organic anion transport by renal cortical slices of harbor seals (phoca vitulina)

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Page 1: Organic anion transport by renal cortical slices of harbor seals (Phoca vitulina)

ORGANIC ANION TRANSPORT BY RENAL CORTICAL SLICES OF HARBOR SEALS (PHOCA VITULINA)

F. J. KOSCHIER, R. W. ELSNER, D. F. HOLLEMAN and S. K. HONG Department of Physiology. School of Medicine. State University of New York at Bufalo.

Buffalo. NY 14214. and Institute of Marine Science. University of Alaska. Fairbanks. AK 99701. U.S.A.

(Received 26 July 1977)

Abstract-i. The uptake of organic anions by renai cortical slices of harbor seals (Phorn riruiitrnt was studied to determine transport characteristics during anoxia.

2. Renal slice uptake of paminohippurate reached a steady-state level of transport within 15-30 min of incubation in an oxygen atmosphere. The steady-state transport of PAH was not altered after 60 min of incubation in a nitrogen atmosphere. In addition, PAH transport was unaffected by glucose or iodocetamide. but was reduced by 2,~dinitrophenoi and increased by acetate.

3. The slice uptake of ~,~dichi~ophenoxyaceta~e did not reach a steady-state level of transport during a 60-min incubation and was depressed significantly by incubation in a nitrogen atmosphere.

4. These results appear to indicate that the harbor seal kidney slice has an organic anion transport system similar to that found in non-diving animals; however. the tolerance of this transport system tb anoxia is greater in the seal.

INTRODUCHON

In the harbor seal, profound ~ardiovascuiar changes occur during breath-hold diving (Elsnet, 1970; Scho- lander, 1940): there are marked reductions in heart rate. cardiac output and peripheral blood flow. How- ever, arterial blood pressure and blood flow through the brain and heart are well maintained. These changes are mediated neurally and result in the con- servation of oxygen for vital functions during diving.

As a result of these diving responses, the blood flow to the kidney decreases markedly during diving (Bradley & Bing, 1942; Elsner et af., 1966; Lowrance et al., 1956; Murdaugh et al., 1961). Accompanying this alteration in renal blood flow are reductions in: vascular oxygen tension, glomerular filtration rate, urine flow, and Na excretion. In addition, increases in the fractional excretion of filtered water and Na are observed (Bradley et al., 1954; Lowrance et al., 1956: Murdaugh et al., 1961). These findings suggest that certain tubular transport mechanisms are affected during diving as a result of the marked reduc- tion in blood flow and the accompanying reduction in available oxygen.

Since harbor seals are engaged in repetitive diving (maximum diving time of about 20-25 minf, it is of interest to study the consequences of prolonged renal ischemia or anoxia on tubular functions. In a recent isolated, perfused kidney study, Halasz et al. (1974) found that the recovery of renal blood flow. urine flow and renal oxygen consumption from a 60-min occlusion of renal blood flow was considerably faster in the harbor seal as compared to the dog. This sug- gests that the kidneys of diving animals have a mech- anism to protect them from detrimental effects of ischemia, to which they are repetitively subjected. In order to gain further insight into this protective mech- anism, the transport of organic ions in the harbor seal kidney slice incubated in a medium containing various metabolic substrates or inhibitors was studied

in the present investigation. The results indicated that the harbor seal kidney slice was indeed capable of carrying out certain transport functions in the absence of oxygen for at least 60 min.

MATERIALS AND METHODS

Five harbor seals (Phoca rirulina) were captured on coas- tal Alaska and reared in Fairbanks. At the time of the experiment, these animals weighed approximately 3&40 kg. The animals were sacrificed instantly by a blow on the head and then exsanguinated. The kidneys were removed immediately and rinsed in ice-cold Krebs-Ringer phosphate buffer @H 7.4). The composition of the buFfer was: 100 mM NaCl. 40 mM KCI. 1.3 mM MeSO,. I mM CaCi, and 5.8 mM NaH,PO, (Koschier & B&n&, 19771. The kidneys were cut into small sections of approximately 2 cm’ and the renal capsule was removed from the lobules. Renal cortical slices (0.3-0.4mm in thickness) were cut free-hand and stored briefly in the ice-cold Krebs-Ringer phosphate buffer.

The uptake of organic anions by the renal cortical slice was determined at 25°C according to the general procedure described by Cross & Taggart (1950). Specifically. 100 mg of the slices were incubated in 3 ml of the phosphate buffer which contained the organic anion p-aminohippurate (PAH) at an initial media concentration of 7.5 x low5 M or 2,4-dichiorophenoxya~tate (2,4-D) at IO-’ M. Incu- bated with the weak organic acids were tracer amounts (0.08 pCi/mi) of 14C labeled compound (Amersham, Arling- ton Heights, IL. and New England Nuclear. Boston. MA). The incubations were performed in a DubnoR metabolic shaker (100 cycles per minf with a 100~, oxygen atmos- phere unless stated otherwise.

