TOXICOLOGY
The
AND
Effect
APPLIED
PHARMACOLOGY
of Potassium
32,40-52 (1975)
Dichromate Processed
on Renal
Tubular
Transport
W. 0. BERNDT~ Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received June 5,1974; accepted October 24,1974
The Effect of Potassium Dichromate on Renal Tubular Transport Processes.BERNDT, W. 0. (1975). Toxicol. Appl. Pharmacol. 32, 40-52. Severalstudieshave beenconductedto examinethe effectsof potassiumdichromateon the morphology of renal tissue,but relatively few studieshave been done on the functional aspectsof the renal damage.This study was designedto examinewith in vitro techniquesthe effectsof dichromateon a number of renal tubular transport processes.The accumulationof paraaminohippurate (PAH) and of tetraethylammonium chloride (TEA) by both rat and rabbit sliceswasinhibited by 10m4M potassiumdichromate added to the bathing solution. On the other hand, the accumulation of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), wasonly modestlyreduced.In addition, the uptake of TEA wasstimulated significantly by low concentrations (lo4 M) of potassiumdichromate. Although, in general, similar results were obtained using renal cortical slices taken from pretreated animals significant differences occurred. For example,at noneof the dosestested(5-20 mg/kg) wasthe accumulation of PAH, TEA, or AIB stimulated.This wastrue whether the measurements weremadeassoonas0.5 hr after administration of potassiumdichromate or as long as 5 days after its administration. At an intermediate doseof potassiumdichromate (10 mg/kg) the lactate-stimulatedaccumulationof PAH was depressed,but the control or nonsubstrate-stimulatedaccumulation was not affected. In addition to the effects on uptake of organic compounds,potassiumdichromate, whether administeredto the intact animal or addedto the extra-cellular fluid compartment,causeda decrease in the intracellular concentration of potassiumand an increase in the intracellular concentration of sodium.From a quantitative point of view, however, the effect of the nephrotoxin administeredto the intact animal was always greater on tissueelectrolytes than when the nephrotoxin was added in vitro. Both the degeneration and regeneration of renal tubular cells resulting from the administration of potassium dichromate and mercuric chloride have been studied in the rat (Baines, 1965; Cuppage et al., 1972; Cuppage and Tate, 1967; Schwartz et al., 1972). These studies showed the development of and recovery from an acute tubular necrosislocated exclusively in the proximal tubule. It was unclear whether the primary locus of damage was in the pars recta or whether the proximal convoluted tubule was * Thiswork wassupportedby USPHS Grant AM 13020. 2 Send reprint requests to: Dr. William 0. Berndt, Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. Copyright 0 1975 by Academic Press, Inc. 40
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in any form reserved.
DICHROMATE
AND
RENAL
TRANSPORT
41
involved. Hirsch’s studies with dichromate (1973) were interpreted to mean that the proximal straight tubule was a major site of damage. Hirsch examined the tubular transport of p-aminohippurate (PAH), N-methylnicotinamide (NMN), and a-aminoisobutyrate (AIB). Although the transport of both PAH and NMN were altered, little influence of dichromate on AIB uptake was noted. It was concluded from these data that the amino acid transporting system of the proximal convoluted tubule were spared. whereas the organic anion and cation systems were affected because the ion is acting at the region of the pars recta, where these transport systems are most active (Tune et al., 1969). However, morphological studies indicate that there is extensive destruction in the proximal convoluted tubule. Furthermore, the micropuncture study of Biber et al. (1968) indicated that dichromate and other nephrotoxins affected the proximal convoluted tubule. That is, the secretion of PAH by the proximal convoluted tubule of the rat, although of lesser magnitude than in the pars recta, was reduced with potassium dichromate. With other nephrotoxins, e.g., mercuric chloride, an effect on the proximal convoluted tubule has been reported. Bank et al. (1967) purported to have shown a leakage of material out of the proximal convoluted segment of the nephron. The demonstration of an effect in the convoluted tubule does not exclude pars recta as a potential site of action. Whether the acute renal failure syndrome as produced by nephrotoxic agents such as potassium dichromate is related to vascular events which disrupt filtration (Oken, 1972) or to a production of proximal tubule damage permitting leakage of tubular fluid into the interstitium (Bank et al., 1967) is still unclear. In part it was the intent of this study to examine this question by using the slice technique and comparing quantitatively the effects of potassium dichromate administered to the intact animal versus the effects of this agent added to renal cortex slices in vitro. METHODS
Sprague-Dawley rats of either sex (Blue Spruce Farms), New Zealand white rabbits (Camm Research), and rabbits of unknown ancestry were used in this study. The animals were sacrificed by cervical dislocation and the kidneys removed immediately. The tissues were stored in a modified Krebs-Ringer’s solution prior to and during the freehand slicing procedure. Also the slices were stored in the same phosphate buffered solution until the incubation was begun. The incubations for the uptake experiments, electrolyte distribution studies, and inulin spaces were done in a Dubnoff metabolic shaker for 2 hr at 25°C in an atmosphere of 100% oxygen. In general, the incubation medium was a modified Krebs-Ringer’s phosphate solution (Umbriet et al., 1957), containing 1.0 mM calcium and 5.0 mM potassium. Either sodium acetate (lo-’ M) or sodium lactate (lo-* M) was used as a substrate in some of these experiments. Acetate was used for the rabbit tissue because of its dramatic stimulatory effect on PAH uptake; lactate produces a similar effect in renal cortical slices, whereas acetate is less effective. The uptake of p-aminohippurate (PAH), tetraethylammonium (TEA), and aaminoisobutyrate (AIB) was studied. In each case sufficient radioactive compound was added to give approximately 0.02 PCi of 14C/ml of bathing solution and in addition enough unlabeled chemical was added to give the following concentrations: PAH
42
W. 0. BERNDT
7 x 10e5, TEA lo+ M, AIB 8 x 10e6 M. At the end of the incubation period, the tissues were blotted, weighed, placed in 1 ml of cold distilled water, and homogenized thoroughly. After appropriate dilution an aliquot of the whole homogenate was placed in a liquid scintillating counting vial and diluted with PCS-Solubilizer (Amersham/ Searle Company). An aliquot of the bathing solution was prepared similarly, and all samples were counted in a Packard liquid scintillation spectrometer using external standard quench correction. Electrolyte analyses were performed on slices incubated for 2 hr in the standard bathing solution containing [carboxyl-14C]inulin. The chemical concentration of inulin varied from approximately 20 to 45 pg/ml, and with a radioactivity concentration of about 0.06 ,&/ml. At the end of the incubation period, the slices were removed, blotted, weighed, and placed in preweighed aluminum foil cups. The tissues were dried at 100°C for 24 hr or until achieving a constant weight. The dried tissues were weighed after which they were extracted with 0.01 or 0.1 N nitric acid for 48 hr. Sodium, potassium, and inulin were determined on the nitric acid extracts. From these data, extracellular and intracellular fluid spaces, and intracellular sodium and potassium concentrations were calculated using standard procedures (Kleinzeller et al., 1969). The potassium dichromate for pretreatment was dissolved in distilled water and administered subcutaneously in various doses. Control animals were given an equivalent volume of distilled water. In the experiments where attempts were made to document the onset of the nephrotoxicity, the rats were placed in metabolism cages in which they had free access to drinking water and food. Urine was collected on a daily basis. Urine pH and volume were measured quantitatively, and various substances (glucose, protein, ketones, blood, and bilirubin) were assessed qualitatively using Bili Labstix (Ames). Oxygen consumption of the renal cortical slices was studied with a Yellow Springs Instrument Company Oxygen Monitor. This technique involved the use of the Clark electrode, and the data are presented as initial rates of oxygen consumption computed from the decrease in oxygen saturation of the bathing solution over the first 5 min of a 15 min incubation. The Krebs-Ringer phosphate bathing solutions were saturated with 100% oxygen. Statistics were done with either a paired comparison t analysis or using Student’s t test, depending upon the experimental design. RESULTS Time course of development of nephrotoxicity. The data in Fig. 1 are from two rats, one that received 20 mg/kg of potassium dichromate and one that received 10 mg/kg. With the higher dose, the animal showed a prompt reduction in urine flow. Some animals responded to this dose by an initial diuresis before the urine flow was reduced. In animals that were severely oliguric, the common finding was marked reduction in the urine concentration with variable changes in pH. In addition, copious amounts of glucose, protein, and blood were found in the urine samples. At this dose it was very unusual for an animal to recover. Note (Fig. 1) that despite the return of urine flow, urinary osmolality did not return to normal. In addition to these patterns of nephrotoxicity, some animals became anuric and died within 3 or 4 days of administration of
DICHROMATE AND RENAL TRANSPORT
43
the dichromate. Although at the lower dose the pattern of response was less predictable, the example presented in Fig. 1 was a common one. Here there was a tendency toward an increase in the urine flow over several days, with a marked decrease in urine osmolality as noted at the higher dose. Also as with the higher dose large amounts of glucose and protein were found in the urine. Although some animals survived for as long as 10 days, many died sooner. The other response to the IO-mg/kg dose of potassium dichromate was very much like that seen with the larger dose, i.e., prompt development of oliguria, except that the animals became less severely oliguric. 30-
2O-
0-O
IO mg/Kg
*--a
20 mg/Kg
ml per 24 hrs. IO-
I
mOsm/L
‘0
010
i
0’
1000 i
\
‘a /\‘““/ \
0
,
I
I
2T
.
