Function
ALFRED
of Autotransplanted Kidneys after Hypothermic Perfusion with Dimethylsulfoxide l SMALL,
NICIIOL.4S
J, FI’I)ITSlir\,”
In order to attain long-tcrin renal preservation for transplantation, freezing, after protection with a cryoprotective compound, has been suggested ,zF a likely method (lS), based on successful cspcriencc with siniplc cell suspensions and tissue (16, lS, 23). Din~ethylsulfosidc (DMSO) has 1m.x used as a cryoprotectivc compound that, in general, is considered to have low cellular tosicity (15). However, some studies have suggcstcd that perfusion of organs with a high concentration of DMSO results in damage which, when added to the prol~al~lc damage of freezing and thawing, would -__Received October 28, 1973. 1 From the Naval \ledical Rcscarch and Devclopmcnt Connnand, SNhIC, Drpartnlcnt of the Navy, Research Task No. hIF,51..524.013.100~5. The opinions and assertions contaimtl lrcwin arc the private ones of tllc arbors anti are not to Ix construed as official or reflrcting the vic\vs of the Navy Department or the naval xrvicc at large. The animals used in this study ww handled in accordance with the provisoes of Pllblic Law 89-14 as amended by Pllblic Law 91-579, the “Animal Welfare .4ct of 1970” and tile principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U.S. Department of Hedth, Educ~~tion and Welfare Publication No. ( KIH) 73-23. 2 Present address of S. J. Feduaka: Transplant Service, Department of Surgery, University of California Medical Center, Sau Francisco, California 94143. 3 Present address of R. S. Filo: l‘ranspl;~nt;ltion Unit, Veterans Administration Hospital, Intlianapolis, Indiana 462%.
Copyright G 1977 by Academic l’ree, Inc. All rights of reproduction in my lorm resrnwl.
r\h,, ROS.-\LD
S. FILO
2
reduce the likelihood of successfully prcserving kidneys by this method (2, 19). In addition, tlic degree of DMSO cquililx~tion bctwccu renal tissue and pcrfusate alld the uuit’ormity of distrilmtion within th kitlncy arc estrcincly important factors f’or c~~xluating the utility of DXlSO as a cr!7)protectivc agent. Yet, measurements of tlmc parainctcrs inndc in scvcral laboratories arc not in good ngrccment (6, 12, 17, 19), p 0551 ::‘l 11,v (1uc to differences in the c~spcrimcntnl niodcl us&, or bccausc of the proccdurc~ used to c5timite D11SO cmccutration. The purpose of this study was twofold. 011~ purpose was to identify the specific rcrml functional processes that may be alkred 1)~. c~sposurc~ of kidneys to DMSO during perfusion. In order to do this, standard renal clearance niethods were used to assess nccuratcly glomcrular and tubular function immediately after, and again 48 hr and 7 da!.s followiug nutotraiis~~lantatioii. Thcsc ineasurcments wcrc rspccted to yield nluch more information than would 1~ ol,taiiied simply 11~following the course of scruin urea or creatinine concentrations, the usual method of evaluatinf; post-trnnsplantation renal function. The second purpose \vxs to obtain additional data concerning the degree of equililmatiou and the uniformity of distribution of DAIS0 iu tissue from such kidneys, using :I radioisotopic lal~l to determine the cou-
JS\Z 0011 2240
SMALL,
21
FEDtJSKA
23
FIG. 1. Protocol of addition of DMSO to and its removal from circulating perfusate. The stages in the procedure, indicated by arabic numerals, are: ( 1) 20-min stabilization period, perfusion apparatus contains 600 ml of perfusate; (2) infusion of 300 ml of perfusate, containing 30 g DMSO/lOO ml, at a rate of 5 ml/mm; (3) 20-min stabilization period at high DhlSO concentration; (4) rapid removal of 300 ml of circulating perfusate, and start of infusion of 600 ml of DhlSO-free perfosate at a rate of 30 ml/min; (5) rapid removal of 600 ml of circulating perfusate, and start of infusion of 600 ml of DMSO-free perfusate at a rate of 30 ml/min; (6) 20-min stabilization period at 1ow DMSO concentration; and (7) removal of kidney from perfusion apparatus.
