissue NaCl (but not urea) concentration in the medulla (14). Newer techniques enable direct continuous measurements of MBF (for review, see Ref. 10). Below we describe a setup for simultaneous measurement in rat renal medulla of tissue ion (mostly NaCl) concentration and tissue perfusion with blood as indicated by a laser-Doppler (LD) signal. The setup offers a unique possibility to follow simultaneously the dynamics of fluctuations in the ionic component of medullary hypertonicity and in the local blood flow and thereby to explore the relationship of the two variables.
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METHODS |
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Simultaneous placement in a rat kidney of our standard set of three needle electrodes for admittance measurement in the outer and inner medulla (15) and, in addition, of an LD probe, would unavoidably cause a serious kidney damage. In all our earlier studies the glomerular filtration rate (GFR) was reduced in the kidney impaled with three admittance electrodes alone. To alleviate the damage, we decided to measure admittance of the inner medulla only, which requires two electrodes. However, only one standard admittance electrode was used, and the cannula housing optical fibers of the LD probe was applied to serve as the second admittance electrode.
The measuring setup. This consists of a needle LD probe (PF 402; Perimed, Jarfalla, Sweden) and of an admittance electrode prepared as described before (15), arranged in parallel at 1-mm distance, both fixed at the base in a common bedplate (Fig. 1). The LD probe is a stainless steel cannula housing two optical fibers for transmission of a laser light beam to and back from the tissue. The tip-to-base length and the diameter of the probe are 5.4 and 0.45 mm, respectively. The admittance electrode is a piece of 75% platinum-25% iridium wire, 6.8 mm in length and 0.3 mm in diameter, sharpened at the tip. The two elements are insulated, except for the top 1.1-mm segment of the LD and 1.3-mm segment of the admittance electrode, respectively. The LD probe/admittance electrode set is connected to an LD flowmeter and to a conductance meter (see below).
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MBF measurement. The LD probe was connected to an LD perfusion monitor (Periflux 4001, Perimed). The system measures tissue perfusion with blood cells (Doppler flux) defined as the product of the number of blood cells moving and their mean velocity, within the area <1 mm3 beneath the tip of the probe; the signal is independent of the direction of cell movement. The results are expressed in arbitrary perfusion units (PU) or in volts of the analogue output (1,000 PU = 10 V). Thus only relative changes are measured but, owing to a calibration procedure the results can be compared among animals. Each probe was calibrated using a motility standard, i.e., a colloidal suspension of latex particles (supplied by the manufacturer). The Brownian motion of the suspension provides the standard value of 250 PU. The biological zero flow was determined at the end of experiments after clamping the renal artery. Application of implanted LD probes for measurement of blood flow in the renal medulla of rats (5, 8) and dogs (17) was described previously.
For simultaneous measurement of renal cortical blood flow (CBF), the fibrous capsule of the kidney was incised, and another LD probe (type PF 415:1; tip diameter, 0.7 mm) was placed on tissue surface.
Admittance measurement. The principle and the main features of the technique were described elsewhere (14, 15). It was shown that admittance (reciprocal impedance) values recorded from the medulla of an in situ kidney are linearly related to all ion and to Na concentration in the extracellular compartment (1, 4). The present setup for admittance measurement differs from the standard one used before, comprising three electrodes of different lengths (labeled electrodes I, II, and III), for measurement in the inner medulla (between electrodes I and II) and outer medulla (electrodes II and III). Instead, measurement is confined to the inner medulla, there is only one standard platinum-iridium electrode, and a segment of the stainless steel cannula housing the LD probe is used as the second electrode (Fig. 1). Similar to in earlier studies, electrodes were connected to a laboratory conductance meter (Mera-Elwro, Wroclaw, Poland) via a programming device that turned on the measuring current for 10 s at 70-s intervals. However, for use with this modified electrode set, the frequency of the measuring current was raised from the usual 3.5 to 24 kHz. During experiments, admittance was recorded along with aortic blood pressure (BP) and medullary and cortical LD flux.