.
3
4
mg ttt +I++ttiltitit*tt+
.-~-w---*-’
.
I
I
,
5
6
7
8
9
DAYS St
-ill-
tt
i+
+
ilt
9
+
20 mg/Kg IO mg/Kg
nq n*
w nq
20mg/Kg IO mg/Kg
Rotein
n.g +
G’“ccse
n.g nrg
it
iit
*
-IIt
Blood
nrg il.0
+ t
+t t
it mg
+I t neg nrg
neg n.g
20 mg/Kg IO mg/Kg
FIG. 1. Typical time courses of nephrotoxicity after subcutaneous administration of two doses potassium dichromate. Each set of data is from a singlerat. The potassium dichromate was administered after the second day (arrow), i.e., first and second days were controls and days 3-9 experimentals.
Because the time course of the nephrotoxic phenomenon often lasted several days, all of the in vitro studies subsequent to the administration of potassium dichromate were also done over several days. Electrolyte and inulin studies. The data in Fig. 2 demonstrate the effect of the adminisstration of potassium dichromate on renal slice electrolyte and water distribution; 1 to 2 days after administration of the nephrotoxin, marked disruption of all of the parameters measured was noted. In general, tissue sodium values rose, tissue potassium values fell, extracellular fluid compartment increased, as did the total tissue water content. The magnitude of the increase in the extracellular fluid compartment could account for the tissue swelling.
44
W.
0. BERNDT
Table 1 presents similar data obtained after the addition of various concentrations of potassium dichromate to normal rat kidney cortical slices. As with the in viva experiments, potassium dichromate disrupted tissue electrolyte balance. However, the magnitude of the effect here was much less than that seen in z&o. That is, no effect on electrolytes or water was noted until a relatively high concentration (10e4 M) was used. Furthermore, none of the effects of potassium dichromate was of a magnitude similar to those produced by 10m4 2,4-dinitrophenol (DNP). RAT RENAL
as Totd l-ii EEA wt.
Na+
CORTEX
SLICES . Control o IO my/Kg KzCr207 A 20 mO/Kq KLCr207
1
03 81 79
0 I
‘\A
90.
0
2 DAYS AFTER
4
6
INJECTION
FIG. 2. The effect of potassium dichromate on renal cortical electrolyte and water content. All animals were injected subcutaneously on day zero. Each point is mean, and bar the SE, of four animals.
Table 2 contains data which show the effects of potassium dichromate on electrolyte and water balance in the slices of rabbit renal cortex. As with the rat, there is no effect seen until the highest concentration of inhibitor is used and again the effect was not as marked as with DNP. Tissueoxygen consumption. The relatively modest effects of potassium dichromate added in vitro on tissue electrolyte distribution was mimicked by oxygen uptake studies. The effects of potassium dichromate on the respiration of normal renal cortical slices of the rat are presented in Table 3. Lactate increased oxygen uptake of these tissue
8 8 6 8
N
76.9 + 0.27 37.7 + 3.47 75.6 (2) 125.3+ 3.0b(5)
-
-
TABLE
1
Lactate lo-’ M 77.6 i 0.23 36.2 + 2.5 63.6 + 2.6 134.65 4.4
lo-” M Lactate lo-’ M
TISSUE WATERELECTRNYTFSIN
77.6 f 0.33 37.6 + 2.11 50.2 IL 6.8 143.6+ 3.9
LACTATEAND~NHIBITORSON
77.5 * 0.17 37.6 + 2.2 71.6 k 10.3 134.2k 3.3
Lactate
Lactate lo-’ M
-
78.1i 0.29 39.4 + 2.3 92.5 + 11.9* 99.4 rt 5.06
lo-'M
lo-"M
10-5M
Potassium dichromate
RAT RENALCORTICALSLICES'
DNP
81.3 (2) 27.6 (2) 134.1(2) 29.9 (2)
lo-'M
Lactate
1o-4 M
3 3 3 3
~--~ ~___.~~
y See Table 1. * D < 0.025 compared to acetate control.
Tissue potassium
Tissuewater (“//utotal wt) Extracellular space Tissuesodium
~
N
76.8 + 0.8 32.1 + 1.4 58.6 L-6.5 109.3+ 4.4*
-
-
76.1 k 0.5 31.7 + 0.6 49.7 + 9.6 135.4+ 1.6
lo-'M
Acetate
-
lo-'M
lo-6 M Acetate
76.8k 0.8 33.2 f 2.7 51.6 _+7.2 130.0+ 2.2
---
76.9k 0.8 35.4 + 2.6 61.0 & 11.9 129.1f 0.6
lo-* M
Acetate
io-5M
78.1 f 1 35.5+ 0.8* 98.2 f 5.5* 77.8+ 1.46
lo-'M
Acetate
10-4M
Potassiumdichromate ._...~_ __~_..
EFFECTSOFACETATEANDINHIBITORSONTISSUEWATERANDELECTROLYTESINRABBITRENALCORTICALSLICES~
TABLE 2
82.4f O.l* 29.2 * 4.1 138.24 17* 24.3 + 3.4*
lo-*M
Acetate
104M
DNP
y The data are expressed as mean + SE. Tissue electrolytes given asmEq/liter of intracellular water. Extracellular space was determined with inulin and is given as a percentage of tissue water. N refers to number of experiments for all measurements in a line, except where a parenthetical value is given. Incubations were carried out for 120 min at 25°C in th: presence of 100% oxygen. *p < 0.05 compared to lactate control.
Extracellular space Tissuesodium Tissuepotassium
Tissue water (% total wt)
EFFKTS~~
% %
$
?
P 8 tz
$
u z 2 s
46
W.
0. BERNDT
slices. No alterations in the control or lactate-stimulated respiration was observed when dichromate was added to the bathing solution, even with concentrations as high
as IO-4
M.
TABLE EFFECT
OF POTASSIUM
Inhibitor -
Dichromate lo4
M
M
3
ON RENAL
CORTICAL
Substrate
N
-
29 29 8 7 6 5 8 6
Lactate
lo-‘M
lo4
DICHROMATE
-
Lactate Lactate Lactate
RESPIRATION’
0 02 1.29 + 1.53 + 1.20 f 1.44 f 1.23 + 1.52 f 1.30 + 1.62 +
0.03 0.04b 0.06 0.05 0.04 0.08 0.06 0.06
’ The mean values are given as ,A O2 hr-’ mg wet tissue-’ + SE. Measurements weremadeat 25°Candrepresentinitial ratesof oxygenconsumption(l-5 min). The Krebs-Ringer’s phosphatesolutionwassaturatedwith 100%oxygen.Lactate,when used,waspresentat lo-’ M. bp < 0.01. The data in Table 4 were obtained from rat renal cortical slices removed from animals pretreated with dichromate. Although no significant effects or tissue respiration were noted with 5-mg/kg of potassium dichromate, with higher doses a significant depression of the renal cortical respiration was noted. This was true at both 2 and 5 days after administration of the nephrotoxin. For example, at the 20-mg/kg dose in the animals tested 5 days after administration of the compound, tissue respiration was depressed by more than 50%. Organic compound transport. The effects of potassium dichromate added to both rabbit and rat renal cortical slices are presented in Tables 5 and 6. The rabbit renal slices showed minimal accumulation of AIB, and accordingly, the uptake was diminished only moderately by high concentrations of potassium dichromate (10m4 M). Both TEA and PAH uptake were depressed by about 50-70 % by high concentrations of potassium dichromate. However, no significant reduction in the accumulation of either the organic acid or the organic base were noted with concentrations below 1o-4
M.