AND
Following p(rfusioti, tlrc kidircty \vas ;,,Itografted into the left iliac fossa, using cntlto-end anastomosis of the common iliac artcry and end-to-side anastomosis of the renal common iliac vein to the rcspcctivc \ressels. In addition, a ureteroneocystostomy was constructed. Contralateral nephrectomy was performed immediately only in animals in experimental series II, but was delayed for 7 days in series I (see below). Perfusion techniques. All kidneys were perfused with cryoprecipitated homologous canine plasma, prepared and supplemented in the manner described by Belzer et al. (1). The systolic perfusion pressure was maintained at 60 mm Hg, using a pump rate of 66 strokes/mm. Because pressure was kept constant, perfusate flow rate was determined solely by renal vascular rcsistawe; in series I, the mean flow rate for 12 animals was 0.713 ml/min,,/g kidney weight (range = 0.494-0.915); in series II, the mean flow rate for 24 animals was 0.710 ml/min,‘g
centration of the cryophylactic agent in plasma and tissue and to correlate alterations in post-transplant function with tissue DMSO levels. hlATERIALS
AND
METHODS
Animals and surgical technique. Adult male mongrel dogs were used in all perfusion studies. Details of anesthesia and surgical technique have been described previously (7). The left kidney was used routinely as the autograft, unless it possessed a double renal artery. Upon removal of the kidney, the renal artery was cannulated and the kidney flushed with heparinized lactated Ringers solution, at 4°C delivered at a pressure of 100 cm HzO, until the renal venous effluent became clear. The kidney was then connected to the perfusion apparatus (Belzer Perfusion Apparatus, Model LI-400; Lifemed Corp.) and treated ‘as described below. The period of warm ischemia was never greater than 10 min.
FILO
kidney
weight
(range
= 0.446-
1.140). Perfusate temperature was maintained between 8 to 10°C; and pH was kept between 7.3 to 7.5 by adjustment of the flow of CO, to the membrane oxygenator. DMSO (Reagent grade, Fisher Scientific Co.) was initially diluted to 30% (v/v) with plasma perfusate on the day prior to the experiment in which it was to be cmployed. In some of the studies, 50 &i [“H]DMSO (87 mCi,~mniolc; 1 &i/nil; New England Nuclear Corp. ) was added to the unlabeled DMSO prior to dilution. On the day of the study, the diluted DMSO was centrifuged at 10,OOOf for 15 min and filtered through a 0.22 ~111hlillipore filter. The protocol for addition and removal of DMSO to the perfusion circuit is illustrated in Fig. 1. After a 20-min stabilization period, 300 ml of 30% DMSO in perfusate was added at a rate of 5 ml/min to the 600 ml of perfusate originally present in the apparatus (final DMSO concentration, approximately
10% ). After
another
20-min
AUTOTRA~SPLA~TATIO~
AFTER PI+:RFUSION WIT11 IllIS
‘)-.)-
clll;ilititati\.c~l!,; arkGal lil0ocl Sali~plcs \VW’ oldainctl at tli(l midpoint of cwli period. At the cmd of the clcarancc study, the al)was sutured and the anidominal wound mal allowc~cl to rwovcr. ,4t 2 and 7 clays after ai1totransplantaticii1, each animal was again anc~stlictizecl, the alxlominal incision reopcwd, and clearanw studies again performed; but man11itol was omittccl from the infusion solution used on the second and seventh da!s. After the clcarancc study on the scvcnth clay, the non-perfused kidney was removed and the wound closed. Thea strum concentrations ot urea and creatinine wcw then detc~rminccl daily for the, first wwk following ncphrectomy and hiwcckly for thcl next 2 wwks. Serum urea was detc~rminccl with a Urea Nitrogen (HUX) Rapid Stat Kit (Pierce Chemical Co., Rockford, Ill. ) and serum creatinine conccntration was determined li!