To check whether admittance measured using the present setup was a reliable index of tissue conductance (which is strictly related to ion concentration), in 16 rats, admittance value recorded at the end of experiment was compared with standard conductance (Gstd) of the inner medulla excised after removal of the kidney, using a method described previously (4). To extend the range of tissue ion concentration, some of animals had received an injection of indomethacin, 10 mg/kg body wt iv, followed by an infusion of 5% saline at 2.4 ml/h (leading to an increase in tissue NaCl), or an infusion of mannitol, given as an intravenous 20% solution or 5% solution into the renal pelvis, at 2.4 and 1.0 ml/h, respectively (leading to "washout" of medullary solutes) (4).
Experimental procedures. Male Wistar rats weighing 270-320 g were anesthetized with intraperitoneal thiobutobarbital (Inactin; Byk Gulden, Constance, Germany; 100 mg/kg body wt). A cannula was placed in the trachea to ensure free airways; the femoral artery and vein were cannulated for measurement of aortic BP and for infusion of fluids and drugs, respectively. The left kidney was exposed from a subcostal flank incision and placed in a plastic holder similar to that used for micropuncture experiments. The inside of the holder was padded in a way that the kidney's dorsal curvature (and not the side surface) was facing upwards. Subsequently, a PE-20 catheter was placed in the ureter, and the set for admittance and MBF measurement was inserted into the kidney along the corticopapillary axis. The dorsal surface was first punctured with the longer, sharp admittance electrode, which was pushed until the shorter LD probe touched the kidney. At this point, the fibrous capsule of the kidney was incised with scissors to facilitate the entry of the cylindrical LD probe, and then the whole set was pushed to penetrate the kidney toward the papilla until the bedplate adhered firmly to the surface. After experiments, the position of the admittance electrode tip (deep in the inner medulla; see Fig. 1) and of the LD probe (close to the border with the outer medulla) was verified at the cross-section.
The set was suspended on a flexible cable of the LD probe fixed to a frame above the kidney. There was no need for rigid fixation. A transient hematuria that followed insertion was comparable to that always observed by us after placement in the kidney of the standard set of admittance electrodes.
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RESULTS AND COMMENTS |
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Measurement of tissue perfusion with blood using LD (single strands of optical fibers) inserted into the cortex and medulla of rat and dog kidneys has been successfully performed and evaluated before (5, 8, 17), and our application of this technique requires no further comment. On the other hand, admittance measurement using the present setup departs from standard conditions defined before in that a stainless steel surface of the cannula encompassing optic fibers of the LD probe is used as one admittance electrode.
Compared with platinum/iridium normally used as electrode material, application of a steel surface would increase electrode polarization impedance arising at the tissue/electrode interface, i.e., of the imaginary component of the impedance measured (14). This component (capacitive reactance) is only slightly dependent on ion concentration in tissue fluid; hence, the admittance measured (reciprocal total impedance) may significantly differ from real tissue conductance, which is a linear function of ion concentration. The consequence could be only a minor change of the admittance measured per any given change of tissue ion concentration (or true conductance), i.e., a "flat" slope relating the two variables; another effect could be poor correlation of these variables. Both factors would result in poor "resolution" of a method estimating tissue ion (mostly NaCl) concentration on basis of tissue admittance.
Electrode polarization impedance decreases with increasing frequency of the measuring current (15, 16); therefore, we raised the frequency from 3.5 kHz used before to 24 kHz. With the latter frequency, admittance measured still relates mostly to the extracellular compartment, as the current bypasses cells (9, 16). The results obtained with the present setup at the frequency of 24 kHz are shown in Fig. 2. There is a good correlation of admittance values measured (Y) and standard conductance (Gstd), defined as a conductance of the excised inner medulla measured with large surface electrodes (no imaginary component) (4). The r value of 0.90 was even distinctly higher than r = 0.76 reported from a similar study using platinum-iridium electrodes and the frequency of 3.5 kHz. Also the slope of Y on Gstd (y = 0.24x, n = 16) was steeper than that observed with the standard setup (y = 0.13x, n = 27) (Dobrowolski, unpublished observations). On the whole, this comparison indicates that increasing the frequency of the measuring current abolished the expected disadvantageous effect of using steel electrode surface and that the modified measuring conditions provide an even better resolution when estimating extracellular ion concentration (conductance) from in situ tissue admittance measurement.