The results with rat renal cortical slices showed a significant reduction in the uptake of AIB at 10m4 M dichromate, although the magnitude of this effect was relatively modest. Lactate depressed AIB uptake and no effect of dichromate was noted on the lactate-regulated uptake of AIB. At the very highest concentration of dichromate (1 mM), a marked reduction of AIB accumulation was observed whether or not lactate was present. On the other hand, TEA and PAH showed marked reductions in uptake at lo-” M dichromate. It is also interesting to note that with low concentrations of dichromate (e.g., 10m6 M), a significant stimulation of uptake of TEA was noted. No significant enhancement of uptake was noted for other compounds.
1.31 k 0.04 1.27+0.03 1.30+0.02
0
1.53 + 0.05 1.65 +0.08 1.60+0.03
5
Lactate ~~______~ 1.28 f0.06 1.63 kO.03 1.35 +0.04 1.69+0.01
_____ -. ~~~~ ~~~ Lactate __~0.82+0.12 0.66f:O.OZ
0.76kO.09 0.53 +0.06
1.13 kO.08 0.84+0.02
-. Lactate
10
Dose @g/kg) 20
0.81 + 0.12 0.57 kO.11
-
Lactate
~_~.._..~ 0.62 +0.08 -
Lactate
-
0.81 kO.03 -
40
3.4 LactatelOM 2.5 15.2 Lactate 1O-2 M 16.2 5.5 Lactate 1O-2 M 13.2
If: 0.26 (9) rf: 0.15 (12)b + 1.2 (9) + 1.7 (11) f 0.43 (9) f 0.88 (11)
Control 2.8 2.4 14.1 14.0 3.6 10.3
+- 0.56 (3) If: 0.40 (3) Ifr 1.6 (3) + 2.2 (3) + 0.38 (3) + 1.4 (3)
lo-’ 2.8 2.4 20.1 22.3 6.0 17.7
-. k 0.17 (3) I!Z0.3 (3) f 0.16 (3)b f 0.43 (3)b f 0.9 (3) f 2.4 (4)
104 2.9 3.0 20.6 21.1 6.4 12.8
+ 0.44 (3) + 0.38 (4) + 1.8 (3) + 1.7 (4) 2~ 0.36 (3) rf: 1.0 (5)
1o-5 2.2 2.5 6.6 8.5 3.3 4.3
2 + f iz rt f
.~ ~~. 0.26 (6)b 0.19 (8) 0.84 (6) 0.99 (8) 0.4 (6) 0.45 (8)
1o-4
M -.......-. .-~~~~~~ .---~~~~~
0.90 1 .o 0.85 1.3 0.92 1 .o
f 0.02 * 0.05 + 0.03 If- 0.32 f 0.03 + 0.09
1o-3 (3)b(3)b (3)C (3) (3) (3)
” Values are S/M ratios + SE, and numbers of experiments given in parentheses. Incubations were carried out for 120 min at 25’C in the presence of lOOg:, oxygen. “p < 0.05 compared to appropriate Control. cp < 0.01 compared to appropriate Control.
PAH uptake
TEA uptake
AIB uptake
5
EFFECT OFPOTASSIUM DICHROMATE ADDEDTORAT RENALCORTICALSLICES"
TABLE
’ The mean Oez values are given as ,ul O2 hr-r mg wet tissue + SE. N varied from 3 to 5. Measurements were done at 25°C and represent initial rates of oxygen consumption (1-5 min). The Krebs-Ringer’s phosphate solution was saturated with 100°A oxygen and lactate, when used, was present at 10e2 M. Control animals were injected with water.
0 2 5
Days after pre- ---~_~_-__treatment -
4
EFFECT OFPOTASSIUM DICHROMATE PRETREATMENT ONRENALCORTICALSLICERESPIRATIONS
TABLE
2 r:
z ;;1 5
i K
5
u ii B 8
W. 0. BERNDT
48
TABLE 6 EFWCTOFPOTASSIUMDICHROMATEADDEDTORABBITRENALCORTICALSLICES~
Potassiumdichromate(M) N AIB uptake TEA uptake PAH uptake
-
10-5
10”
10-4
6 6
1.7 + 0.2
1.7 * 0.2
1.6 f 0.2
1.2 + O.lb
M
1.5 If: 0.1
1.5 f 0.1
1.6 f 0.2
1.2+ 0.2b
M Acetate 10m2
6 6
17.3 f 1.3
15.6 f 1.2
15.1 f: 1.2
19.8f 1.3
20.5 f 1.8
19.9f 2.2
8.7 t 1.4b 7.7 + l.6b
Acetate lo-*
5 5
6.1 f 1.4 13.3f 1.5
5.7 * 1.3 13.3f 1.3
5.3 + 1.2 12.6+ 1.5
3.3 f 0.8b 3.8 f l.3b
Acetate lo-’
M
a Values are S/M ratios f SE and Nrefers to number of experiments. Slices were incubated for 120 min at 25°C in the presence of lOOok oxygen. bp < 0.05 compared to appropriate control.
In Fig. 3, data are presented for the corresponding pretreatment experiments. The smaller dose reduced TEA significantly as early as 2 hr, but a more marked effect was seen at 24 hr and a somewhat greater depression was observed at 48 hr. The uptake returned to an intermediate level of depression for the remaining 5 days of the experiment. With the higher dose of dichromate, the significant reduction at 24 hr was followed by a further reduction at 48 hr with the maximal effect seen at 3 or 4 days. RAT
RENAL
CORTEX
SLICES
NO Substrate . .
Lactate 0 control A IOmq/Kg K&O, 0 2Omg/KP K&q
OOT
2
I
3
Hours Time
ottsr
4 WS
5
6
7
Pretreatment
FIG. 3. The effect of potassium dichromate on renal cortical slice uptake of tetraethylammonium. Each point is the mean, and bar the SE, of four animals. All animals were injected subcutaneously on day 0.
DICHROMATE
AND
RENAL
49
TRANSPORT
PAH uptake was stimulated by lactate (Fig. 4), and it was this lactate-stimulated accumulation of PAH which was depressed by IO-mg/kg of potassium dichromate. No consistent or marked effect of dichromate on the resting uptake of PAH was noted at this dose of nephrotoxin. However, at the higher dose of dichromate both the substrate-stimulated and the nonstimulated accumulation of the organic anion were depressed significantly. Data for AIB uptake by slices from pretreated animals are not presented, since they were negative. That is, neither dose of dichromate affected AIB accumulation at any of the times used in this study. RAT
RENAL
CORTEX
SLICES NO Substrate . *
04-l-l-F 0 2 4 Hours
6
-8 I Time
of
FIG. 4. The effect of data as in Fig. 3.
2
3
after
Pretreatment
4 DW
5
6
Lactate 0 Control 6 IOm(l~g
K&C+
7
potassium dichromate on renal cortical slice uptake of p-aminohippurate.
