The Urinary Concentrating Defect

in the Gunn Strain of Rat

ROLEOF BILIRUBIN

N. BRANSONCALL and C. CRAIG TISHER

From the Division of Nephrology, Department of Medicine and the Departmentof Pathology, Duke University Medical Center, Durham, North Carolina 27710

A B S T R A C T The role of high serum and tissuelevels of unconjugated bilirubin in the pathogenesis ofthe impaired urinary concentrating ability was investi-gated in homozygous (fi) Gunn rats with the con-genital absence of hepatic glucuronyl transferase. Con-tinuous phototherapy with blue fluorescent lights at awave length of 460 nm or oral cholestyramine feedingor both reduced serum levels of unconjugated bili-rubin to levels consistently below 3.0 mg/100 ml forseveral weeks in both weanling and adult fi Gunnrats. The renal concentrating defect was already presentin weanling jj Gunn rats by 21 days of age. In treatedweanling jj animals, maximum concentrating abilityand the concentration of urea and nonurea solutes inthe papilla and medulla, determined after 24 h of fluiddeprivation, were normal when compared to unaffectedheterozygous (Jj) littermates. Solute-free water re-absorption which is reduced in jaundiced fi Gunn ratswas restored to normal in treated weanling ji rats.The tissue concentration of unconjugated bilirubin wasreduced throughout the papilla and inner and outermedulla in the treated jj rats in comparison with un-treated jj littermates. The defect in urinary concen-trating ability was only partially reversible and some-times irreversible in adult ji rats, probably because ofpermanent renal parenchymal damage occurring sec-ondary to massive crystalline deposits in the papillaand medulla. It is concluded that unconjugated bili-

Dr. Tisher is the recipient of Research Career Develop-ment Award HL 440074 from the U. S. Public HealthService.

Dr. Call's present address is: University of Utah Medi-cal Center, University of Utah, Salt Lake City, Utah.

Received for publication 17 July 1974 and in revised formn2 October 1974.

rubin is directly involved in the pathogenesis of theconcentrating defect in jaundiced ji Gunn rats.

INTRODUCTIONThe chronically-jaundiced homozygous Gunn rat (jj),a mutant of the normal Wistar strain, has a persistentunconjugated hyperbilirubinemia secondary to the con-genital absence of the hepatic enzyme glucuronyl trans-ferase (1-4). Urine concentration at both high andlow rates of osmolar clearance has been found to beimpaired in these animals (5, 6). A causal relationshipbetween the hyperbilirubinemia and the renal defecthas been postulated and several observations indirectlysupport such a relationship. In those animals with im-paired urine concentrating ability, tissue concentrationsof bilirubin are markedly elevated throughout the kid-ney, especially in the papilla (4-7). In the adult jjanimals, massive crystalline deposits of bilirubin arefound in the renal papilla and are often associatedwith papillary necrosis (8). Concentrations of ureaand nonurea solutes are greatly reduced in the medullaof adult jj rats when compared to normal controls(5). Both free water clearance (CH20) ' and solute-free water reabsorption (TcH20) are reduced in adultji animals when compared to normal controls (5, 6).This indirect evidence has been used to support thehypothesis that unconjugated bilirubin damages thetransport capacity of the ascending limb of Henle,thereby altering the countercurrent system, thus lead-ing to the observed defect in urine concentration (5, 6).

1Abbreviations uscd in this paper: CH2o, free waterclearance; COSm, osmolar clearance; TcH20, solute-free waterreabsorption; Unax, maximum urine concentrating ability.

The Journal of Clinical Investigation Volume 55 February 1975-319-329 319

The purpose of the present study was to criticallyexamine the role of unconjugated bilirubin in thepathogenesis of the urine concentrating defect in thehomozygous jj Gunn rat. Serum levels of unconjugatedbilirubin were lowered to near normal levels in bothweanling and adult animals for prolonged periods oftime through the combined use of phototherapy (9-11)and oral cholestyramine administration (12). In thetreated jj rats, maximum urine concentrating abilityand the concentration of urea and nonurea solutes inthe medulla, determined after 24 h of fluid deprivation,were normal. TCH20 was restored to normal and theconcentration of bilirubin was markedly reducedthroughout the renal medulla. Thus, according to allmeasured parameters, the defect in urine concentratingability was completely reversed with reduction in serumand tissue levels of bilirubin, directly implicating un-conjugated bilirubin as the nephrotoxic agent in thisexperimental animal model.

METHODSOffspring of jj male and heterozygous (Ji) female Gunnrats obtained from our own breeding colony were employedin all experiments. The animals ranged in age from 21 daysto 8 mo. Except where otherwise indicated, the animalswere allowed free access to standard rat chow (RalstonPurina Co., St. Louis, Mo.) and tap water. Animals treatedwith phototherapy were shaved and placed in special cagesequipped with overhead blue fluorescent lights (F20T12/BB, Westinghouse Electric Corp., Fairmont, W. Va.) withan illumination of 1,000 MW/cm' at a wavelength of 460nm. The animals were illuminated continuously day andnight throughout the period of experimentation. Oral cho-lestyramine (Questran, Meade Johnson Laboratories, Evans-ville, Ind.) was fed to certain animals to lower the serumbilirubin concentration. The animals received standard ratchow ground to a fine powder to which cholestyraminewas added in a concentration of 5% by weight. Otheranimals received both modalities of therapy simultaneously.Base-line measurements of serum bilirubin2 and maximumurine concentrating ability (Umax), the latter determinedafter 24 h of fluid deprivation, were obtained in all animalsupon their entry into the study. In most animals, repeatdeterminations of serum bilirubin and Umax were obtainedweekly throughout the duration of the study, unless other-wise indicated.

