Human Bence Jones protein toxicity in rat proximal tubule epithelium in vivo

Kidney International, Vol. 32 (1987), pp. 851—861

Human Bence Jones protein toxicity in rat proximal tubule epithelium in vivo PAUL W. SANDERS, GUILLERMO A. HERRERA, and JOHN H. GALLA

with the technical assistance of JAMES R. HANCOCK and ROBERT L. LOTT Nephrology Research and Training Center and Division of Nephrology, Department of Medicine; Department of Pathology, University of Alabama at Birmingham; and Veterans Administration Medical Center, Birmingham, Alabama, USA

Human Hence Jones protein toxicity in rat proximal tubule epithelium in vho. To investigate the direct toxicity of human Bence Jones protein (BJP), individual nephrons of male Sprague-Dawley rats were perfused in vivo at 20 nl/min with an artificial tubule fluid (ATF) that contained no protein, a human kappa BJP (5 gIdl), or bovine serum albumin (5 gIdl), and proximal convoluted tubule function and morphology were

examined. Perfusion with BJP perfusate for 5 minutes produced no changes (P = NS) in absorption of water, J,,, (1.09 0.20 vs. 1.50 0.25 nI/mm/mm), chloride, Jci, (95 47 vs. 123 41 pEq/min/mm), and glucose, J0, (39 3 vs. 40 5 pmol/min/mm) compared to perfusions with only ATF. However, perfusion for at least 20 minutes with the same BJP perfusate produced decreased (P < 0.025) in J (0.58 0.12 vs. 1.15 0.14 nI/mm/mm) and J0 (27 3 vs. 38 3 pmol/min/mm) compared to perfusions with ATF alone; the decrease in Jci (64 47 vs. 27 pEq/min/mm) did not reach statistical significance. Perfusion 119 for 20 minutes with ATF containing albumin resulted in no changes in J (1.22 0.21 vs. 1.15 0.14 nI/mm/mm), Jci (207 29 vs. 119 27 1 vs. 38 3 pmol/minlmm), when compEq/mmn/mm), and J0 (31 pared to the ATF perfusions. Immunocytochemistry, immunofluores-

cence and immunoelectron microscopy of the BJP-perfused tubules demonstrated the kappa light-chain protein in endosomes and activated lysosomes. In addition, cellular desquamation and fragmentation, prominent cytoplasmic vacuolation, and focal loss of the microvillus border were found in the BJP-perfused tubules, but not in the albumin-perfused tubules. In conclusion, these functional and morphologic data show that a human kappa light-chain is toxic to the proximal convoluted tubule of the rat. This toxicity occurred in a time-dependent fashion when the lysosomal system was markedly activated. Direct damage of the tubule

studies from our laboratory [8, 9] and others [10—131 have shown that both acute and chronic exposure of kidneys of experimental animals to BJP's can produce tubule damage that histologically resembles the human disease [8]. The pathogenetic mechanism(s) of this toxicity of BJP's is undetermined, but there are currently at least two postulated processes of tubule injury: direct toxicity to the epithelial cells of the nephron and intratubular obstruction [14]. These mechanisms are not mutually exclusive and may produce renal damage in concert. Using in vivo single nephron perfusion in the rat and light, electron and immunofluorescence microscopy, this study investigates the hypothesis that human BJP's are directly toxic to the epithelium of the proximal convoluted tubule. Methods

Animal preparation The preparation for micropuncture was similar to the method described previously [15]. Male Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts, or Taconic Farms, Inc., Germantown, New York, USA), which weighed 230 to 360 g and were maintained on standard rat chow (Agway/Prolab 1000, Syracuse, New York, USA) and tap

epithelium by BJP's may be involved in the development of the water ad libitum, were anesthetized with thiobutabarbitol (Intubulointerstitial nephropathy associated with multiple myeloma.