Rest
The time course of the effects of the dichromate on organic compound transport should be emphasized. Although 2 hr after a dose of 20 mg/kg, large concentrations of dichromate appear in kidney cortex (0.2-0.5 mM, N = 6), maximal inhibitory effects on the transport of the organic compounds was not noted until later. The same or even lower concentration of dichromate (0.1 mM) added in vitro did reduce PAH and TEA accumulation (Tables 5 and 6). DISCUSSION
In general, the data reported here agree with the earlier report of Hirsch (1973). However, there are certain important areas of disagreement which deserve comment. In addition, although some of the data are in agreement, it is possible to interpret them differently than Hirsch did. For example, it seems clear that the transport of AIB is altered only slightly by dichromate administered in vim or added in vitro (except for extremely high concentrations,
i.e., 1 mM). Hirsch has interpreted this as evidence of an
50
W.
0.
BERNDT
anatomical localization of the action of dichromate, i.e.: an effect on the pars recta where PAH transport is maximal, but AIB transport is not. This suggestion does not agree with the autoradiographic studies of Wedeem and Thier (1971), who reported extensive accumulation of [3H]AIB in the straight portions of rat proximal tubules. Although anatomical localization of dichromate is a possible explanation for differential effects, it is not unreasonable to suggest that the effect is related to the failure of dichromate to affect the AIB transport carrier. This proposal is consistent with the multiplicity of effects noted by Hirsch (1972, 1973) and reported here. Furthermore, several anatomical studies (Oliver and MacDowell, 1963; Biter et al., 1968; Kramp et al., 1974) complicate this problem additionally. All of these investigators demonstrated that potassium dichromate produced a selective necrosis of the pars convoluta of the proximal tubule. Only minimal damage to the pars recta was noted and the microscopic data were supported by micropuncture experiments. The studies of Tune et al. (1969) on isolated perfused tubules indicated that maximal transport of PAH occurs in the pars recta and therefore, one might expect only minimal effects on PAH transport after dichromate since this nephrotoxin leaves the pars recta intact. The severe depression of PAH uptake by renal slices might be interpreted as an effect of dichromate on the renal transport in the pars convoluta as well, perhaps unrelated to the production of necrosis. These data also raise the question of the value of purely microscopic studies in the determination of tubular sites of nephrotoxicity. In this study the accumulation of PAH, TEA, and AIB was examined both in the presence and absence of metabolic substrate. Lactate had little effect on the uptake of TEA, but stimulated PAH uptake markedly and depressed AIB uptake significantly. The lactate effect on AIB was not noted in the presence of dichromate. Renal slices prepared from animals pretreated with the low dose of dichromate showed a reduced level of lactate stimulation of PAH uptake, although the nonstimulated accumulation was unaffected. This differential effect may be related to interference with energy metabolism, since this dose of dichromate did depress tissue respiration significantly. However, the depressed respiration occurred whether or not lactate was present in the bathing solution. Therefore, this generalized metabolic effect does not appear to account for the specificity of the dichromate effect on PAH uptake. The mechanism of lactate stimulation is unknown (Schachter et al., 1955; Kim and Hook, 1973). If the dichromate effect is related to the intracellular actions of lactate in promoting PAH transport, it may prove useful for understanding the mechanism(s) of lactate stimulation. On the other hand, the effect may be related to blockade of lactate entry into the tissue. There was an apparent attempt at recovery of tissue electrolytes over several days after administration of potassium dichromate to the animals. Total tissue water and tissue potassium concentrations tended toward control values 4 or 5 days after the nephrotoxin was given, especially after the lower dose (10 mg/kg). The significance of this is not clear, since none of the high-dose animals survived even 5 days and many of the animals that received the low dose did not survive 10 days. It is noteworthy that marked quantitative differences appeared depending on how the nephrotoxin was administered. Quantitatively, the alterations in tissue electrolytes and water were never as large after in vitro addition as after in vivo administration, even when the tissue concentrations of dichromate were approximately the same.
DlCHROMATE
AND
RENAL
TRANSPORT
51
It is tempting to suggest that these quantitative data can be used to help distinguish between the different theories for the pathogenesis of acute renal failure. The effect of dichromate was consistently more dramatic after in viuo administration than after in vitro addition, at least by 6-24 hr after injection of the nephrotoxin. The effect at 2 hr, which corresponds to the in vitro time course, was not as dramatic but still a greater effect was noted in vivo than in vitro. Given that tissue concentrations of dichromate are approximately the same, these data would appear to support an indirect effect on renal transport, e.g., in vivo the nephrotoxin altered vascular events in such a way to compromise renal transport mechanisms. On the other hand, if the in vice action was solely through vascular mechanisms, it seems unlikely that any specificity of effect would be noted, e.g., lack of effect on AIB transport or blockade of substratestimulated PAH uptake only. Hence, although the quantitative differences between in uivo and in vitro effects supports a pathogenic mechanism related to interruption of vascular events, the specificity of the effects on the transport processes does not. This argument gains support from the experiments where 10m3 M dichromate was used in vitro. At this very high concentration all transport processes for the organic compounds used in this study were disrupted. In addition, the different effects may be related to the particular chemical species of chromate ion present. In vitro dichromate is added to a protein-free solution, wherein the usual chemical equilibria would be expected to exist (Cotton and Wilkinson, 1972): H+ + CrO,’ + HCrO,- + Cr207=. The first step occurs with acidification and the second as a result of a dimerization. For example, at pH 7.4, CrO,= is the predominant species with the ratios of Cr04= : HCrO,- : Cr,07= being 200: 10 : 1. Presumably, under in vitro conditions CrO,= is predominant and may be the active form of the chromate ion. In vivo, the species which predominates is not clear. Although the pH of plasma, extracellular fluids, etc., are about 7.4, problems of plasma protein binding must be involved. Perhaps Cr,07’ is bound to plasma proteins and thereby “protected” from the usual equilibria. This might mean that in vivo Cr,O,= is delivered to the renal site(s) of action, whereas in vitro CrO; is the active species. Similar distinctions have been suggested by Schuchter et al. (1973) for the action of chromate ion on water and solute permeability of the toad bladder. ACKNOWLEDGMENTS
The author is most appreciative of the excellent technical assistance of Bonnie Bemdt and Ellen Costello. REFERENCES
A. D. (1965). Cell renewal following dichromate induced renal tubular necrosis.
BAINES,
Amer. J. Pathol. 47, 851-876. BANK, N., MUTZ, B. F. AND AYNEDJIAN,
H. S. (1967). The role of “leakage” of tubular fluid in anuria due to mercury poisoning. J. Clin. Invest. 46,695-704.
BIBER, M.
T.