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In a kidney impaled with an admittance electrode and an LD probe, a significant fraction of nephrons is rendered nonfunctional. Our probe-electrode set is implanted in such a way that only the thinner (admittance) component enters a substantial portion of the inner medulla (Fig. 1); this limits the damage of the area under investigation. Microscopic examination of the critical area detects a damage zone of <0.1 mm in thickness surrounding the electrodes. When the distance of ~1.7 mm between them is considered (note that the active surfaces are not at the same depth in the kidney), the contribution of the damaged tissue to measurement can be regarded as minor. This conclusion is supported by additional functional studies. In six rats, the GFR (clearance of [3H]inulin) was determined and found to be 0.85 ± 0.06 ml/min (mean ± SE) for the exposed kidney bearing the present set, compared with 1.01 ± 0.12 ml/min for the contralateral organ. Such a 16% reduction of the filtration rate on the manipulated side compares favorably with 20-30% reduction observed in the kidney impaled with three standard admittance electrodes as used in our previous studies. We checked also that electrode impalement did not affect total renal blood flow (renal vein outflow recording); in anesthetized rats, the kidney bearing the electrode set was able to concentrate urine up to at least 1,600 mosmol/kgH2O and dilute it after an intravenous water load to 100 mosmol/kgH2O.
A limitation of the present setup is that, unlike with the standard three-electrode set, no data are obtained for the outer medulla. However, in all our previous studies (8 publications), admittance changes in this zone were in the same direction as in the inner medulla but less pronounced and or not significant. Thus omission of the third electrode and lack of data for the outer medulla would probably not entail loss of important information.
Tissue admittance during spontaneous changes in MBF. During many hours of simultaneous recording of Y, MBF, CBF, and BP in anesthetized rats, a spontaneous change in MBF was occasionally seen that was not accompanied by any change in BP and CBF, at least not for ~10 min. A sample record of such an event (Fig. 3) shows a decrease in MBF associated with an inverse change in Y, followed by a recovery of both variables. As a rule, a change in the former preceded that in the latter by 1-3 min. Such observations suggest impaired dissipation of medullary tissue ions due to a reduced tissue perfusion with blood. The stability of BP and CBF (the latter reflecting cortex perfusion and, probably, indirectly, GFR) is an essential condition for the above interpretation, since both variables could modify NaCl load delivered to the medullary ascending limb of Henle's loop and alter Y independent of the influence of vasa recta blood flow.
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A collective analysis of 15 spontaneous changes in MBF (4 increases and 10 decreases) and associated changes in Y is shown in Fig. 4. A significant inverse correlation was seen (with the linear regression line running through the origin of the coordinate system), which suggests that the rate of evacuation of solutes (including ions) from the medullary interstitium varies directly with the MBF. Admittedly, the effect of MBF changes on tissue ion concentration was small: Fig. 4 shows that a 50% decrease in the flow would cause an ~7% increase in Y, equivalent to ~15 mmol NaCl/kg tissue. The changes in Y given in Fig. 4 occurred within 10-30 min from the start of the decrease in MBF. Unfortunately, with time, even when MBF continued to change or remained reduced or elevated, total renal CBF and/or BP also started to change, and the conditions for interpretation of Y changes were no longer fulfilled. Therefore, it remains to be established whether a prolonged moderate change in MBF can induce a progressing increase or decrease in medullary hypertonicity and thereby substantially influence urine concentration.