actin, Promonta, Hamburg, FRG), 100 mg/kg body weight (body wt), intraperitoneally. After placement on a servo-con-

trolled heated table, tracheostomy was performed with PE-240 polyethylene tubing followed by insertion of a right external Renal failure is a major complication of multiple myeloma, jugular line using PE-50 tubing. Ringer-bicarbonate (0.15 M) occurring in about half of the patients with this disease; its with 2% polyfructosan (mutest, Laevosan, Linz, Austria) was appearance is associated with a markedly-shortened patient infused at 2.0 ml/100 g body wt/hr after a 1 ml bolus. The right survival [1—3]. Although several different renal lesions may femoral artery was cannulated to monitor arterial pressure and develop, the most commonly recognized is a tubulointerstitial to sample blood. The bladder was catheterized with PE-50 nephropathy often referred to as "myeloma kidney" [3]. This tubing for urine collections. The left kidney was exposed tubule damage may develop insidiously or acutely [4] and is through a subcostal incision and, after separation from the usually associated with the presence of urinary Bence Jones perirenal fat and adjacent adrenal gland, was placed in a proteins (BJP) [4—6], which consist of immunoglobulin light Lucite® cup. A well was formed on the surface of the kidney chains in monomeric, dimeric, and aggregate forms [7]. Several with 4% agar and was filled with water-equilibrated mineral oil. Received for publication December 10, 1986 and in revised form June 1, 1987

© 1987 by the International Society of Nephrology

Microperfusion protocol Approximately one hour after the intravenous infusion was started, arterial blood samples were collected for the determination of hematocrit, osmolality, and plasma sodium, potas851

852

Sanders et a!

sium, chloride, and protein concentrations. The micropuncture period was then begun. A pipette filled with an artificial tubule fluid (ATF) was inserted into an early surface, proximal convoluted tubule (PCT) segment and a small bolus of fluid outlined

During the microperfusion period, two 30-minute inulin clear-

NaHCO3, 5 KC1, 0.6 CaCI2, 3.1 NaH2PO4, 14.5 Na2HPO4, 5 D-glucose; this fluid was tinted with Food, Drug and Cosmetic (FD&C) Green #3 (Keystone Aniline and Chemical Co., Chi-

able for study if the arterial pressure fell below 100 mm Hg or if the inulin clearance was less than 500 j.d/min. Tubule perfusions

inserted into the earliest accessible surface proximal convolu-

ratios

ances were determined and, at the completion of micropuncture, arterial blood was sampled for determination of hematocrit, osmolality, and plasma sodium, potassium, chloride, prothe nephron. The ATF contained (in mM): 100 NaC1, 25 tein, and glucose concentrations. Rats were considered unsuit-

were accepted for study if they had: 1) calculated perfusion cago, Illinois, USA). A pipette filled with bone wax was rates between 15 and 23 nI/mm, 2) collectate to perfusate inulin 1.0,

and 3) perfused tubule lengths between 0.7 and 4.0

tion and a small cast 4 to 5 tubule diameters in length was mm. injected into the tubule with a microdrive unit (Trent Wells,

South Gate, California, USA). A pipette attached to a microperfusion pump (W-P Instruments, Inc., New Haven,

Morphologic and immunocytochemical studies Histologic examination of individual in vivo perfused neph-

rons was performed. In these experiments, a single inulin wax block and the remaining nephron was perfused at 20 clearance and packed cell volume were collected during the nI/mm. The composition of the perfusion solutions was the microperfusion period. Control and experimental perfusates same as described above except for the addition of [3H]- contained albumin, 5 g/dl, or BJP, 5 g/dl, respectively. These methoxyinulin (269.5 mCi/g, New England Nuclear, Boston, perfusates were similar to those used in the microperfusion Massachusetts, USA), which had been extensively dialyzed experiments except for omission of the [3H]-methyoxyinulin Connecticut, USA) was inserted into the tubule just distal to the

against distilled water, and a trace amount of D-['4C]glucose (257.7 mCilmmol, New England Nuclear). The experimental perfusate contained a human BJP, 5 gIdl. This concentration of BJP is in excess of the reported range of serum BJP concentration in humans who have multiple myeloma and renal failure