Ail inhibitors and substrates were added to the bathing solution without changing the final incubation volume. the Na’ concentration, or the total osmoiaiity in the bathing solution. At the end of the incubation period (15-60 min). the tissues were quickly removed from the bathing solution and blotted on filter paper. Their wet weight was deter- mined and they were then digested in I N NaOH for at least 36 hr. An aiiquot of the medium and tissue digest was assayed for ‘*C using standard liquid scintillation

Page 2: Organic anion transport by renal cortical slices of harbor seals (Phoca vitulina)

Fig. I. Time course of uptake of PAH by harbor seal (left) and rat (right) kidney cortical slices in the presence of oxygen or nitrogen. Each point represents the mean value k SE. obtained from 3-5 experiments. Accumulation of PAH by rat renal cortical slices was performed exactly as described for the seal (see Materials and Methods). Adult male Sprague-Dawley rats (200-300 g) were used in

these experiments.

techniques (Bromsone. 1970: Kobayashl & Maudsley, 1974). A Nuclear-Chicago Mark I liquid scintillation spec- trometer with external standardization capabilities was used for the radioassay. The renal slice uptake data are expressed as the slice/medium (S/M) ratio, i.e. the tissue concentration of radioactivity (dpm/gm wet tissue) divided by that of the medium (dpm/ml medium).

The data were analyzed statistically using Student’s r-test, either paired or unpaired depending on experimental design. The level of significance was chosen as P < 0.05.

RESULTS

PA H uptake

The time course of PAH uptake in the presence of 100’? O2 or N, is shown in Fig. 1. In harbor seal kidney slices (left panel), a rapid attainment of the steady-state transport of PAH was measured; however, PAH was concentrated to a relatively low level. Moreover, there was no significant difference, at a given incubation time, in the level of PAH uptake when slices were incubated in either an O2 or N, atmosphere. In contrast, PAH accumulation by the rat kidney slice (right panel) continued to increase as a function of the incubation time in the presence of 0,. and reached an S/M ratio of 6.0 at 60 min. However, this greater capacity to accumulate PAH in the rat kidney slice required the presence of 02. as indicated by a continuous decline in PAH accumu- lation after 15 min of incubation in N,. The S/M ratio of PAH was approximately 1.0 at the end of 60 min of incubation, which strongly suggestes a com- plete breakdown of the active transport mechanism.

In order to characterize further the PAH transport system in the harbor seal kidney, the effects of various substrates or inhibitors (some of which are known to modify the PAH transport in non-diving animals) were studied and the results were summarized in Table 1. Addition of acetate (10 mM), a positive effec- tor of PAH transport in most mammals, to the incu- bation medium raised PAH accumulation at 60 min two-fold in the presence of 02. However, acetate

Table 1. Effects of various substrates and inhlbltors on the uptake of organic anions by the harbor seal kidnc\

slice

Substrates or inhibitors

None

Acetate (IO mM)

Glucose (IOmM)

Iodoacetamide (0.1 mM)

2.4 DNP (0.1 mM)

S/M PAH S’M ‘-4-D __

2.40 * 0.35 9.45 f I.2 2.46 i 0.14 5.98 + 0.9X’ 5.26 k O.55t 2.47 _+ 0.20* 2.48 f 9.09 9.23 2.1 I f 0.29 5.15 2.09 * 0. IX

0.974 + 0.03t

* Significantly different from O2 controls. t Significantly different from PAH uptake in Oz and no

substrate. Data are presented as mean values i S.E. of four cxpcrl-

merits, except for 2,4-D with glucose which represents one experiment. Cortical slices were incubated for 60 min in the designated atmosphere.

failed to increase PAH transport in the presence of N2. Addition of glucose (10 mM) (a substrate for gly- colysis) to the medium had no effect on’PAH accumu- lation in either an O2 or Nz atmosphere. Similarly. iodoacetamide (0.1 mM), a glycolytic inhibitor, had no effect on PAH uptake. On the other hand. the addition of 2,4_dinitrophenol (0.1 mM). an uncoupler of oxidative phosphorylation, in the presence of O2 decreased the S/M ratio to approximately 1.0.