U. L.,
MYLLE,
M.,
BAINES,
A. D.,
GOTTSCHALK,
C. W.,
OLIVER,
J. AND MACDOWELL,
C. (1968). A study of micropuncture and microdissection of acute renal damage in rats. Amer. J. Med. 44, 664-705.
52
W. 0. BERNDT
COTTON, F. A. AND WILKINSON, G. (1972).AdvancedInorganic Chemistry.3rd edition, Inter-
sciencePublishers,New York. CUPPAGE, F. E., CHIGA,M. ANDTATE, A. (1972). Cell cycle studiesin the regeneratingrat nephron following injury with mercuric chloride. Lab. Invest. 26, 122-126. CUPPAGE, F. E. AND Tam, A. (1967).Repair of the nephron following injury with mercuric chloride. Amer. J. Pathol. 51,405-429. HIRSCH,G. H. (1973).Differential effectsof nephrotoxic agentson renal organicion transport and metabolism.J. Pharmacol.Exp. Ther. l&593-599. HIRSCH,G. H. (1972).Stimulation of renal organic basetransport by uranyl nitrate. Can.J. Physiol. Pharmacol.50, 533-538. KIM, J. K. AND HOOK,J. B. (1972).On the mechanismof acetateenhancementof renal paminohippuratetransport. Biochim.Biophys.Acta 290, 368-374. KRAMP,R. A., MACDOWELL,M., GOTTSCHALK, C. W. AND OLIVER, J. R. (1974).A study of microdissectionand micropuncture of the structure and function of the kidneys and the nephronsof rats with chronic renal damage.Kidney Inter. 5, 147-176. OLIVER,J. AND MACDOWELL,M. (1963). Studiesby microdissectionand micropuncture of the abnormal kidney: The structural aspect. In Proceedingsof the SecondInternational Congress OfNephrology,Excerpta Medica International CongressSeriesNo. 78, pp. 13-20, Prague. OKEN, D. E. (1972).Modern Conceptsof the role of nephrotoxic agentsin the pathogenesis of acuterenal failure. In DrugsAffecting Kidney FunctionandMetabolism(K. D. Edwards, Ed.), Prog. Biochem.Pharmacol.7,219-247. KLEINZELLER, A., KOSTYUK,P. G., KOTYK, A. ANDLEV,A. A. (1969).Determination of intracellular ionic concentrations and activities. In Laboratory Techniquesin Membrane Biophysics(H. Passowand R. Stampfli, Eds.), pp. 69-84. Springer-Verlag,New York. SCHWARTZ, R. H., LEWIS, R. A. AND SIZHENK, E. A. (1972).Tamm-Horsfall mucoprotein. III. Potassiumdichromate-inducedrenal tubular damage.Lab. Invest. 27, 214-217. SCHACHTER, D., MANIS, J. G. AND TAGGART, J. V. (1955).Renal synthesis,degradationand active transport of aliphatic aryl aminoacids.Relationshipto p-aminohippuratetransport. Amer. J. Physiol. 182, 537-544. SHUCHTER, S. H., FRANKI, N. ANDHAYS,R. M. (1973).The effect of tanning agentson the permeability of the toad bladder to water and solutes.J. Memb. BioZ.14, 177-191. TUNE, B. M., BURG, M. B. AND PATLAK, C. S. (1969).Characteristicsofp-aminohippurate transport in proximal renal tubules.Amer. J. Physiol.217, 1057-1063. UMBRIET, W. E., BIJRRIS, R. H. AND STAIJFFER, J. F. (1957).Manometric Techniques. Burgess, Minneapolis. WEDEEM, R. P. AND THIER, S. 0. (1971). Intrarenal distribution of nonmetabolized amino acidsin vivo. Amer. J. Physiol.220, 507-512.
The
AND
Effect
APPLIED
PHARMACOLOGY
of Potassium
32,40-52 (1975)
Dichromate Processed
on Renal
Tubular
Transport
W. 0. BERNDT~ Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received June 5,1974; accepted October 24,1974
The Effect of Potassium Dichromate on Renal Tubular Transport Processes.BERNDT, W. 0. (1975). Toxicol. Appl. Pharmacol. 32, 40-52. Severalstudieshave beenconductedto examinethe effectsof potassiumdichromateon the morphology of renal tissue,but relatively few studieshave been done on the functional aspectsof the renal damage.This study was designedto examinewith in vitro techniquesthe effectsof dichromateon a number of renal tubular transport processes.The accumulationof paraaminohippurate (PAH) and of tetraethylammonium chloride (TEA) by both rat and rabbit sliceswasinhibited by 10m4M potassiumdichromate added to the bathing solution. On the other hand, the accumulation of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), wasonly modestlyreduced.In addition, the uptake of TEA wasstimulated significantly by low concentrations (lo4 M) of potassiumdichromate. Although, in general, similar results were obtained using renal cortical slices taken from pretreated animals significant differences occurred. For example,at noneof the dosestested(5-20 mg/kg) wasthe accumulation of PAH, TEA, or AIB stimulated.This wastrue whether the measurements weremadeassoonas0.5 hr after administration of potassiumdichromate or as long as 5 days after its administration. At an intermediate doseof potassiumdichromate (10 mg/kg) the lactate-stimulatedaccumulationof PAH was depressed,but the control or nonsubstrate-stimulatedaccumulation was not affected. In addition to the effects on uptake of organic compounds,potassiumdichromate, whether administeredto the intact animal or addedto the extra-cellular fluid compartment,causeda decrease in the intracellular concentration of potassiumand an increase in the intracellular concentration of sodium.From a quantitative point of view, however, the effect of the nephrotoxin administeredto the intact animal was always greater on tissueelectrolytes than when the nephrotoxin was added in vitro. Both the degeneration and regeneration of renal tubular cells resulting from the administration of potassium dichromate and mercuric chloride have been studied in the rat (Baines, 1965; Cuppage et al., 1972; Cuppage and Tate, 1967; Schwartz et al., 1972). These studies showed the development of and recovery from an acute tubular necrosislocated exclusively in the proximal tubule. It was unclear whether the primary locus of damage was in the pars recta or whether the proximal convoluted tubule was * Thiswork wassupportedby USPHS Grant AM 13020. 2 Send reprint requests to: Dr. William 0. Berndt, Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. Copyright 0 1975 by Academic Press, Inc. 40
All rights of reproduction Printed in Great Britain
in any form reserved.
DICHROMATE
AND
RENAL
TRANSPORT
41
involved. Hirsch’s studies with dichromate (1973) were interpreted to mean that the proximal straight tubule was a major site of damage. Hirsch examined the tubular transport of p-aminohippurate (PAH), N-methylnicotinamide (NMN), and a-aminoisobutyrate (AIB). Although the transport of both PAH and NMN were altered, little influence of dichromate on AIB uptake was noted. It was concluded from these data that the amino acid transporting system of the proximal convoluted tubule were spared. whereas the organic anion and cation systems were affected because the ion is acting at the region of the pars recta, where these transport systems are most active (Tune et al., 1969). However, morphological studies indicate that there is extensive destruction in the proximal convoluted tubule. Furthermore, the micropuncture study of Biber et al. (1968) indicated that dichromate and other nephrotoxins affected the proximal convoluted tubule. That is, the secretion of PAH by the proximal convoluted tubule of the rat, although of lesser magnitude than in the pars recta, was reduced with potassium dichromate. With other nephrotoxins, e.g., mercuric chloride, an effect on the proximal convoluted tubule has been reported. Bank et al. (1967) purported to have shown a leakage of material out of the proximal convoluted segment of the nephron. The demonstration of an effect in the convoluted tubule does not exclude pars recta as a potential site of action. Whether the acute renal failure syndrome as produced by nephrotoxic agents such as potassium dichromate is related to vascular events which disrupt filtration (Oken, 1972) or to a production of proximal tubule damage permitting leakage of tubular fluid into the interstitium (Bank et al., 1967) is still unclear. In part it was the intent of this study to examine this question by using the slice technique and comparing quantitatively the effects of potassium dichromate administered to the intact animal versus the effects of this agent added to renal cortex slices in vitro. METHODS
Sprague-Dawley rats of either sex (Blue Spruce Farms), New Zealand white rabbits (Camm Research), and rabbits of unknown ancestry were used in this study. The animals were sacrificed by cervical dislocation and the kidneys removed immediately. The tissues were stored in a modified Krebs-Ringer’s solution prior to and during the freehand slicing procedure. Also the slices were stored in the same phosphate buffered solution until the incubation was begun. The incubations for the uptake experiments, electrolyte distribution studies, and inulin spaces were done in a Dubnoff metabolic shaker for 2 hr at 25°C in an atmosphere of 100% oxygen. In general, the incubation medium was a modified Krebs-Ringer’s phosphate solution (Umbriet et al., 1957), containing 1.0 mM calcium and 5.0 mM potassium. Either sodium acetate (lo-’ M) or sodium lactate (lo-* M) was used as a substrate in some of these experiments. Acetate was used for the rabbit tissue because of its dramatic stimulatory effect on PAH uptake; lactate produces a similar effect in renal cortical slices, whereas acetate is less effective. The uptake of p-aminohippurate (PAH), tetraethylammonium (TEA), and aaminoisobutyrate (AIB) was studied. In each case sufficient radioactive compound was added to give approximately 0.02 PCi of 14C/ml of bathing solution and in addition enough unlabeled chemical was added to give the following concentrations: PAH