Effect of phototherapy on urine concentrating ability inweanling jj Gunn rats. The ability of phototherapy toreverse the unconj ugated hyperbilirubinemia and preventthe defect in urine concentration was studied in male andfemale weanling ji Gunn rats. All of the offspring fromfive pairings of ij Gunn males and Ji Gunn females were

2 Initially, total and direct reacting serum bilirubin con-centrations were determined in the ji rats. The differencebetween the two measurements represents the fraction ofunconjugated bilirubin present in the serum. However, sincedirect reacting bilirubin represented less than 2% of thetotal serum bilirubin concentration, the total serum bilirubinconcentration was used as a measure of unconjugated bili-rubin in all subsequent experiments unless otherwise in-dicated.

weaned at 21 days and base-line U.a. and serum bilirubinconcentrations were determined. The ii animals from eachlitter were randomly divided into two groups; one group(it = 9) was subjected to continuous phototherapy whilethe second group (n = 14) served as untreated ji controlanimals. Ten Jj littermates which have normal serum bili-rubin concentrations and do not exhibit hyposthenuria weremaintained as normal Ji control animals. Weekly measure-ments of serum bilirubin and Uma. were obtained in thetwo groups of ji rats and Umax only in the JI animals fora period of 20 wk.

Effect of cholestyramine on urine concentrating abilityin weanling ij Gunn rats. The ability of oral cholestyra-mine to reverse the unconjugated hyperbilirubinemia andthe urine concentrating defect was next evaluated in fourweanling ji Gunn rats. Base-line determinations of serumbilirubin and Umax were obtained and the animals werethen allowed free access to standard rat chow containing5% cholestyramine by weight. Measurements of serumbilirubin and Umax were obtained weekly for a period of20 wk.

Effect of phototherapy and cholestyramine on urine con-centrating ability in weanling ji Gunn rats. The effective-ness and feasibility of combined therapy was studied in23 weanling ji Gunn rats subjected to both phototherapyand oral cholestyramine feeding for a period of 20 wk. 22ji Gunn rats served as untreated controls and 23 JI rats asnormal controls. The usual pretreatment and periodicmeasurements of serum bilirubin and Uma. were determinedin the three groups of animals.

Effect of phototherapy and cholestyramine on urine con-centrating ability in adult ji Gunn rats. To determine thereversibility of the urine concentrating defect in adult jiGunn rats, six chronically jaundiced jj female rats werechosen at random from the colony and subjected to 20 wkof continuous phototherapy and oral cholestyramine feed-ing. Weekly measurements of serum bilirubin concentrationand Uma. were obtained for comparison with similar pre-treatment values. The animals were sacrificed at the con-clusion of the period of observation by stunning and thekidneys were examined grossly.

Measurement of tissue solute and bilirubin concentration.To determine the completeness of the correction of theurine concentrating defect in the weanling ji Gunn rat,urea and nonurea solute concentrations were determinedin the kidneys of 11 weanling ji Gunn rats treated witheither phototherapy alone or both phototherapy and oralcholestyramine. Ten weanling ji Gunn littermates servedas untreated controls and eight Ji littermates served asnormal controls. Weekly measurements of serum bilirubinand Umax were obtained in the two groups of ji Gunn ratsand Umax only in the JI animals. After 24 h of fluid de-privation and the attainment of a urine sample, each animalwas sacrificed by stunning. The left kidney was quicklyexcised and samples of tissue were immediately removedfrom the papilla, inner medulla, outer medulla, and cortexand placed in thin preweighed polyethylene dishes whichwere precooled to -80'C on dry ice. Sample size wasdetermined by tare weight. The frozen tissue was thentransferred into a small known volume of cold deionizeddistilled water and homogenized in preparation for mea-surement of sodium, potassium, urea, and NH3 content.Small samples of tissue were also removed from the same

regions of the kidney in some animals for the determina-tion of tissue water content. The samples were dried at1100F for 1 wk and weighed by tare to constant weight.

Tissue bilirubin content was determined on the contra-

320 N. B. Call and C. C. Tisher

lateral kidney of six untreated j5 Gunn rats and seventreated j5 Gunn rats previously utilized for tissue soluteanalysis. The kidney was bisected and small pieces of tissuewere removed from the papilla, inner medulla, outer me-dulla, and cortex. Sample size was determined by tareweight. The samples were then homogenized for approxi-mately 2 min in a solution containing 2 ml of chloroformand 1 ml of 0.01 N HC1. The sample was then centrifugedat 3,000 rpm for 10 min and the bilirubin content wasmeasured in the supernatant layer.