2.4-D uptake

The time course of 2,4-D uptake by the harbor seal kidney slice in the presence of glucose (10 mM) and O2 or N, is shown in Fig. 2. In the presence of 0,. the S/M ratio of 2,4-D increased to 6.0 in 15 min. after which it continued to increase slowly and reached 9.0 at 60 min. Moreover, 2,4-D transport at a given incubation time was approximately 50% lower in the presence of Nz than that in OZ. The same inhibitory effect of N, was observed in the absence of glucose in the medium (Table 1).

DISCUSSION

The present results appear to indicate that the har- bor seal kidney has the ability to transport organic

lo-

a- 0

lncubatlon Time, minutes

Fig. 2. Time course of uptake of 2.4-D by harbor seal kid- ney cortical slices in the presence of oxygen or nitrogen. Each point represents value obtained from I experiment.

Page 3: Organic anion transport by renal cortical slices of harbor seals (Phoca vitulina)

Organic anion transport in harbor seals 291

anions such as PAH and 24-D. Althou~ the general characteristics of PAH transport in this animal appear to be similar to those in other (non-diving) animals, the following species differences may be pointed out. Firstly, PAH uptake in the harbor seal kidney slice reached a steady-state level of transport much faster than in the rat (Fig. 1). Secondly, the steady-state transport in the harbor seal renal slice was much lower than that in the rat for the given incubation medium. Lastly, PAH uptake in the har- bor seal renal dice was not affected for at least 60 min on replacement of O2 with N, (Fig. 1). Since seal renal slice accumulation of PAH virtually ceased in the presence of 2PDNP (Table I), the N,-insensitive baseline PAH uptake appears to be an active trans- port process. In contrast, the rat kidney slice began to lose its ability to accumulate PAH after only 15 min incubation in N2 medium (Fig. 1). Even though the kidneys from non-diving mammals can recover normal renal cortical transport capabilities after a brief period of anoxia (Berndt, 1976; Coleman et al., 1976; Randall, 1972), the present data appear to indicate that the baseline PAH transport system in the harbor seal renal slice is far more resistant to anoxia than in the rat.

Glucose added to the incubation medium had no stimulatory effect on baseline PAH uptake by harbor seal kidney slices in an O2 or N, atmosphere (Table I), which is consistent with results observed in other animals (e.g. Cross & Taggart, 1950). However, it should be noted that glucose has been implicated in supporting the transport or metabolism of hypoxan- thine in slices of the renal medulla under anaerobic conditions (Berndt & Miller, 1972). The significant stimulation of PAH transport caused by acetate was observed only in an oxygen atmosphere. When renal slices were incubated with acetate in an Nz atmos- phere, the PAH S/M ratio was reduced to 2.47 + 0.2, which was virtually identical to the baseline PAH value. This finding is in contrast to the observation made in the rabbit kidney slice in which N,, decreases the PAH S/M ratio to virtually 1.0 even in the pres- ence of acetate (e.g. Cross & Taggart, 1950).

The overall results on PAW: uptake obtained in the present work thus indicate that the harbor seal kidney slice has the ability to sustain a basal level of PAH transport for at least 60 min even in the absence of 0,. Possibly, energy produced by an anaerobic path- way was sufficient to maintain such a tolerance to anoxia. One alternative explanation is that PAH is bound extensively to renal tissue or intra~~lular pro- teins, and that this binding is unaffected by tissue anoxia. Such a high degree of binding is evident with the organic anion 2,4$trichlorophenoxyacetate in nondiving mammals (Berndt & Koschier, 1973; Hong er ai., 1977).

Limited studies on the transport of 2,4-D, a com- pound which has a high degree of binding, indicate that the harbor seal kidney slice is able to accumulate this anion to the same extent as the rat or rabbit kidney slice (Berndt & Koschier, 1973; Koschier & Berndt, 1976), and that glucose in the medium has no effect on the uptake of this anion (Table l), as in the case of rabbit (Berndt & Koschier, 1973). Althou~ 2,4-D uptake was decreased sibilantly in the presence of N2, the S/M ratio was still 5-6 at

60 min incubation in N, (Table 1, Fig. 2). In the rabbit kidney slice, the latter value is approximately 2.0 (Berndt & Koschier, 1973). This preliminary find- ing again supports the conclusion that the harbor seal kidney is better able to sustain the organic anion anion transport system in the absence of 0, than the rat or rabbit kidney slice.

Acknowledgements-This investigation was supported by Public Health Service Grants HL-16020 and AM-05437. The authors would like to thank Sally Dunker for techni- cal assistance and, along with Howard Ferren and Susan Ashwell-Erickson, for care of the seals. This manuscript was prepared by Mary Lou Taggart, Jean Wilhelmsen and Phyliis Parisi.

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