~ the method of Folin and \Yu ( 8 ) . Each urine and plasma sample was analyzed for the following substances, using the method indicated: sodium and potas( flame photometry) ; Series 1: Detailed rend function studies. sium concentration chloride concentration (potentiometric tiIn this series of six control and six esperitration); in&n (method of Fiihr et al. (9); mental dogs, DMSO perfusions were alPL4H (method of Smith et al. (24); glucose ternated with control stud&. Following (cnz)matic assay; Bochringer Manheim autotransplantation, tlie uwters of both the Corp., New York); ancl osmolality (freezperfused and contralateral kidnc)zs, as well ing-point depression osmometry ). These as a femoral artery, were cannulated with data permitted calculation of glomerular polyethylene tubing. Intravenous priming doses of i11ulin (50 mg kg) and p-aminofiltration rate (CFR; equal to inulin clearhippuratc (PAH; 115 mg, kg) in 150 ml of nnw 1; niasiniu11~ tubular transport rate normal saline were acln~inistc~rcd owr a (T,,,) for PAH; p ercentage of tulxlar rulo-min period. The priming doses wcw foljcction of sodium, chloride, and water; lowed b,- a sustainecl intrave11ous infusioii (percentage of tubular rejection of X = [X crcretcd f X filterecll .lOO); free water of the same substances (6.8 mg inulin, ‘nil; ( T“r12c,); 15 mg PAH/ml) pl us maniiitol ( 100 mg / clearance (C,,,,,) or r&sorption ml) in normal saline solution at a rate of and tubular rejection of glucose. In the case of free water reabsorption, the value 0.2 ml,‘min/kg. The priming ancl ilrfusion solutions had l~cw~ filtered througl1 a 0.2 was espreswd as the ratio of T”I-IZO to the pm Mllipore filter prior to use. After a 60- os1nolar clearance (T“ I12~/C~,s1\I), since the to BO-min equilibration period, the first of absolute value of T“n,o is a function of filthree or four 20-min clearance periods was trate delivery to the loop of Henle, which begun. During every period, the urine fro111 is cstimatccl by Cosar (21). In each animal, each kiclwy was collected separately and a single valrw for each functional parameter
staliilizatioll pclriocl, 300 1111of pc?rfw’tc! \v;u rapidly drained from tl1c apparatllS, a1rc1addition of 600 1111Dh’ISO-frcw pcarfusate was started at a rate of 30 iiil,‘min. L4t the end of that phase, the DMSO conccntration of the pcrfr1satc was approrimatel) 5%‘. Then, a 600-ml volumc~ of the pcrfusate was rapidly rcwovcd, followc~d by infusion of an additbnal 600-1111 v0h~11e of DMSO-free perfusate, again at a rate of 30 pcrfusatc in a final ml, ‘min, resulting DMSO concentration of approximatel) 2.5%. Following a final 20-min stabilization period, the kidney was rcmovcd and rcimplanted. Control kidneys wcrc trcatetl in a similar niainwr, cscept that DhlSO-frcC perfusate, rather than 30% DMSO, was added during the 20- to SO-min period, wit11 no further removal or addition of pcrfusate cluri11g the remainclcr of tl1c 160-min perfusion. In both csperimeiital series, a complete autopsy was performed in the case of every animal that died lIefore the end of the observation period.