Determination of TcH20 in j5 and J1 rats. T0H2o wasmeasured in four treated j5 Gunn rats, five untreated j5Gunn rats, and three normal J1 littermates. Upon intro-duction into the study, all animals were 21 days old. Thetreated j5 animals received continuous phototherapy andoral cholestyramine as described above. All j5 rats werefollowed with weekly measurements of serum bilirubin con-centration and Urn.. Urns only was determined just beforeacute experiments in the J1 rats. All rats were deprivedof food, but allowed free access to water during the nightpreceding the study. On the evening preceding the study,each animal received a subcutaneous injection of 1 U ofvasopressin (Pitressin tannate in oil; Parke, Davis & Com-pany, Detroit, Mich.). The rats were lightly anesthetizedwith sodium pentobarbital (32 mg/kg body wt), andpolyethylene catheters were inserted into the femoral ar-tery and vein. A polyethylene 160 catheter was insertedthrough a small suprapubic incision into the urinary blad-der. Estimated surgical blood loss was replaced with iso-tonic saline (approximately 2 ml). The animals were thenplaced in a restraining cage and allowed to awaken. Meanarterial pressure was monitored throughout the entireprocedure with a Sanborn 267 BC pressure transducerconnected to a Sanborn recorder (Hewlett-Packard Co.,Waltham Div., Waltham, Mass.). After completion ofsurgery, each animal received an i.v. inulin prime suf-ficient to achieve a plasma concentration of approximately50 mg/100 ml. During the initial hour of the experiment,each animal received two separate i.v. infusions: a 1.2%saline solution administered initially at a rate of 0.02 ml/min and a 0.85% saline infusion administered at a constantrate ranging between 0.0123 and 0.0185 ml/min dependingon the weight of the animal. The latter infusion containedaqueous vasopressin (25 mU/rnl) and inulin sufficient tomaintain a blood level of approximately 50 mg/100 ml.After a 60-min period of equilibration, the 1.2% saline in-fusion was increased to 0.1 ml/min and three 20-min urinespecimens and midpoint blood samples (200 ul/sample)were collected for determination of inulin clearance, urineflow, and urine osmolality. The infusion of 1.2% salinewas gradually increased in a stepwise manner to levelsas high as 2.3 ml/min so that the infusion rate always ex-ceeded urine flow. Two or more urine and blood collec-tions were obtained at each level of infusion.

Analytical methods. Inulin concentrations in plasma andurine were measured by the anthrone method of Fiihr,Kaczmarczk, and Kruittgen (13). The concentration ofsodium and potassium in urine, blood, and tissue homoge-nates was determined by standard flame photometry. Allosmolalities were determined by the method of freezing-point depression with an Advanced osmometer (AdvancedInstruments, Inc., Needham Heights, Mass.). Urine vol-umes were determined gravimetrically. Serum concentra-tions of bilirubin were determined with an ultramicroanalytical system (model 150, Beckman Instruments, Inc.,Fullerton, Calif.) adapted from the method of Malloy andEvelyn (14). Bilirubin measurements in supernates of tissue

homogenates were also performed with a modification ofthe method of Malloy and Evelyn (14). Urea and am-monia measurements were determined with a Uni-test kit(Hyland Div., Travenol Laboratories, Inc., Costa Mesa,Calif.), which employs a modification of a method describedby Chaney and Marbach (15).

Statistical methods. Where applicable, data have beenanalyzed by Student's t test. Data are expressed as mean+standard error unless indicated otherwise and P valuesgreater than 0.05 are considered to be nonsignificant.

RESULTS

Effect of phototherapy on Um,. in weanling ji Gunnrats. The effects of phototherapy alone on reducingthe concentration of serum bilirubin and increasingUma1 in jj Gunn rats are depicted in Table I and Fig.1. The average pretreatment serum bilirubin concen-tration was 11.2±0.8 mg/100 ml in treated jj rats(n = 9), 12.3±0.4 mg/100 ml in untreated jj rats(n = 14), and 0.46±0.02 mg/100 ml in the jj litter-mates (n = 10). The difference in serum bilirubinconcentrations between Ji and jj animals was highlysignificant (P < 0.001). The mean maximum urineosmolality obtained after 24 h of fluid deprivation inthe same three groups of animals was 1,467±108,1,506±+152, and 2,498±+129 mosmol/kg H20, respec-tively. Again differences between Jj and jj rats werehighly significant (P < 0.001). Thus, contrary to aprevious report (7), the jaundiced weanling jj ratsalready had a defect in maximum urine concentratingability at 21 days of age when compared to their nor-mal heterozygous littermates. The average serum bili-rubin concentration throughout the 20-wk period ofobservation fell to 2.1±0.06 mg/100 ml in the treatedjj rats and remained elevated at 7.0±0.10 mg/100 mlin the untreated jj littermates (P < 0.001). In thetreated jj animals the mean maximum urine osmo-lality over the 20-wk period of observation was 2,531±46 mosmol/kg H20, which compared quite favorablywith a mean value of 2,765±52 mosmol/kg H20achieved by the Jj littermates. Umas in the untreatedjj littermates fell slightly from the initial values of1,506+152 to 1,322±32 mosmol/kg H20. Fig. 1 de-picts the typical course of the treated and untreated j5rats during the 20 wk of observation. Within 1 wkafter initiation of phototherapy, the elevated serumbilirubin concentration had fallen to less than 2.5 mg/100 ml, but the defect in Um11 was only partially cor-rected. After 2 wk of continuous phototherapy, how-ever, the treated j5 rats could attain a Uma1 within thenormal range. In contrast, Umax in the untreated ijrats continued to slowly decline over the 20-wk period.The average serum bilirubin concentration in theuntreated ii rats declined early to approximately 7.0mg/100 ml where it remained.