42
W. 0. BERNDT
7 x 10e5, TEA lo+ M, AIB 8 x 10e6 M. At the end of the incubation period, the tissues were blotted, weighed, placed in 1 ml of cold distilled water, and homogenized thoroughly. After appropriate dilution an aliquot of the whole homogenate was placed in a liquid scintillating counting vial and diluted with PCS-Solubilizer (Amersham/ Searle Company). An aliquot of the bathing solution was prepared similarly, and all samples were counted in a Packard liquid scintillation spectrometer using external standard quench correction. Electrolyte analyses were performed on slices incubated for 2 hr in the standard bathing solution containing [carboxyl-14C]inulin. The chemical concentration of inulin varied from approximately 20 to 45 pg/ml, and with a radioactivity concentration of about 0.06 ,&/ml. At the end of the incubation period, the slices were removed, blotted, weighed, and placed in preweighed aluminum foil cups. The tissues were dried at 100°C for 24 hr or until achieving a constant weight. The dried tissues were weighed after which they were extracted with 0.01 or 0.1 N nitric acid for 48 hr. Sodium, potassium, and inulin were determined on the nitric acid extracts. From these data, extracellular and intracellular fluid spaces, and intracellular sodium and potassium concentrations were calculated using standard procedures (Kleinzeller et al., 1969). The potassium dichromate for pretreatment was dissolved in distilled water and administered subcutaneously in various doses. Control animals were given an equivalent volume of distilled water. In the experiments where attempts were made to document the onset of the nephrotoxicity, the rats were placed in metabolism cages in which they had free access to drinking water and food. Urine was collected on a daily basis. Urine pH and volume were measured quantitatively, and various substances (glucose, protein, ketones, blood, and bilirubin) were assessed qualitatively using Bili Labstix (Ames). Oxygen consumption of the renal cortical slices was studied with a Yellow Springs Instrument Company Oxygen Monitor. This technique involved the use of the Clark electrode, and the data are presented as initial rates of oxygen consumption computed from the decrease in oxygen saturation of the bathing solution over the first 5 min of a 15 min incubation. The Krebs-Ringer phosphate bathing solutions were saturated with 100% oxygen. Statistics were done with either a paired comparison t analysis or using Student’s t test, depending upon the experimental design. RESULTS Time course of development of nephrotoxicity. The data in Fig. 1 are from two rats, one that received 20 mg/kg of potassium dichromate and one that received 10 mg/kg. With the higher dose, the animal showed a prompt reduction in urine flow. Some animals responded to this dose by an initial diuresis before the urine flow was reduced. In animals that were severely oliguric, the common finding was marked reduction in the urine concentration with variable changes in pH. In addition, copious amounts of glucose, protein, and blood were found in the urine samples. At this dose it was very unusual for an animal to recover. Note (Fig. 1) that despite the return of urine flow, urinary osmolality did not return to normal. In addition to these patterns of nephrotoxicity, some animals became anuric and died within 3 or 4 days of administration of
DICHROMATE AND RENAL TRANSPORT
43
the dichromate. Although at the lower dose the pattern of response was less predictable, the example presented in Fig. 1 was a common one. Here there was a tendency toward an increase in the urine flow over several days, with a marked decrease in urine osmolality as noted at the higher dose. Also as with the higher dose large amounts of glucose and protein were found in the urine. Although some animals survived for as long as 10 days, many died sooner. The other response to the IO-mg/kg dose of potassium dichromate was very much like that seen with the larger dose, i.e., prompt development of oliguria, except that the animals became less severely oliguric. 30-
2O-
0-O
IO mg/Kg
*--a
20 mg/Kg
ml per 24 hrs. IO-
I
mOsm/L
‘0
010
i
0’
1000 i
\
‘a /\‘““/ \
0
,
I
I
2T
.
.
3
4
mg ttt +I++ttiltitit*tt+
.-~-w---*-’
.
I
I
,
5
6
7
8
9
DAYS St
-ill-
tt
i+
+
ilt
9
+
20 mg/Kg IO mg/Kg
nq n*
w nq
20mg/Kg IO mg/Kg
Rotein
n.g +
G’“ccse
n.g nrg
it
iit
*
-IIt
Blood
nrg il.0
+ t
+t t
it mg
+I t neg nrg
neg n.g
20 mg/Kg IO mg/Kg
FIG. 1. Typical time courses of nephrotoxicity after subcutaneous administration of two doses potassium dichromate. Each set of data is from a singlerat. The potassium dichromate was administered after the second day (arrow), i.e., first and second days were controls and days 3-9 experimentals.
Because the time course of the nephrotoxic phenomenon often lasted several days, all of the in vitro studies subsequent to the administration of potassium dichromate were also done over several days. Electrolyte and inulin studies. The data in Fig. 2 demonstrate the effect of the adminisstration of potassium dichromate on renal slice electrolyte and water distribution; 1 to 2 days after administration of the nephrotoxin, marked disruption of all of the parameters measured was noted. In general, tissue sodium values rose, tissue potassium values fell, extracellular fluid compartment increased, as did the total tissue water content. The magnitude of the increase in the extracellular fluid compartment could account for the tissue swelling.
44
W.
0. BERNDT
Table 1 presents similar data obtained after the addition of various concentrations of potassium dichromate to normal rat kidney cortical slices. As with the in viva experiments, potassium dichromate disrupted tissue electrolyte balance. However, the magnitude of the effect here was much less than that seen in z&o. That is, no effect on electrolytes or water was noted until a relatively high concentration (10e4 M) was used. Furthermore, none of the effects of potassium dichromate was of a magnitude similar to those produced by 10m4 2,4-dinitrophenol (DNP). RAT RENAL
as Totd l-ii EEA wt.
Na+
CORTEX
SLICES . Control o IO my/Kg KzCr207 A 20 mO/Kq KLCr207
1
03 81 79
0 I
‘\A
90.
0
2 DAYS AFTER
4
6
INJECTION
FIG. 2. The effect of potassium dichromate on renal cortical electrolyte and water content. All animals were injected subcutaneously on day zero. Each point is mean, and bar the SE, of four animals.
Table 2 contains data which show the effects of potassium dichromate on electrolyte and water balance in the slices of rabbit renal cortex. As with the rat, there is no effect seen until the highest concentration of inhibitor is used and again the effect was not as marked as with DNP. Tissueoxygen consumption. The relatively modest effects of potassium dichromate added in vitro on tissue electrolyte distribution was mimicked by oxygen uptake studies. The effects of potassium dichromate on the respiration of normal renal cortical slices of the rat are presented in Table 3. Lactate increased oxygen uptake of these tissue
8 8 6 8
N
76.9 + 0.27 37.7 + 3.47 75.6 (2) 125.3+ 3.0b(5)
-
-
TABLE
1
Lactate lo-’ M 77.6 i 0.23 36.2 + 2.5 63.6 + 2.6 134.65 4.4
lo-” M Lactate lo-’ M
TISSUE WATERELECTRNYTFSIN
77.6 f 0.33 37.6 + 2.11 50.2 IL 6.8 143.6+ 3.9
LACTATEAND~NHIBITORSON
77.5 * 0.17 37.6 + 2.2 71.6 k 10.3 134.2k 3.3
Lactate
Lactate lo-’ M
-
78.1i 0.29 39.4 + 2.3 92.5 + 11.9* 99.4 rt 5.06
lo-'M
lo-"M
10-5M
Potassium dichromate
RAT RENALCORTICALSLICES'
DNP
81.3 (2) 27.6 (2) 134.1(2) 29.9 (2)
lo-'M
Lactate
1o-4 M
3 3 3 3
~--~ ~___.~~
y See Table 1. * D < 0.025 compared to acetate control.
Tissue potassium
Tissuewater (“//utotal wt) Extracellular space Tissuesodium
~
N
76.8 + 0.8 32.1 + 1.4 58.6 L-6.5 109.3+ 4.4*
-
-
76.1 k 0.5 31.7 + 0.6 49.7 + 9.6 135.4+ 1.6
lo-'M
Acetate
-
lo-'M
lo-6 M Acetate
76.8k 0.8 33.2 f 2.7 51.6 _+7.2 130.0+ 2.2
---
76.9k 0.8 35.4 + 2.6 61.0 & 11.9 129.1f 0.6
lo-* M
Acetate
io-5M
78.1 f 1 35.5+ 0.8* 98.2 f 5.5* 77.8+ 1.46
lo-'M
Acetate
10-4M
Potassiumdichromate ._...~_ __~_..