wiis calculated by averaging tlic values in all clearance periods. Series II: DMSO perfusion and autotransplantation with immediate contralateral nephrectomy. The experiments of series II were performed in order to determine if either the presence of a contralateral kidney or the induction of mannitol diuresis immediately post-transplantation had an effect ‘on the viability or life-supportive capability of the kidneys. In series II, contralateral nephrectomy was performed at the time of removal of the experimental kidney for perfusion. In this series, four treatment groups were studied: Kidneys were either exposed or not exposed to Dl\/ISO during perfusion; and animals received a postreimplantation infusion of either mannitol (20 mg,/min/kg ) in saline, or of lactated Ringers solution; both solutions were infused at a rate of 0.2 ml/min/ kg. Six animals were included in each of the groups, treated as follows: group A: perfused with DMSO-free medium but not infused with mannitol after reimplantation; group 13: perfused with DMSO and infused with mannitol; group C: perfused with DblSO-free medium and infused with mannitol; and group D: perfused with DMSO, but not infused with mannitol. The protocol for addition and removal of DMSO, and the rate and duration of mannitol infusion were the snmc as in series I, but the mannitol infusion solution did not contain inulin or PAH. Following autotransplantation, the only measurements made in the animals of series II were serial determination of plasma urea and creatinine: daily for the first week, and biweekly for the next 3 weeks. The order in which animals were assigned to the various treatment groups was dictated by a stratified random design. Determination of DMSO concentration. In experiments in which [3H] DMSO was added to the unlabeled compound (either scrics I or series II), samples of perfusate
WCTC
tiikt31
iIt
oacll
st;lgc:
of
tllc:
infllsiol~
and removal of D_\ISO. In addition, in many cxpcriments of scrics II in which [“H] DR,fSO was used, the contralatcral and experimental kidneys were perfused simultaneo~~sly; but the contralateral kidney, removed cithcr at 100 min of perfusion (peak D&IS0 concentration ) or at the end of perfusion (minimum DMSO concentration), was used to dctermine tissue DMSO. Ten samples each (100 to 200 mg, wet weight) of outer cortex, inner cortex, and medulla, representing at least three widely separated regions of the organ, were weighed and digested in Soluene-100 (Packard Instrument Co., Downers Grove, Ill.). Also, samples (about 500 mg) of each of the three anatomical levels were weighed before ancl after drying at 100°C for 15 hr in order to estimate tissue water content. In every experiment of series II employing [“HI DMSO, all urine produced during the 140 min following revascularization of the kidney was collected in order to dcterminc the amomlt of DMSO that could be rccovered. DMSO concentration in perfusate, tissue digest, or urine was calculated from the radioactivity observed in a liquid scintillation counting system, after correction for quenching, and was based on the radioactivity present in the undiluted DMSO. Statistical analysis. In series I, the effect of perfusion in either the DMSO or control groups was assessed as the difference bctween the function of the perfused kidney and that of its nonperfused mate, and was tested for statistical significance by means of a paired t test. The influence of DMSO was assessed by comparing the effect of perfusion in the DMSO group with that in the control group, using a group t test. In series II, peak urea or creatinine levels in the four treatment groups were subjected to analysis of variance to assess the effects of the two variables.
AIITOTR.4~SPLANT.\TIOr
I\FTER
Statisticd ~u~ul~~.st~s wcw p(~+ort~~~d ;tc’cording to the nicthotls gi~~;n 1,~ Sncd(0x (25). The criterion for rcjcctiou of the null hypothesis was a value of P S 0.05. Unless otherwise stated, numerical values given in the text arc lllfallS 2 stalKlarcl ~‘1’1‘01’ of the 111ca11.
RESULTS
Series I. Survival of animals in the DMSO-treated group of series I was not substantially different than in the control group. Of six control animals, four survived the full 30&y study period without an) sign of illness. One animal died on the fifth day post-transplantatioll due to failure of
Flul~lioll
\VITII
*)’ -I
DMSO
111~renal artt~rial anastonlosis. A110tht:r alli1na1 was sacrificed whm it was found that its pcrfnsd kidney had ccascd to function as a result of a renal venous thrombosis. The thrombosis was probably due to damage caused bv repeated catheterizatioii, \vhich was performed in an attempt to cstimate renal blood flow from the measured extraction of PAR. Such catheterizations wc’rc discontinued after being performed in the first two animals of each group, and no further incidence of venous thrombosis was O1~servt’d* In the DhlSO group, only two animals survived for 30 days. One was sacrificed after 7 da!5 bccansc of venous thrombosis.
Tr\l%l,l‘: I:wd
PERFITSIOS
1
~le:t.~l~rcnlenl :, ill I’c~~f~~~cdi~\~~lotla~~spl;~r~l etl :mtl N011prrf11,~;etl (‘clllt~:tlwtrl~;~l ~~ltilolit
I):\> (
sorl~,erlrlse~! 0
2 7
4.03 i I.O(i 2.87 + 1 .oo 1.89 It O.i!i
0 2 ‘i
21.1 i :i.ci :;o. 1 * ::.7 21 .(i f .i.o
0 2 7
10.2 s. I 6.i
i i i
0 2 7
10.0 s.2 6.:;
0 2 7
10.7 x.0 7.2
0 2
1)~lHO
:;.ii3 * 0.62 2.70 f 0.79 O.i,j f 0.2.7 I(i.0 24.2 22.0
i 2.0 zk 4.(i f :i.ci
.i.s .i.2 .i.!)