Effect of cholestyramine on urine concentrating abil-

Reversibility of Urinary Concentrating Defect in Gunn Rats 321

TABLE IResponse of Weanling jj Gunn Rats to Phototherapy, Cholestyramine, or Combined Therapy*

Serum bilirubin concentration Urine osmolalitytNo. of

Therapy animals Before therapy During therapy Before therapy During therapy

mg/100 ml mosmol/kg H20Phototherapy

jj treated 9 11.2 40.8 2.1 ±0.06 1,467±108 2,531446P < 0.001¶ P < 0.001

jj untreated 14 12.3 40.4 7.0 ±0.10 1,5064152 1,322432NS§ P < 0.001§ NS§ P < 0.001§

Jj control 10 0.46±0.02 0.63±0.04 2,498±129 2,765452P < 0.001§ P < 0.001§ P < 0.001§ P < 0.01§

Cholestyraminejj treated 4 10.8 40.3 3.3 ±0.1 1,4744124 2,330±86

NS§ P < 0.001§,jj,¶ NS§ P < 0.05§,I1l; P < 0.01¶

Combined therapyjjtreated 23 11.3 ±0.7 1.6 ±0.02 1,380464 2,635±69

P < 0.001§,¶ NS§; P < 0.001¶jj untreated 22 10.5 40.2 6.7 40.3 1,363±88 1,099449

NSII P < 0.00111 NSII P < 0.0111

Jj control 23 0.4540.09 0.4840.03 2,4454161 2,552±4135P < 0.00111 P < 0.00111 P < 0.00111 NSII

* ±Standard error of the mean.Determined after 24 h of fluid deprivation.

§ Compared to treated jj rats receiving phototherapy only.Compared to treated jj rats receiving combined therapy.

¶ Compared to pretreatment value.

ity in weanling jj Gunn rats. With initiation of oralcholestyramine therapy, the mean serum bilirubin con-centration fell from a pretreatment level of 10.8±0.3mg/100 ml to a mean value of 3.3±0.1 mg/100 ml

15

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FIGURE 1 SeriAl measurements of unconjugated serumbilirubin and Umax in treated and untreated weanling jiGunn rats over a period of 20 wk. Treated animals wereexposed to continuous phototherapy. Brackets indicate +SEof the group.

over a 20-wk period (P < 0.001) (Table I). Withlowering of the serum bilirubin concentration, meanUmax rose from a pretreatment value of 1,474±124mosmol/kg H20 to a mean value of 2,330+-86 mosmol/kg H20 over the same period of time (P <0.01). Incomparison with phototherapy alone, significant dif-ferences in the response of the serum bilirubin level(P <0.001) and Umas (P <0.05) were noted duringcholestyramine feeding, despite the fact that pretreat-ment values for serum bilirubin and Umax were not sig-nificantly different in the two groups. Thus, oral cho-lestyramine administered as 5% of the total weight ofthe dietary intake was not as effective as continuousphototherapy in reducing the serum bilirubin concen-tration and reversing the defect in urine concentrationat low rates of osmolar clearance.

Effect of phototherapy and cholestyramine on urineconcentrating ability in weanling jj Gunn rats. Thecombination of oral cholestyramine and phototherapyproved to be more effective than either modality alonein maintaining a reduced level of unconjugated serum

bilirubin in the weanling jj rats (Table I). Serumlevels of unconjugated bilirubin averaged 1.6±0.02

322 N. B. Call and C. C. Tisher

TABLE I IResponse of Adult jj Gunn Rats to Cholestyramine and Phototherapy*

Age at

Serum bilirubinconcentration

Urine osmolalitytAnimal start of Before During

no. therapy therapy therapy Before therapy During therapy

mO mg/100 ml mosmol/kg H20

48 8 7.4±0.2 2.4--0.1 777-+38 5274-22 P < 0.001§49 8 9.0±-0.6 2.9±40.2 917--43 1,011±-31 P < 0.05§68 4 7.6±0O.1 2.9±-0.2 1,240±-61 1,560±-61 P < 0.005§69 4 9.3±-0.3 2.9±-0.2 1,093±-191 2,074±-89 P < 0.001§70 4 8.0±-0.4 2.9±-0.2 1,306±-152 1,958±-113 P < 0.005§75 3 8.1±-0.4 3.0±-0.1 1,240±E201 1,715±168 P < 0.025§

P <0.001§

* ±Standard error of the mean.Determined after 24 h of fluid deprivation.

§ Compared with pretreatment values.

mg/100 ml in rats receiving combined therapy versus2.1+0.06 mg/100 ml in rats receiving phototherapyalone (P < 0.001) and 3.3+0.1 mg/100 ml in ratsgiven cholestyramine alone (P < 0.001). There wasno significant difference between Um.. achieved inanimals on combined therapy versus phototherapy alone(P = NS); however, a significant differences in U.,,was observed between animals receiving combinedtherapy and oral cholestyramine alone (P <0.05).

Effect of phototherapy and cholestyramine in adultjj Gunn rats (Table II). The pretreatment serumbilirubin concentration in the six adult jj rats rangedbetween 7.4±0.2 and 9.3±0.3 mg/100 ml. The pre-treatment Um.. in the four animals that were 3-4 moof age varied between 1,093±191 and 1,306+152 mos-mol/kg H20 compared to 777±38 and 917±43 mosmol/kg I-LO in the two animals that were 8 mo of age.After initiation of therapy all six animals showedsignificant decreases in serum bilirubin levels (P <0.001), which were comparable in degree, but a modestimprovement in Um.. was noted in only one of the twoolder animals, while all four of the younger animalsdemonstrated significant though partial improvementfrom their pretreatment values (see Table II). Oneof the older animals (no. 48) actually demonstrated adecline in U... over the period of therapy (from 777±38 to 527±22 mosmol/kg, P <0.001).