EFFECTSOFACETATEANDINHIBITORSONTISSUEWATERANDELECTROLYTESINRABBITRENALCORTICALSLICES~
TABLE 2
82.4f O.l* 29.2 * 4.1 138.24 17* 24.3 + 3.4*
lo-*M
Acetate
104M
DNP
y The data are expressed as mean + SE. Tissue electrolytes given asmEq/liter of intracellular water. Extracellular space was determined with inulin and is given as a percentage of tissue water. N refers to number of experiments for all measurements in a line, except where a parenthetical value is given. Incubations were carried out for 120 min at 25°C in th: presence of 100% oxygen. *p < 0.05 compared to lactate control.
Extracellular space Tissuesodium Tissuepotassium
Tissue water (% total wt)
EFFKTS~~
% %
$
?
P 8 tz
$
u z 2 s
46
W.
0. BERNDT
slices. No alterations in the control or lactate-stimulated respiration was observed when dichromate was added to the bathing solution, even with concentrations as high
as IO-4
M.
TABLE EFFECT
OF POTASSIUM
Inhibitor -
Dichromate lo4
M
M
3
ON RENAL
CORTICAL
Substrate
N
-
29 29 8 7 6 5 8 6
Lactate
lo-‘M
lo4
DICHROMATE
-
Lactate Lactate Lactate
RESPIRATION’
0 02 1.29 + 1.53 + 1.20 f 1.44 f 1.23 + 1.52 f 1.30 + 1.62 +
0.03 0.04b 0.06 0.05 0.04 0.08 0.06 0.06
’ The mean values are given as ,A O2 hr-’ mg wet tissue-’ + SE. Measurements weremadeat 25°Candrepresentinitial ratesof oxygenconsumption(l-5 min). The Krebs-Ringer’s phosphatesolutionwassaturatedwith 100%oxygen.Lactate,when used,waspresentat lo-’ M. bp < 0.01. The data in Table 4 were obtained from rat renal cortical slices removed from animals pretreated with dichromate. Although no significant effects or tissue respiration were noted with 5-mg/kg of potassium dichromate, with higher doses a significant depression of the renal cortical respiration was noted. This was true at both 2 and 5 days after administration of the nephrotoxin. For example, at the 20-mg/kg dose in the animals tested 5 days after administration of the compound, tissue respiration was depressed by more than 50%. Organic compound transport. The effects of potassium dichromate added to both rabbit and rat renal cortical slices are presented in Tables 5 and 6. The rabbit renal slices showed minimal accumulation of AIB, and accordingly, the uptake was diminished only moderately by high concentrations of potassium dichromate (10m4 M). Both TEA and PAH uptake were depressed by about 50-70 % by high concentrations of potassium dichromate. However, no significant reduction in the accumulation of either the organic acid or the organic base were noted with concentrations below 1o-4
M.
The results with rat renal cortical slices showed a significant reduction in the uptake of AIB at 10m4 M dichromate, although the magnitude of this effect was relatively modest. Lactate depressed AIB uptake and no effect of dichromate was noted on the lactate-regulated uptake of AIB. At the very highest concentration of dichromate (1 mM), a marked reduction of AIB accumulation was observed whether or not lactate was present. On the other hand, TEA and PAH showed marked reductions in uptake at lo-” M dichromate. It is also interesting to note that with low concentrations of dichromate (e.g., 10m6 M), a significant stimulation of uptake of TEA was noted. No significant enhancement of uptake was noted for other compounds.
1.31 k 0.04 1.27+0.03 1.30+0.02
0
1.53 + 0.05 1.65 +0.08 1.60+0.03
5
Lactate ~~______~ 1.28 f0.06 1.63 kO.03 1.35 +0.04 1.69+0.01
_____ -. ~~~~ ~~~ Lactate __~0.82+0.12 0.66f:O.OZ
0.76kO.09 0.53 +0.06
1.13 kO.08 0.84+0.02
-. Lactate
10
Dose @g/kg) 20
0.81 + 0.12 0.57 kO.11
-
Lactate
~_~.._..~ 0.62 +0.08 -
Lactate
-
0.81 kO.03 -
40
3.4 LactatelOM 2.5 15.2 Lactate 1O-2 M 16.2 5.5 Lactate 1O-2 M 13.2
If: 0.26 (9) rf: 0.15 (12)b + 1.2 (9) + 1.7 (11) f 0.43 (9) f 0.88 (11)
Control 2.8 2.4 14.1 14.0 3.6 10.3
+- 0.56 (3) If: 0.40 (3) Ifr 1.6 (3) + 2.2 (3) + 0.38 (3) + 1.4 (3)
lo-’ 2.8 2.4 20.1 22.3 6.0 17.7
-. k 0.17 (3) I!Z0.3 (3) f 0.16 (3)b f 0.43 (3)b f 0.9 (3) f 2.4 (4)
104 2.9 3.0 20.6 21.1 6.4 12.8
+ 0.44 (3) + 0.38 (4) + 1.8 (3) + 1.7 (4) 2~ 0.36 (3) rf: 1.0 (5)
1o-5 2.2 2.5 6.6 8.5 3.3 4.3
2 + f iz rt f
.~ ~~. 0.26 (6)b 0.19 (8) 0.84 (6) 0.99 (8) 0.4 (6) 0.45 (8)
1o-4
M -.......-. .-~~~~~~ .---~~~~~
0.90 1 .o 0.85 1.3 0.92 1 .o
f 0.02 * 0.05 + 0.03 If- 0.32 f 0.03 + 0.09
1o-3 (3)b(3)b (3)C (3) (3) (3)
” Values are S/M ratios + SE, and numbers of experiments given in parentheses. Incubations were carried out for 120 min at 25’C in the presence of lOOg:, oxygen. “p < 0.05 compared to appropriate Control. cp < 0.01 compared to appropriate Control.
PAH uptake
TEA uptake
AIB uptake
5
EFFECT OFPOTASSIUM DICHROMATE ADDEDTORAT RENALCORTICALSLICES"
TABLE
’ The mean Oez values are given as ,ul O2 hr-r mg wet tissue + SE. N varied from 3 to 5. Measurements were done at 25°C and represent initial rates of oxygen consumption (1-5 min). The Krebs-Ringer’s phosphate solution was saturated with 100°A oxygen and lactate, when used, was present at 10e2 M. Control animals were injected with water.
0 2 5
Days after pre- ---~_~_-__treatment -
4
EFFECT OFPOTASSIUM DICHROMATE PRETREATMENT ONRENALCORTICALSLICERESPIRATIONS
TABLE
2 r:
z ;;1 5
i K
5
u ii B 8
W. 0. BERNDT
48
TABLE 6 EFWCTOFPOTASSIUMDICHROMATEADDEDTORABBITRENALCORTICALSLICES~
Potassiumdichromate(M) N AIB uptake TEA uptake PAH uptake
-
10-5
10”
10-4
6 6
1.7 + 0.2
1.7 * 0.2
1.6 f 0.2
1.2 + O.lb
M
1.5 If: 0.1
1.5 f 0.1
1.6 f 0.2
1.2+ 0.2b
M Acetate 10m2
6 6
17.3 f 1.3
15.6 f 1.2
15.1 f: 1.2
19.8f 1.3
20.5 f 1.8
19.9f 2.2
8.7 t 1.4b 7.7 + l.6b
Acetate lo-*
5 5
6.1 f 1.4 13.3f 1.5
5.7 * 1.3 13.3f 1.3
5.3 + 1.2 12.6+ 1.5
3.3 f 0.8b 3.8 f l.3b
Acetate lo-’
M
a Values are S/M ratios f SE and Nrefers to number of experiments. Slices were incubated for 120 min at 25°C in the presence of lOOok oxygen. bp < 0.05 compared to appropriate control.
In Fig. 3, data are presented for the corresponding pretreatment experiments. The smaller dose reduced TEA significantly as early as 2 hr, but a more marked effect was seen at 24 hr and a somewhat greater depression was observed at 48 hr. The uptake returned to an intermediate level of depression for the remaining 5 days of the experiment. With the higher dose of dichromate, the significant reduction at 24 hr was followed by a further reduction at 48 hr with the maximal effect seen at 3 or 4 days. RAT
RENAL
CORTEX
SLICES
NO Substrate . .