zt 2.2 i 1.!I i 1.2
* 2.:; It 2.4 f 2.0
7,s 1o.:i 2.::
f- 2.4 rt 2.0 * 0.4
f %..i XII 2.7 f 3.0
‘i.!J *2.:3 I I .:: f 2..i 1.4 I 0.4
*A.!) :;.2 2.1
0.17 f 0.0s 0.29 + 0. I*
\vlth
)‘
I’rrfllwl
7
O.l(i f 0.14
-0. I!1 & 0.0X” 0.2:; 0.0,w f 0,:;s & 0. I .i”
0 2 7
I .4(i * 0.x: O..X i 0.4.5 I.!hs + 4.95
2.:10 * 0.x: 0.37 f 0.2:: 2.2s i 2.2.i
s
ALFRED
of Autotransplanted Kidneys after Hypothermic Perfusion with Dimethylsulfoxide l SMALL,
NICIIOL.4S
J, FI’I)ITSlir\,”
In order to attain long-tcrin renal preservation for transplantation, freezing, after protection with a cryoprotective compound, has been suggested ,zF a likely method (lS), based on successful cspcriencc with siniplc cell suspensions and tissue (16, lS, 23). Din~ethylsulfosidc (DMSO) has 1m.x used as a cryoprotectivc compound that, in general, is considered to have low cellular tosicity (15). However, some studies have suggcstcd that perfusion of organs with a high concentration of DMSO results in damage which, when added to the prol~al~lc damage of freezing and thawing, would -__Received October 28, 1973. 1 From the Naval \ledical Rcscarch and Devclopmcnt Connnand, SNhIC, Drpartnlcnt of the Navy, Research Task No. hIF,51..524.013.100~5. The opinions and assertions contaimtl lrcwin arc the private ones of tllc arbors anti are not to Ix construed as official or reflrcting the vic\vs of the Navy Department or the naval xrvicc at large. The animals used in this study ww handled in accordance with the provisoes of Pllblic Law 89-14 as amended by Pllblic Law 91-579, the “Animal Welfare .4ct of 1970” and tile principles outlined in the “Guide for the Care and Use of Laboratory Animals,” U.S. Department of Hedth, Educ~~tion and Welfare Publication No. ( KIH) 73-23. 2 Present address of S. J. Feduaka: Transplant Service, Department of Surgery, University of California Medical Center, Sau Francisco, California 94143. 3 Present address of R. S. Filo: l‘ranspl;~nt;ltion Unit, Veterans Administration Hospital, Intlianapolis, Indiana 462%.
Copyright G 1977 by Academic l’ree, Inc. All rights of reproduction in my lorm resrnwl.
r\h,, ROS.-\LD
S. FILO
2
reduce the likelihood of successfully prcserving kidneys by this method (2, 19). In addition, tlic degree of DMSO cquililx~tion bctwccu renal tissue and pcrfusate alld the uuit’ormity of distrilmtion within th kitlncy arc estrcincly important factors f’or c~~xluating the utility of DXlSO as a cr!7)protectivc agent. Yet, measurements of tlmc parainctcrs inndc in scvcral laboratories arc not in good ngrccment (6, 12, 17, 19), p 0551 ::‘l 11,v (1uc to differences in the c~spcrimcntnl niodcl us&, or bccausc of the proccdurc~ used to c5timite D11SO cmccutration. The purpose of this study was twofold. 011~ purpose was to identify the specific rcrml functional processes that may be alkred 1)~. c~sposurc~ of kidneys to DMSO during perfusion. In order to do this, standard renal clearance niethods were used to assess nccuratcly glomcrular and tubular function immediately after, and again 48 hr and 7 da!.s followiug nutotraiis~~lantatioii. Thcsc ineasurcments wcrc rspccted to yield nluch more information than would 1~ ol,taiiied simply 11~following the course of scruin urea or creatinine concentrations, the usual method of evaluatinf; post-trnnsplantation renal function. The second purpose \vxs to obtain additional data concerning the degree of equililmatiou and the uniformity of distribution of DAIS0 iu tissue from such kidneys, using :I radioisotopic lal~l to determine the cou-
JS\Z 0011 2240
SMALL,
21
FEDtJSKA
23
FIG. 1. Protocol of addition of DMSO to and its removal from circulating perfusate. The stages in the procedure, indicated by arabic numerals, are: ( 1) 20-min stabilization period, perfusion apparatus contains 600 ml of perfusate; (2) infusion of 300 ml of perfusate, containing 30 g DMSO/lOO ml, at a rate of 5 ml/mm; (3) 20-min stabilization period at high DhlSO concentration; (4) rapid removal of 300 ml of circulating perfusate, and start of infusion of 600 ml of DhlSO-free perfosate at a rate of 30 ml/min; (5) rapid removal of 600 ml of circulating perfusate, and start of infusion of 600 ml of DMSO-free perfusate at a rate of 30 ml/min; (6) 20-min stabilization period at 1ow DMSO concentration; and (7) removal of kidney from perfusion apparatus.