Gross examination of the kidneys in these animalsprovided a possible explanation for the difference inresponse. Despite treatment, bilirubin deposition inthe papillae of both of the older animals was still quiteevident and was associated with varying degrees ofdestruction of the papilla and inner medulla. In addi-tion, mild to moderate hydronephrosis was also evi-dent in the kidneys of one of the older animals andwas associated with a calculus (Fig. 2). Marked inter-

stitial scarring which often extended from the medullato the cortical surface was also present. In contrast,crystalline bilirubin deposits were much less obvious in

FIGURE 2 Gross appearance of the left kidney of an 8-mo-old jj Gunn rat (no. 48) demonstrating severe destructionof the renal papilla and inner medulla and deep corticalscarring. A large calculus (below) filled the entire renalpelvis. Magnification X 3.5.

Reversibility of Urinary Concentrating Defect in Gunn Rats 323

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TABLConcentration of Tissue Bilirubin in Treated and Untreated jj Gunn Rats*

Bilirubin concentration

Inner OuterGroup Papilla medulla medula Cortex

jug/mg tissue

Untreated jj rats (n = 6) 5.19A2.50 0.12±0.07 0.13:40.06 0.04±0.005

Treatedjj rats (n = 7) 0.32±0.15 0.04±0.01 0.03±0.004 0.044±0.010P < 0.05 P < 0.10 P < 0.10 P < 0.30

* Mean4standard error.

the younger adult animals receiving treatment andstructural abnormalities in the cortex and medullawere less severe than in the older animals. Thus, itwould appear that permanent structural damage occursafter prolonged elevation of serum and tissue concen-trations of bilirubin in adult animals which becomesincreasingly severe with age, finally reaching a pointat which the defect in Um8. is no longer reversibledespite prolonged lowering of the serum bilirubin con-centration.

Tissue urea and nonvurea solute concentrations. Theresults of the tissue solute analyses are shown inTable III. Total tissue osmolality (calculated as 2[Na'+ K+ + NH3] + urea) was significantly reduced in therenal papilla and inner medulla of untreated jj Gunnrats when compared to values obtained in normal Jjlittermates (P < 0.001). The decreased tissue osmo-lality was due to a significant reduction in the con-centration of both sodium and urea (P < 0.001).Highly significant differences in total tissue osmolalityand sodium and urea concentrations were also notedbetween untreated and treated weanling jj rats in thepapilla, inner medulla, and outer medulla (P <0.01 orgreater). With treatment the reduction in the con-centration of sodium and urea and total osmolality inthe papilla and inner medulla of ji rats was restored tothose levels obtained in the normal Jj littermates(treated jj vs. Jj littermates; P = NS). Thus, thereduction in the serum concentration of unconjugatedbilirubin with the resultant correction of the defect inUma during treatment was accompanied by a normaliza-tion of the urea and nonurea solute profile in the papillaand inner medulla of the jj Gunn rats when comparedto their normal Jj littermates. The failure to demon-strate a difference in total tissue omolality betweenuntreated ij rats and their Jj littermates in the outermedulla may reflect a sampling error. In both un-treated and treated jj Gunn rats the anatomical boun-daries of the outer and inner medulla are less distinctthan in Jj rats. It is quite possible that samples fromthe outer medulla actually included small amounts of

tissue from the inner medulla as well, sufficient to raisethe concentration of sodium and urea and hence thetotal tissue osmolality in both treated and untreatedjj rats. It should be noted, however, that a significantdifference in total tissue osmolality and sodium andurea concentrations did exist in the outer medulla oftreated versus untreated jj rats (P < 0.01 or greater),thus demonstrating the effectiveness of reducing theserum and tissue concentration of bilirubin in theseanimals. The concentrations of NH8 and potassium inthe papilla, inner medulla, and outer medulla were com-parable in the three groups of animals (P = NS).

Tissue bilirubin concentration. The bilirubin con-centrations measured in the papilla, inner and outermedulla, and cortex in seven treated and six untreatedjj rats are shown in Table IV. In all regions of thekidney except the cortex there was a reduction in theconcentration of bilirubin in the treated animals; how-ever, only in the papilla where the mean concentrationwas 5.2 tkg/mg of wet tissue in untreated rats versus0.32 /sg/mg of wet tissue in treated animals did thedifferences become statistically significant (P < 0.05).The wide range of values obtained in these analyses,especially in the papilla and inner medulla of the un-treated rats was due in part to the problem of lossof bilirubin deposits during tissue harvesting. Thelarge aggregates of bilirubin in the papilla and innermedulla were easily dislodged from the tissue andwhen this occurred the fragments of bilirubin were notincluded in the analyses, thus falsely lowering the finalvalues.

TcH.O formation in treated and untreated jj Gunnrats and Jj littermates (Table V and Fig. 3). Therelationship between TCH2o and osmolar clearance(C08m) in the three groups of animals is depicted inFig. 3. Within all ranges of C08m, TcH20 was signifi-cantly less in untreated jj rats when compared totheir normal Jj littermates. In contrast, no significantdifference was noted in T'H2o at any level of C0.mbetween treated ji rats and their normal Jj littermates.