and D-['4C]glucose. After perfusion of the nephron distal to the

surface-proximal convoluted tubule, so the earliest to latest accessible surface convolutions were micropunctured with perfusion and collection pipettes, respectively. One to three perfusions were obtained in each animal. After the collections were complete, the perfused segments were injected with Microfil (Canton Bio-Medical Products, Inc., Boulder, Colorado, USA), the kidney was removed and dissolved in 25% NaOH, and the perfused segments were dissected and their

trimmed until the nephron that contained the India ink was identified. Successive 3 sections were cut with a microtome (Reichert Jung, Buffalo, New York, USA) and stained with hematoxylin and eosin (H&E) for light microscopy or with an avidin-biotin complex (ABC) method that used anti-kappa or

lengths measured directly with a micrometer scale. Five different groups of perfused nephrons were studied. The first experimental group (BJP5) of nephrons and a corresponding control group (ATF5) were perfused for five minutes or less. The other three groups consisted of: 1) an experimental group (BJP2O) of perfusions that used ATF containing the BJP; 2) another group (ALB2O) that consisted of tubules perfused using ATF that contained albumin (bovine albumin, fraction V, purity >96%, Sigma Chemical Co., St. Louis, Missouri, USA), that had been prepared similarly to the BJP and was present in the same concentration, 5 g/dl; and 3) control perfusions (ATF2O)

pletely.

wax block with either control or experimental perfusate for approximately 20 minutes, the tubules were perfused immedi-

ately for three to four minutes with Carson-Millonig's solution, pH 7.0, that was tinted with filtered India ink (Faber-Castell (0.3 to 3.2 g/dl) [8, 16], but was chosen to enhance the Corp., Lewisburg, Tennessee, USA). In each experiment, two possibility of detection of toxicity since the contact time (perfu- tubules were perfused with either control or experimental sion duration) was relatively short. The final pH of each perfusates, the order of which was alternated daily. The animals perfusion solution was adjusted to 7.40 by bubbling a mixture of were then sacrificed, the perfused tubules were partially dis95% 02/ 5% CO2 into the solution just before aspiration of the sected, and the remaining tissue placed in untinted Carsonfluid into the perfusion pipette. Mter perfusion of the tubule for Millonig's solution, pH 7.0. The India ink was not removed by either 5 or 20 minutes, a collection pipette filled with tinted the fixation procedure and therefore allowed accurate identifimineral oil was inserted into the latest accessible, surface cation of the nephron that had been perfused. The tissue was proximal convolution, a mineral oil block was injected, and a processed for light microscopy in standard fashion and was complete timed collection of tubule fluid was made for three to embedded in paraffin (Ameraffin, Stephens Scientific, Oak five minutes. We perfused the maximum available length of Ridge, New Jersey, USA) for sectioning. The specimens were

anti-lambda antibodies, dilution 1/5000 (Accurate Chemical and Scientific Corp. [Dakopatts], Westbury, New York) [17]. This

process was continued until the nephron was sectioned comIn another group of experiments, after perfusion of a single nephron with the BJP solution for 20 minutes, this nephron was perfused for three to four minutes With FD&C tinted ATF that did not contain BJP, so that excess BJP remaining in the lumen of the nephron was removed. The animal was sacrificed and the perfused nephron was rapidly dissected and immediately em-

bedded in Tissue-Tek Optimum Cutting Temperature compound (Miles Laboratory, Naperville, Illinois,USA) and snapfrozen in liquid nitrogen. The tissue was mounted, and 3 /L serial sections of the entire perfused nephron were carefully made and

that used ATF alone. These groups were perfused for 20 stained with fluorescein isothiocyanate-labeled anti-kappa or minutes and differed from the first two groups only in the anti-lambda antisera (Accurate Chemical and Scientific Corp). duration of perfusion before which the tubule fluid samples In the final series of experiments, after perfusion for 20 were obtained. The ALB2O group served as a control for the minutes with either control (albumin) or experimental (BJP) presence of intraluminal protein.