AND
Following p(rfusioti, tlrc kidircty \vas ;,,Itografted into the left iliac fossa, using cntlto-end anastomosis of the common iliac artcry and end-to-side anastomosis of the renal common iliac vein to the rcspcctivc \ressels. In addition, a ureteroneocystostomy was constructed. Contralateral nephrectomy was performed immediately only in animals in experimental series II, but was delayed for 7 days in series I (see below). Perfusion techniques. All kidneys were perfused with cryoprecipitated homologous canine plasma, prepared and supplemented in the manner described by Belzer et al. (1). The systolic perfusion pressure was maintained at 60 mm Hg, using a pump rate of 66 strokes/mm. Because pressure was kept constant, perfusate flow rate was determined solely by renal vascular rcsistawe; in series I, the mean flow rate for 12 animals was 0.713 ml/min,,/g kidney weight (range = 0.494-0.915); in series II, the mean flow rate for 24 animals was 0.710 ml/min,‘g
centration of the cryophylactic agent in plasma and tissue and to correlate alterations in post-transplant function with tissue DMSO levels. hlATERIALS
AND
METHODS
Animals and surgical technique. Adult male mongrel dogs were used in all perfusion studies. Details of anesthesia and surgical technique have been described previously (7). The left kidney was used routinely as the autograft, unless it possessed a double renal artery. Upon removal of the kidney, the renal artery was cannulated and the kidney flushed with heparinized lactated Ringers solution, at 4°C delivered at a pressure of 100 cm HzO, until the renal venous effluent became clear. The kidney was then connected to the perfusion apparatus (Belzer Perfusion Apparatus, Model LI-400; Lifemed Corp.) and treated ‘as described below. The period of warm ischemia was never greater than 10 min.
FILO
kidney
weight
(range
= 0.446-
1.140). Perfusate temperature was maintained between 8 to 10°C; and pH was kept between 7.3 to 7.5 by adjustment of the flow of CO, to the membrane oxygenator. DMSO (Reagent grade, Fisher Scientific Co.) was initially diluted to 30% (v/v) with plasma perfusate on the day prior to the experiment in which it was to be cmployed. In some of the studies, 50 &i [“H]DMSO (87 mCi,~mniolc; 1 &i/nil; New England Nuclear Corp. ) was added to the unlabeled DMSO prior to dilution. On the day of the study, the diluted DMSO was centrifuged at 10,OOOf for 15 min and filtered through a 0.22 ~111hlillipore filter. The protocol for addition and removal of DMSO to the perfusion circuit is illustrated in Fig. 1. After a 20-min stabilization period, 300 ml of 30% DMSO in perfusate was added at a rate of 5 ml/min to the 600 ml of perfusate originally present in the apparatus (final DMSO concentration, approximately
10% ). After
another
20-min
AUTOTRA~SPLA~TATIO~
AFTER PI+:RFUSION WIT11 IllIS
‘)-.)-
clll;ilititati\.c~l!,; arkGal lil0ocl Sali~plcs \VW’ oldainctl at tli(l midpoint of cwli period. At the cmd of the clcarancc study, the al)was sutured and the anidominal wound mal allowc~cl to rwovcr. ,4t 2 and 7 clays after ai1totransplantaticii1, each animal was again anc~stlictizecl, the alxlominal incision reopcwd, and clearanw studies again performed; but man11itol was omittccl from the infusion solution used on the second and seventh da!s. After the clcarancc study on the scvcnth clay, the non-perfused kidney was removed and the wound closed. Thea strum concentrations ot urea and creatinine wcw then detc~rminccl daily for the, first wwk following ncphrectomy and hiwcckly for thcl next 2 wwks. Serum urea was detc~rminccl with a Urea Nitrogen (HUX) Rapid Stat Kit (Pierce Chemical Co., Rockford, Ill. ) and serum creatinine conccntration was determined li!