Glomerular filtration rate determined during maxi-

Reversibility of Urinary Concentrating Defect in Gunn Rats 325

TABLE VTCH20 Formation in Treated and Untreated jj Gunn Rats and Jj Littermates*

Range of CoW(pl/min)

0-200 200-400 400-600 600-800

Group CoSm TCH20 C00. T'H20 CoM TCH2o Coem TCH2O

pil/min P/min pi/min pi/minNormal Jj rats 124.0±18.6 95.01:12.4 303.1±23.1 169.0:19.9 509.61:14.1 200.8±6.2 686.3416.3 218.3±13.8

(n = 3) (n =6) (n= 9) (n = 13) (n =7)

Untreated jj rats 94.0 16.6 52.7 ±8.8 278.0-±14.1 113.5 ±7.3 494.7 ±13.3 148.7-4 10.0 680.9 ± 13.9 149.3 12.6(n = 5) (n= 15) (n= 15) (n = 17) (n= 13)

NS$ P < 0.01$ NS* P < 0.0005t NS* P < 0.0005: NSJ P < 0.0005*Treated jj rats 152.1 ±13.5 109.7 ±8.5 305.6416.2 160.4±4.8 513.9±18.9 194.9+4.2 703.8 ±4.2 202.643.8

(n = 4) (n= 7) (n= 19) (n = 13) (n =12)NSt NS$ NS* NS* NSt NS* NS* NS*

* MeanLstandard error.$ Compared to normal Jj rats.

mumTCH20 formation, that is at Co.m of 600-800 A1/min, was similar in the three groups of animals andaveraged 2.60±0.11 ml/min in Jj rats versus 2.43±0.17 ml/min in treated jj rats (P = NS) and 1.84±0.16ml/min in untreated jj rats (treated versus untreatedjj rats, P < 0.05). The lower GFR in untreated jjrats can probably be ascribed to the loss of renal paren-chyma secondary to severe cortical and medullaryscarring which was observed on gross inspection ofthe kidneys of the untreated jj animals.

DISCUSSIONFour findings of significance can be derived from thedata compiled in the present study. First, as previouslysuspected, unconjugated bilirubin can be directly im-

HETEROZYGOUSCONTCO'C(n -3)

200- _= HOMOZYGOUSTREATED(n .4)

I/mn0 HOMOZYGOUSUNTREATED

100/,,

200 400 600 800

C,,,, uLI/min

FIGURE 3 Comparison of the relationship between TCH2oand Co.m in heterozygous Jj Gunn rats and treated anduntreated jj Gunn rats. Each point represents the mean ofall values for T'H2o and Cosm that fell within each of thefour ranges of Comm as demarcated by the broken verticallines. The vertical and horizontal bars through the pointsrepresent + 1 SE of mean T'H2o and Cosm, respectively. Dataused to derive the curves are presented in Table V. At allranges of Cosm, TeH2o was depressed in untreated jj Gunnrats (lower curve). With treatment (middle curve), TCH2owas comparable to that measured in normal heterozygouslittermates (upper curve).

plicated in the urine concentrating defect previouslydescribed in the Gunn strain of rat. Second, the defectin urine concentration is only partially reversible andsometimes irreversible in adult jj Gunn rats, probablybecause of secondary parenchymal destruction of therenal medulla. Third, the urine concentrating defect isalready present in weanling jj Gunn rats by 21 daysof age, contrary to a previously published report (7).Fourth, a combination of phototherapy using blue fluo-rescent lights and oral cholestyramine feeding is themost effective means presently known to lower serumand tissue concentrations of unconjugated bilirubin inthe jaundiced jj Gunn rat.

The results of the present study provide direct evi-dence that increased concentrations of unconjugatedbilirubin in the serum and tissue are causally related tothe defect in urine concentration in the ji Gunn rat.Although earlier studies had documented an associa-tion between hyperbilirubinemia and the renal impair-ment, a direct cause-and-effect relationship had notbeen established (2, 5, 6). In the present study, aprolonged reduction in the serum bilirubin level inweanling jj Gunn rats, induced through the use ofphototherapy or oral cholestyramine or both, was asso-ciated with reversal of the impairment in urine con-centration and TCH20 formation. The reduced concen-trations of urea and nonurea solutes in the papilla andinner and outer medulla of untreated jj Gunn rats wererestored to normal in treated jj littermates. The low-ering of the hyperbilirubinemia with the attendant nor-

malization of renal function was associated with a

marked reduction in the concentration of bilirubin inthe papilla and medulla. Thus, a direct cause-and-effectrelationship appears well established.

The defect in urine concentrating ability was onlypartially reversible in young adult jj Gunn rats and es-

326 N. B. Call and C. C. Tisher

sentially irrevessible in older adult ii animals despitethe fact that the concentration of serum bilirubin waslowered to levels comparable to the weanling jj ratsin which the defect was totally reversible (Table II).Despite therapy, gross examination of the kidneys re-vealed persistent deposits of bilirubin in the papilla andinner medulla which were associated with varying de-grees of parenchymal damage. Often the damage wasso severe, especially in the oldest animals, that hydro-nephrosis of one or both kidneys was evident as wellas extensive scarring which extended from the medullato the cortex. Investigators have previously describedextensive bilirubin deposition in untreated adult jj ratswhich was frequently associated with papillary necro-sis (8). In the 3- and 4-mo-old animals in which onlypartial correction in the renal impairment was ob-served, definite, albeit less extensive parenchymal de-struction and bilirubin deposition was evident. Thus,at some point in time between 21 days and 3-4 moof age irreversible parenchymal damage occurs andappears to be slowly progressive in nature in the ab-sence of therapy directed toward the reduction ofserum and tissue concentrations of bilirubin.