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Tissue admittance and indomethacin-induced decrease in
MBF. There is a sound evidence that, in the
anesthetized rat subjected to a trauma of acute surgery, the renal
medullary circulation is under tonic vasodilator influence of
prostaglandins (11, 12). Therefore, to reduce MBF in 10 Inactin-anesthetized rats, we administered indomethacin (5 mg/kg body
wt iv) infused during 5-8 min. Because this invariably resulted in
an increase in systemic BP and usually some transient increase in CBF,
renal perfusion pressure was maintained constant by a suprarenal
screw-controlled snare placed on the aorta. The data of Fig. 4 indicate
that the decreases in MBF and increases in
Y observed after indomethacin were
distinctly correlated, as during spontaneous variations described above. However, when MBF decreased 26-66% (mean change,
43%), Y increased considerably
11-34% (mean, 23%). Dissimilarly, it can be read from the
regression line for spontaneous variations (Fig. 4) that a mean
spontaneous MBF decrease of 43% would be associated with a
Y increase of 6% only, fourfold less
than was observed with 43% MBF decrease after indomethacin. This
indicates existence of a major component of indomethacin action on
tissue ion concentration unrelated to a decrease in MBF. Most probably, the drug abolished the inhibitory action of prostaglandins on the
vasopressin-dependent NaCl transport in the ascending limb of Henle's
loop (2, 3). However, the apparent difference in the slope of
Y dependence on MBF between the
spontaneous variations and the indomethacin series suggests that the
response of Y to prostaglandin
blockade was not simply a sum of a fixed effect on loop NaCl transport
(i.e., delivery of ions to the interstitium) and a blood flow-dependent
washout of interstitial solutes.
Tissue admittance and glibenclamide-induced changes in
MBF. Glibenclamide, an inhibitor of ATP-dependent K
channels, was recently reported to selectively depress MBF (11). In
nine Inactin-anesthetized rats, glibenclamide (Research Biochemicals,
Natick, MA) was infused through an aortic cannula with the tip
positioned just above the origin of the left renal artery. Within
20-30 min of glibenclamide infusion at a rate of 7.2 µg · h1 · kg
body wt
1, MBF decreased
14.8 ± 3.2% (means ± SE) compared with the preceding infusion
of the drug solvent (P < 0.01).
Tissue Y did not change significantly
(+0.6 ± 2.3%); changes in the two variables were not correlated.
Because glibenclamide administration tends to increase systemic BP, the
renal perfusion pressure was maintained constant as described above for
the indomethacin series. There was no significant change in CBF (+3.2 ± 2.2%). Thus the data failed to support the dependence of
medullary tissue electrolyte concentration on medullary perfusion with
blood. Possibly, the effect cannot be demonstrated with modest changes
in MBF, as suggested by the data on spontaneous flow variations.
Moreover, a possible vascular effect of glibenclamide (increased
Y due to reduced evacuation of solutes
with the blood) could have been offset by a direct effect on the
epithelium of the ascending limb of Henle's loop. The blockade of
ATP-dependent K channels would reduce loading of the luminal Na-K-2Cl
cotransporter (reviewed recently in Ref. 13) and limit NaCl
reabsorption and delivery to the medullary interstitium.
Collectively, the data of the spontaneous MBF variations and indomethacin and glibenclamide series provide first experimental evidence suggesting dependence of medullary interstitial ion concentration on the rate of tissue perfusion with blood. At the first sight, the dependence seemed demonstrable with major MBF changes only, probably in >20-30%. Further studies with simultaneous recording of MBF and medullary tissue admittance are needed to define the relationship of the two variables more precisely. This may not be easy: the difficulties with interpretation described above for indomethacin (tubular transport and vascular changes acting in the same direction) and glibenclamide (possible offsetting of the two effects) exemplify the fact that it is extremely difficult to alter renal vascular status without changing tubular function. Selective manipulation of medullary circulation without changing cortical blood flow is another challenge. Administration of vasoactive agents directly to the renal medulla (7) could be attempted to achieve this goal.
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ACKNOWLEDGEMENTS |
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The study was supported by the National Committee for Scientific Research (KBN) Grant no. 4PO5A 01308.
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FOOTNOTES |
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Address for reprint requests: J. Sadowski, Laboratory of Renal and Body Fluid Physiology, Medical Res. Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland.
Received 3 March 1997; accepted in final form 5 June 1997.
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