~ the method of Folin and \Yu ( 8 ) . Each urine and plasma sample was analyzed for the following substances, using the method indicated: sodium and potas( flame photometry) ; Series 1: Detailed rend function studies. sium concentration chloride concentration (potentiometric tiIn this series of six control and six esperitration); in&n (method of Fiihr et al. (9); mental dogs, DMSO perfusions were alPL4H (method of Smith et al. (24); glucose ternated with control stud&. Following (cnz)matic assay; Bochringer Manheim autotransplantation, tlie uwters of both the Corp., New York); ancl osmolality (freezperfused and contralateral kidnc)zs, as well ing-point depression osmometry ). These as a femoral artery, were cannulated with data permitted calculation of glomerular polyethylene tubing. Intravenous priming doses of i11ulin (50 mg kg) and p-aminofiltration rate (CFR; equal to inulin clearhippuratc (PAH; 115 mg, kg) in 150 ml of nnw 1; niasiniu11~ tubular transport rate normal saline were acln~inistc~rcd owr a (T,,,) for PAH; p ercentage of tulxlar rulo-min period. The priming doses wcw foljcction of sodium, chloride, and water; lowed b,- a sustainecl intrave11ous infusioii (percentage of tubular rejection of X = [X crcretcd f X filterecll .lOO); free water of the same substances (6.8 mg inulin, ‘nil; ( T“r12c,); 15 mg PAH/ml) pl us maniiitol ( 100 mg / clearance (C,,,,,) or r&sorption ml) in normal saline solution at a rate of and tubular rejection of glucose. In the case of free water reabsorption, the value 0.2 ml,‘min/kg. The priming ancl ilrfusion solutions had l~cw~ filtered througl1 a 0.2 was espreswd as the ratio of T”I-IZO to the pm Mllipore filter prior to use. After a 60- os1nolar clearance (T“ I12~/C~,s1\I), since the to BO-min equilibration period, the first of absolute value of T“n,o is a function of filthree or four 20-min clearance periods was trate delivery to the loop of Henle, which begun. During every period, the urine fro111 is cstimatccl by Cosar (21). In each animal, each kiclwy was collected separately and a single valrw for each functional parameter
staliilizatioll pclriocl, 300 1111of pc?rfw’tc! \v;u rapidly drained from tl1c apparatllS, a1rc1addition of 600 1111Dh’ISO-frcw pcarfusate was started at a rate of 30 iiil,‘min. L4t the end of that phase, the DMSO conccntration of the pcrfr1satc was approrimatel) 5%‘. Then, a 600-ml volumc~ of the pcrfusate was rapidly rcwovcd, followc~d by infusion of an additbnal 600-1111 v0h~11e of DMSO-free perfusate, again at a rate of 30 pcrfusatc in a final ml, ‘min, resulting DMSO concentration of approximatel) 2.5%. Following a final 20-min stabilization period, the kidney was rcmovcd and rcimplanted. Control kidneys wcrc trcatetl in a similar niainwr, cscept that DhlSO-frcC perfusate, rather than 30% DMSO, was added during the 20- to SO-min period, wit11 no further removal or addition of pcrfusate cluri11g the remainclcr of tl1c 160-min perfusion. In both csperimeiital series, a complete autopsy was performed in the case of every animal that died lIefore the end of the observation period.