In the present study it was observed that the jaun-diced weanling jj rats already had a significant im-pairment in U.ax at 21 days of age. After 24 h of fluiddeprivation, the jj animals only attained a Um.. ofapproximately 1,500 mosmol/kg H20, while their nor-mal Jj littermates without the defect achieved a U...of approximately 2,500 mosmol/kg H20 (see Table I).These results conflict with previously published ob-servations of Odell (7), who reported finding no sig-nificant difference in the urea and nonurea solute con-centrations of the inner medulla in weanling jj and Jjrats and concluded that jaundiced weanling jj rats upto 21 days of age have normal medullary function.However, Odell (7) did not determine urine osmo-

lality in the two groups of animals, which would havebeen a more sensitive method of estimating urine con-centrating ability, and this may explain, at least inpart, his failure to detect a defect in U.as in the jaun-diced weanling jj rats. Possibly of equal importancein explaining the discrepancy in results between thetwo studies is the fact that the solute analyses wereperformed on the entire inner medulla, not just thepapillary tip (7). As a result the average values forurea and nonurea solute concentrations that obtainedundoubtedly underestimated the maximum solute con-centration that existed at the papillary tip. Finally,in the present study the presence of a significant im-pairment in Unax in jaundiced weanling jj rats wasfurther supported by the finding that ii rats receivingtreatment to lower the concentration of unconjugated

serum bilirubin increased their Um.. by at least 1,000mosmol/kg H20 over a period of 2 wk.

The use of both phototherapy and oral cholestyra-mine to lower the serum concentration of unconju-gated bilirubin in the jaundiced jj Gunn rats provedto be more effective than either modality alone (TableI). The use of phototherapy alone has been employedin several studies involving ji Gunn rats (10, 11, 16-18), but renal functional parameters were never deter-mined. Phototherapy has also been used extensively inmany neonatal intensive care units to lower the concen-tration of unconjugated serum bilirubin in newbornand especially premature infants (19), thereby mini-mizing the chances for the development of kernicteruswith attendant brain damage. When the light sourceis of the proper wavelength, photodegradation of bili-rubin in the skin and cutaneous capillaries occursprobably by a process of photo-oxidation resulting inthe formation of water-soluble substances which areexcreted principally in the bile, but to a lesser extentin the urine (18). Ostrow (18, 20) has demonstratedthat in the jj Gunn rat subjected to phototherapy, thewater-soluble bilirubin derivatives are chromatographi-cally identical with those normally found in the bileunder normal fluorescent lighting conditions, althoughthe exact nature of all of the photoderivatives stillremains in doubt. The effectiveness of the therapy de-pends, at least in part, on the flux of the radiant energyincident on the sample (20, 21). Blue fluorescent lampswith wavelength emission from 420 to 475 nm are themost effective light source and the degree of photo-degradation is proportional to the energy flux emitted inthe blue region of the visible spectrum (20, 21). Ithas also been noticed that during phototherapy thereis a marked increase in the biliary excretion of un-conjugated bilirubin, an amount which accounts fornearly half of the bilirubin that is catabolized duringphototherapy (18, 20). Previous studies have demon-strated that cholestyramine, when ingested orally, isalso capable of lowering unconjugated bilirubin bybinding the pigment in the intestine, thereby prevent-ing its reabsorption (12). These two pieces of evidenceformed the basis for utilizing the combination of photo-therapy and cholestyramine feeding to maintain low-ered bilirubin levels in our animals. If previous ob-servations were correct it was anticipated that theincreased unconjugated bilirubin excreted by the biliarysystem in response to phototherapy should be bound bythe cholestyramine in the gut, thus preventing its re-absorption. Although the fecal excretion of bilirubinwas not measured, the two modalities were more ef-fective than either one alone in reducing the level ofserum bilirubin suggesting that cholestyramine did, in

Reversibility of Urinary Concentrating Defect in Gunn Rats 327

fact, increase total fecal excretion of unconjugatedbilirubin.

The present study was not primarily designed toexamine either the mechanism whereby unconjugatedbilirubin alters renal concentrating ability or the siteof the defect. However, previous studies in adult jjGunn rats have documented the presence of alterationsin both CH2o and TCH20 (6), suggesting that the pri-mary renal defect probably involves the ascending limbof Henle. As previously suggested (5, 6) bilirubincould interfere directly with active solute transportout of the ascending limb of Henle or alter the perme-ability of the segment to water. Either mechanismcould explain the impairment in CH2o and TcH20. Invitro data (22, 23) do suggest that unconjugated bili-rubin in concentrations comparable to those that havebeen measured in the papilla of the jj Gunn rat (6)are capable of uncoupling oxidative phosphorylation.However, whether the concentration of bilirubin in theouter medulla, where active solute transport occurs,is sufficient to uncouple oxidative phosphorylation isopen to question. Diamond and Schmid (24) wereunable to demonstrate uncoupling of oxidative phos-phorylation in mitochondria isolated from the brainsof guinea pigs in which the total tissue concentrationof exogenous "4C-labeled bilirubin infused into theanimals was 10.6 /g/g of tissue. In the present studythe concentration of unconjugated bilirubin was 130ug/g of tissue in the outer medulla of untreated jjGunn rats and fell to 30 Ag/g of tissue with treatment.In addition, Martinez-Maldonado, Suki, and Schenker(6) were unable to demonstrate diminished concentra-tions of ATP in the medulla of jaundiced adult jjGunn rats, suggesting that if bilirubin does interferewith solute transport in the renal medulla, the mecha-nism may not be dependent on ATP. Whatever themore acute and potentially reversible effect(s) of bili-rubin may be, it is evident from the results of the

present study that massive deposits of crystalline bili-rubin which develop with time within the papilla andouter medulla result in irreversible parenchymal de-struction including papillary necrosis, hydronephrosis,and severe medullary and cortical fibrosis. In such a

setting the defect in urine concentration is no longerreversible.