solutions, the tubules were immediately perfused for three to

Renal toxicity of Bence Jones protein

four minutes with either FD&C tinted, phosphate-buffered, 2% glutaraldehyde solution, pH 7.3, or FD&C tinted, Carson-Mi!lonig's solution, pH 7.0. Both fixatives also contained either 15 or 40 nm colloidal-gold particles (Janssen Pharmaceutica N.Y., Beerse, Belgium) which served as a marker for ultrastructural identification of the perfused tubule. One control and experi-

mental tubule were obtained in each experiment; their order was alternated daily. The rat was sacrificed and the tubules partially dissected and placed in either untinted, phosphatebuffered, 2% glutaraldehyde solution, pH 7.3, or untinted Carson-Millonig's solution, pH 7.0, until preparation for electron microscopy. After initial fixation, the samples were postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA) and embedded either in araldite resin (Electron Microscopy Sciences) or L,R. White resin (Polysciences Inc., Fort Valley, Pennsylvania USA). Thick sections of the plastic-embedded material were stained with toluidine blue (Fisher Scientific Co., Fair Lawn, New Jersey, USA) for light microscopy survey. Ultrathin sections were postcontrasted with uranyl acetate and lead citrate (Fisher Scientific Co.). Sections that were embedded in the L.R. White

resin were not post-fixed. After fixation, they were washed briefly in Tris buffer, pH 7.6, and were blotted. They were then

processed through several solutions of increasing concentrations of ethyl alcohol: 15 minutes in 60%, 15 minutes in 80%,

and 20 minutes in 95% (2 exchanges). In order to avoid excessive shrinkage, the tissues were also incubated in a 2:1

mixture of L.R. White/95% ethanol for one hour at room temperature. Following these procedures, the specimens were blotted and infiltrated with pure L.R. White resin overnight at 4°C. The tissues were then incubated for one hour in fresh resin, allowing further infiltration of the tissues, and the resin was polymerized over 18 to 24 hours at 50°C (temperatures greater then 60°C were avoided). The specimens were examined with a transmission electron microscope (Model H-600, Nissei Sangyo America, Ltd., Hitachi Scientific Instruments, Tokyo, Japan). The perfused tubules were identified by the presence of the colloidal gold in their lumens. Tissue for immunoelectron microscopy was prepared using two indirect immunogold staining procedures that utilized similar methodologies [18, 19]. In the first method, osmicated ultrathin sections in the araldite/epon resin were cut from the block and collected on uncoated 300 mesh nickel grids (Electron Microscopy Sciences) and dried overnight at room temperature. Grid-mounted sections were etched in 10% hydrogen

853

processed similarly. The second indirect immunogold staining technique used tissue that was fixed in the Carson Millonig's solution and embedded in the L.R. White resin. This method

was very similar to the first, except that the tissue was not osmicated and was not etched with hydrogen peroxide. In addition, the tissue was incubated with polyclonal antibodies to kappa and lambda light chains, 1:1000 and 1:2000 dilutions, for only 16 hours.

BJP preparation Using standard techniques, the protein was isolated from the urine of a patient who had had multiple myeloma and renal failure. Initially, ammonium sulfate was added to the urine to achieve a 70% saturation. The precipitate was redissolved in distilled water and then dialyzed at least 24 hours using cellulose dialysis tubing (Spectrapor Membrane Tubing, Fisher Scientific) that had a 12,000 to 14,000 dalton cutoff, against distilled water and then 0.01 M sodium phosphate buffer, pH 7.6. Ion exchange chromatography using a 2.5 x 30 cm DEAE52 column that had been equilibrated in 0.01 M sodium phosphate buffer, pH 7.6, was then employed to further purify the BJP. The protein was desalted with a G-10 column and lyophilized. Immunoelectrophoresis confirmed that the BJP was a kappa light chain. Purity of the protein was shown to be greater than 95% by electrophoresis on 7 to 209o polyacrylamide gradient slab-gels containing 0.1% sodium dodecyl sulfate (SDS-PAGE), using the discontinuous buffer system described by Laemmli [20]. The majority of the protein consisted of monomeric and dimeric forms that had molecular weights of 23,400 and 46,800, with only a small fraction present as aggre-

gates. In addition, prior to use, the protein was dialyzed extensively against distilled water at 4°C using cellulose dialysis

tubing. The isoelectric point of the kappa light chain was determined using the standard techniques described in the LKB Application Note 250.