Lactate 0 control A IOmq/Kg K&O, 0 2Omg/KP K&q
OOT
2
I
3
Hours Time
ottsr
4 WS
5
6
7
Pretreatment
FIG. 3. The effect of potassium dichromate on renal cortical slice uptake of tetraethylammonium. Each point is the mean, and bar the SE, of four animals. All animals were injected subcutaneously on day 0.
DICHROMATE
AND
RENAL
49
TRANSPORT
PAH uptake was stimulated by lactate (Fig. 4), and it was this lactate-stimulated accumulation of PAH which was depressed by IO-mg/kg of potassium dichromate. No consistent or marked effect of dichromate on the resting uptake of PAH was noted at this dose of nephrotoxin. However, at the higher dose of dichromate both the substrate-stimulated and the nonstimulated accumulation of the organic anion were depressed significantly. Data for AIB uptake by slices from pretreated animals are not presented, since they were negative. That is, neither dose of dichromate affected AIB accumulation at any of the times used in this study. RAT
RENAL
CORTEX
SLICES NO Substrate . *
04-l-l-F 0 2 4 Hours
6
-8 I Time
of
FIG. 4. The effect of data as in Fig. 3.
2
3
after
Pretreatment
4 DW
5
6
Lactate 0 Control 6 IOm(l~g
K&C+
7
potassium dichromate on renal cortical slice uptake of p-aminohippurate.
Rest
The time course of the effects of the dichromate on organic compound transport should be emphasized. Although 2 hr after a dose of 20 mg/kg, large concentrations of dichromate appear in kidney cortex (0.2-0.5 mM, N = 6), maximal inhibitory effects on the transport of the organic compounds was not noted until later. The same or even lower concentration of dichromate (0.1 mM) added in vitro did reduce PAH and TEA accumulation (Tables 5 and 6). DISCUSSION
In general, the data reported here agree with the earlier report of Hirsch (1973). However, there are certain important areas of disagreement which deserve comment. In addition, although some of the data are in agreement, it is possible to interpret them differently than Hirsch did. For example, it seems clear that the transport of AIB is altered only slightly by dichromate administered in vim or added in vitro (except for extremely high concentrations,
i.e., 1 mM). Hirsch has interpreted this as evidence of an
50
W.
0.
BERNDT
anatomical localization of the action of dichromate, i.e.: an effect on the pars recta where PAH transport is maximal, but AIB transport is not. This suggestion does not agree with the autoradiographic studies of Wedeem and Thier (1971), who reported extensive accumulation of [3H]AIB in the straight portions of rat proximal tubules. Although anatomical localization of dichromate is a possible explanation for differential effects, it is not unreasonable to suggest that the effect is related to the failure of dichromate to affect the AIB transport carrier. This proposal is consistent with the multiplicity of effects noted by Hirsch (1972, 1973) and reported here. Furthermore, several anatomical studies (Oliver and MacDowell, 1963; Biter et al., 1968; Kramp et al., 1974) complicate this problem additionally. All of these investigators demonstrated that potassium dichromate produced a selective necrosis of the pars convoluta of the proximal tubule. Only minimal damage to the pars recta was noted and the microscopic data were supported by micropuncture experiments. The studies of Tune et al. (1969) on isolated perfused tubules indicated that maximal transport of PAH occurs in the pars recta and therefore, one might expect only minimal effects on PAH transport after dichromate since this nephrotoxin leaves the pars recta intact. The severe depression of PAH uptake by renal slices might be interpreted as an effect of dichromate on the renal transport in the pars convoluta as well, perhaps unrelated to the production of necrosis. These data also raise the question of the value of purely microscopic studies in the determination of tubular sites of nephrotoxicity. In this study the accumulation of PAH, TEA, and AIB was examined both in the presence and absence of metabolic substrate. Lactate had little effect on the uptake of TEA, but stimulated PAH uptake markedly and depressed AIB uptake significantly. The lactate effect on AIB was not noted in the presence of dichromate. Renal slices prepared from animals pretreated with the low dose of dichromate showed a reduced level of lactate stimulation of PAH uptake, although the nonstimulated accumulation was unaffected. This differential effect may be related to interference with energy metabolism, since this dose of dichromate did depress tissue respiration significantly. However, the depressed respiration occurred whether or not lactate was present in the bathing solution. Therefore, this generalized metabolic effect does not appear to account for the specificity of the dichromate effect on PAH uptake. The mechanism of lactate stimulation is unknown (Schachter et al., 1955; Kim and Hook, 1973). If the dichromate effect is related to the intracellular actions of lactate in promoting PAH transport, it may prove useful for understanding the mechanism(s) of lactate stimulation. On the other hand, the effect may be related to blockade of lactate entry into the tissue. There was an apparent attempt at recovery of tissue electrolytes over several days after administration of potassium dichromate to the animals. Total tissue water and tissue potassium concentrations tended toward control values 4 or 5 days after the nephrotoxin was given, especially after the lower dose (10 mg/kg). The significance of this is not clear, since none of the high-dose animals survived even 5 days and many of the animals that received the low dose did not survive 10 days. It is noteworthy that marked quantitative differences appeared depending on how the nephrotoxin was administered. Quantitatively, the alterations in tissue electrolytes and water were never as large after in vitro addition as after in vivo administration, even when the tissue concentrations of dichromate were approximately the same.
DlCHROMATE
AND
RENAL
TRANSPORT
51
It is tempting to suggest that these quantitative data can be used to help distinguish between the different theories for the pathogenesis of acute renal failure. The effect of dichromate was consistently more dramatic after in viuo administration than after in vitro addition, at least by 6-24 hr after injection of the nephrotoxin. The effect at 2 hr, which corresponds to the in vitro time course, was not as dramatic but still a greater effect was noted in vivo than in vitro. Given that tissue concentrations of dichromate are approximately the same, these data would appear to support an indirect effect on renal transport, e.g., in vivo the nephrotoxin altered vascular events in such a way to compromise renal transport mechanisms. On the other hand, if the in vice action was solely through vascular mechanisms, it seems unlikely that any specificity of effect would be noted, e.g., lack of effect on AIB transport or blockade of substratestimulated PAH uptake only. Hence, although the quantitative differences between in uivo and in vitro effects supports a pathogenic mechanism related to interruption of vascular events, the specificity of the effects on the transport processes does not. This argument gains support from the experiments where 10m3 M dichromate was used in vitro. At this very high concentration all transport processes for the organic compounds used in this study were disrupted. In addition, the different effects may be related to the particular chemical species of chromate ion present. In vitro dichromate is added to a protein-free solution, wherein the usual chemical equilibria would be expected to exist (Cotton and Wilkinson, 1972): H+ + CrO,’ + HCrO,- + Cr207=. The first step occurs with acidification and the second as a result of a dimerization. For example, at pH 7.4, CrO,= is the predominant species with the ratios of Cr04= : HCrO,- : Cr,07= being 200: 10 : 1. Presumably, under in vitro conditions CrO,= is predominant and may be the active form of the chromate ion. In vivo, the species which predominates is not clear. Although the pH of plasma, extracellular fluids, etc., are about 7.4, problems of plasma protein binding must be involved. Perhaps Cr,07’ is bound to plasma proteins and thereby “protected” from the usual equilibria. This might mean that in vivo Cr,O,= is delivered to the renal site(s) of action, whereas in vitro CrO; is the active species. Similar distinctions have been suggested by Schuchter et al. (1973) for the action of chromate ion on water and solute permeability of the toad bladder. ACKNOWLEDGMENTS
The author is most appreciative of the excellent technical assistance of Bonnie Bemdt and Ellen Costello. REFERENCES
A. D. (1965). Cell renewal following dichromate induced renal tubular necrosis.
BAINES,
Amer. J. Pathol. 47, 851-876. BANK, N., MUTZ, B. F. AND AYNEDJIAN,
H. S. (1967). The role of “leakage” of tubular fluid in anuria due to mercury poisoning. J. Clin. Invest. 46,695-704.
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