ACKNOWLEDGMENTSThe authors express their appreciation to Dr. Myron Wol-barsht, Department of Ophthalmology, Duke UniversityMedical Center, for helpful discussions during the conductof this study and to Dr. Roger Yeary, Department ofVeterinary Physiology and Pharmacology, The Ohio StateUniversity, Columbus, Ohio, who kindly supplied theoriginal breeding stock of Gunn rats for our colony. The

technical assistance of Mrs. Helen Parks is gratefullyacknowledged.

This work was supported in part by grants AM 13845and AM 10844 from the U. S. Public Health Service.

REFERENCES1. Gunn C. H. 1938. Hereditary acholuric jaundice in a

new mutant strain of rats. J. Hered. 29: 137-139.2. Gunn, C. H. 1944. Hereditary acholuric jaundice in the

rat. Can. Med. Assoc. J. 50: 230-237.3. Lathe, G. H., and M. Walker. 1958. The synthesis of

bilirubin glucuronide in animal and human liver. Bio-chem. J. 70: 705-712.

4. Schmid, R., J. Axelrod, L. Hammaker, and R. L.Swarm. 1958. Congenital jaundice in rats, due to adefect in glucuronide formation. J. Clin. Invest. 37:1123-1130.

5. Odell, G. B., J. C. Natzschka, and G. N. B. Storey.1967. Bilirubin nephropathy in the Gunn strain of rat.Am. J. Physiol. 212: 931-938.

6. Martinez-Maldonado, M., W. N. Suki, and S. Schenker.1969. Nature of the urinary concentrating defect inthe Gunn rat. Am. J. Physiol. 216: 1386-1391.

7. Odell, G. B. 1968. The response of jaundiced and non-jaundiced weanling rats to thirsting. Pediatr. Res. 2:237-242.

8. Bernstein, J., and B. H. Landing. 1962. Extraneurallesions associated with neonatal hyperbilirubinemia andkernicterus. Am. J. Pathol. 40: 371-391.

9. Cremer, R. J., P. W. Perryman, and D. H. Richards.1958. Influence of light on hyperbilirubinaemia of in-fants. Lancet. 1: 1094-1097.

10. Broughton, P. M. G., E. J. R. Rossiter, C. B. M.Warren, G. Goulis, and P. S. Lord. 1965. Effect ofblue light on hyperbilirubinaemia. Arch. Dis. Child.40: 666-671.

11. Ballowitz, L., R. Heller, J. Natzschka, and M. Ott.1970. The effect of blue light on infant Gunn rats.Birth Defects Orig. Artic. Ser. 6: 106-113.

12. Lester, R., L. Hammaker, and R. Schmid. 1962. Anew therapeutic approach to unconjugated hyperbili-rubinaemia. Lancet. 2: 1257.

13. Fiuhr, J., J. Kaczmarczk, and C. D. Kruttgen. 1955.Eine einfache colorimetrische Method zur Inulinbestim-mung fur Nieren clearance-Untersuchungen bei Stoff-wechselgesunden und Diabetrikern. Klin. Wochenschr.33: 729-730.

14. Malloy, H. T., and K. A. Evelyn. 1937. The determina-tion of bilirubin with the photoelectric colorimeter. J.Biol. Chem. 119: 481490.

15. Chaney, A. L., and E. P. Marbach. 1962. Modified re-agents for the determination of urea and ammonia.Clin. Chem. 8: 130-132.

16. Ballowitz, L. 1971. Effects of blue and white light oninfant (Gunn) rats and on lactating mother rats. Biol.Neonate. 19: 409425.

17. Butcher, R. E., C. V. Vorhees, C. W. Kindt, and W. J.Keenan. 1972. An experimental evaluation of photo-therapy for hyperbilirubinemia in the Gunn rat. Am.J. Dis. Child. 123: 576-578.

18. Ostrow, J. D. 1971. Photocatabolism of labeled bilirubinin the congenitally jaundiced (Gunn) rat. J. Clin. In-vest. 50: 707-718.

328 N. B. Call and C. C. Tisher

19. Lucey, J. F., M. Ferreiro, and J. Hewitt. 1968. Preven-tion of hyperbilirubinemia of prematurity by photother-apy. Pediatrics. 41: 1047-1054.

20. Ostrow, J. D. 1972. Mechanisms of bilirubin photo-degradation. Semin. Hematol. 9: 113-125.

21. Davies, R. E., and S. J. Keohane. 1970. Some aspectsof the photochemistry of bilirubin. Boll. Chim. Farm.109: 589-598.

22. Cowger, M. L., R. P. Igo, and R. F. Labbe. 1965. The

mechanism of bilirubin toxicity studied with purifiedrespiratory enzyme and tissue culture systems. Bio-chemistry. 4: 2763-2:770.

23. Zetterstrom, R., and L. Ernster. 1956. Bilirubin, anuncoupler of oxidative phosphorylation in isolated mito-chondria. Nature (Lond.). 178: 1335-1337.

24. Diamond, I., and R. Schmid. 1967. Oxidative phos-phorylation in experimental bilirubin encephalopathy.Science (Wash. D. C.). 155: 1288-1289.

Reversibility of Urinary Concentrating Defect in Gunn Rats 329