wiis calculated by averaging tlic values in all clearance periods. Series II: DMSO perfusion and autotransplantation with immediate contralateral nephrectomy. The experiments of series II were performed in order to determine if either the presence of a contralateral kidney or the induction of mannitol diuresis immediately post-transplantation had an effect ‘on the viability or life-supportive capability of the kidneys. In series II, contralateral nephrectomy was performed at the time of removal of the experimental kidney for perfusion. In this series, four treatment groups were studied: Kidneys were either exposed or not exposed to Dl\/ISO during perfusion; and animals received a postreimplantation infusion of either mannitol (20 mg,/min/kg ) in saline, or of lactated Ringers solution; both solutions were infused at a rate of 0.2 ml/min/ kg. Six animals were included in each of the groups, treated as follows: group A: perfused with DMSO-free medium but not infused with mannitol after reimplantation; group 13: perfused with DMSO and infused with mannitol; group C: perfused with DblSO-free medium and infused with mannitol; and group D: perfused with DMSO, but not infused with mannitol. The protocol for addition and removal of DMSO, and the rate and duration of mannitol infusion were the snmc as in series I, but the mannitol infusion solution did not contain inulin or PAH. Following autotransplantation, the only measurements made in the animals of series II were serial determination of plasma urea and creatinine: daily for the first week, and biweekly for the next 3 weeks. The order in which animals were assigned to the various treatment groups was dictated by a stratified random design. Determination of DMSO concentration. In experiments in which [3H] DMSO was added to the unlabeled compound (either scrics I or series II), samples of perfusate
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and removal of D_\ISO. In addition, in many cxpcriments of scrics II in which [“H] DR,fSO was used, the contralatcral and experimental kidneys were perfused simultaneo~~sly; but the contralateral kidney, removed cithcr at 100 min of perfusion (peak D&IS0 concentration ) or at the end of perfusion (minimum DMSO concentration), was used to dctermine tissue DMSO. Ten samples each (100 to 200 mg, wet weight) of outer cortex, inner cortex, and medulla, representing at least three widely separated regions of the organ, were weighed and digested in Soluene-100 (Packard Instrument Co., Downers Grove, Ill.). Also, samples (about 500 mg) of each of the three anatomical levels were weighed before ancl after drying at 100°C for 15 hr in order to estimate tissue water content. In every experiment of series II employing [“HI DMSO, all urine produced during the 140 min following revascularization of the kidney was collected in order to dcterminc the amomlt of DMSO that could be rccovered. DMSO concentration in perfusate, tissue digest, or urine was calculated from the radioactivity observed in a liquid scintillation counting system, after correction for quenching, and was based on the radioactivity present in the undiluted DMSO. Statistical analysis. In series I, the effect of perfusion in either the DMSO or control groups was assessed as the difference bctween the function of the perfused kidney and that of its nonperfused mate, and was tested for statistical significance by means of a paired t test. The influence of DMSO was assessed by comparing the effect of perfusion in the DMSO group with that in the control group, using a group t test. In series II, peak urea or creatinine levels in the four treatment groups were subjected to analysis of variance to assess the effects of the two variables.
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Statisticd ~u~ul~~.st~s wcw p(~+ort~~~d ;tc’cording to the nicthotls gi~~;n 1,~ Sncd(0x (25). The criterion for rcjcctiou of the null hypothesis was a value of P S 0.05. Unless otherwise stated, numerical values given in the text arc lllfallS 2 stalKlarcl ~‘1’1‘01’ of the 111ca11.
RESULTS
Series I. Survival of animals in the DMSO-treated group of series I was not substantially different than in the control group. Of six control animals, four survived the full 30&y study period without an) sign of illness. One animal died on the fifth day post-transplantatioll due to failure of
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111~renal artt~rial anastonlosis. A110tht:r alli1na1 was sacrificed whm it was found that its pcrfnsd kidney had ccascd to function as a result of a renal venous thrombosis. The thrombosis was probably due to damage caused bv repeated catheterizatioii, \vhich was performed in an attempt to cstimate renal blood flow from the measured extraction of PAR. Such catheterizations wc’rc discontinued after being performed in the first two animals of each group, and no further incidence of venous thrombosis was O1~servt’d* In the DhlSO group, only two animals survived for 30 days. One was sacrificed after 7 da!5 bccansc of venous thrombosis.
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