Analytical methods Hematocrit was estimated with a microhematocrit capillary

tube reader (Spiracrit, Lancer, St. Louis, Missouri, USA).

Plasma sodium and potassium concentrations were determined with a flame photometer (Model IL-943, Instrumentation Laboratories, Inc.). Plasma chloride concentration and osmolality were determined arnperometrically (Buehler digital chloridometer, Buehler Instruments, Inc., Fort Lee, New Jersey, USA) and by vapor pressure osmometry (Wescor, Inc., Logan, Utah, peroxide for 10 minutes, washed in distilled water for five USA), respectively. Plasma protein concentration was determinutes, and then incubated at room temperature in normal mined by refractometry (American Optical Corp., Buffalo, New goat serum (Vector Laboratories, Inc., Burlingame, California, York, USA). Values obtained at the start and completion of the USA), 1:30 dilution in 0.05 M Tris-buffered saline (TBS), for 30 micropuncture period were averaged for each rat. Inulin conminutes. The grids were then incubated with polyclonal anti- centration in urine and plasma samples was determined using bodies to kappa and lambda light chains (Accurate Chemical the anthrone colorimetric method [211. Clearance of inulin (C111) and Scientific Corp.) 1:1000 and 1:2000 dilutions in TBS, for 48 was calculated from these values and was factored by body hours at 4°C, and then washed twice in TBS and once in TBS weight. Plasma glucose concentration was determined by the that contained 1% BSA, 10 minutes each. The grids were then glucose oxidase method on a model SBA-300 batch analyzer incubated for one hour at room temperature in gold-labeled (Gilford Co., Oberlin Ohio, USA). The volume of fluid collected from the PCT was measured goat, anti-rabbit IgG serum (Janssen Pharmaceutica N.Y.), 1/15 in 0.02 M TBS containing 1% BSA, pH 8.2. The samples were using constant bore glass tubing and a microslide comparator again washed twice in TBS and twice in distilled water, 10 (Gaertper Scientific Corp., Chicago, Illinois, USA). Chloride minutes each. The sections were counterstained with uranyl concentrations of the perfusate and collectate samples were acetate and lead citrate, Sections of the control tubules were determined by electrometric titration [22] (model FT-2230

854

Sanders et at Table 1. Characteristics of the perfusion groups Perfusion rate

Group BJP5 ATF5

18.8

20.1

ATF2O

18.6

BJP2O

18.5

P valuea ALB20

P valueb

1.3 1.7

0.5 0.8

1.6 2.0

0.1 0.3

107

302 2 292 5

0.2 0.3

104

1

299 4

104

1

307

NS

NS 19.0

Perfusate osmolality mOsm/kg

mm

0.8 0.6

NS

P value

Perfusate chloride mEq/li:er

Tubule length

nI/mm

0.5

NS

NS

0.2

1.9

1

107 2 NS

NS

NS

Table 2. Absorption characteristics of the perfusion groups

NS

104

1

NS

I

Number of perfusions/ rats

Group BJP5

11/6

ATF5 P value

8/5

ATF2O BJP2O

18/13

9/8

NS

P valuea

NS

P valu&'

288 4

a Comparison of data of the BJP2O and ATF2O groups b Comparison of data of the ALB2O and ATF2O groups

ALB2O

15/10

Volume absorption ni/mm/mm 1.09 1.50

0.20 0.25

Chloride absorption pEq/min/mm

95 47

123 41

NS

NS

0.14 0.58 0.12 <0.025 1.22 0.21 NS

119 27

1.15

Glucose absorption pmoI/min/mm

64 43 NS

207 29 NS

39 3 40 5 NS

38 3 27

3

<0.025 31

1

NS

a Comparison of data of the BJP2O and ATF2O groups b Comparison of data of the ALB2O and ATF2O groups

microtitrator, W-P Instruments, Inc., New Haven, Connecticut, USA). Osmolalities of the nanoliter samples were determined by freezing point depression (nanoliter osmometer, Clifton Technical Physics, Hartford, New York, USA). The [3H] and [14C] activities of measured volumes of the collected samples and perfusate were determined using a liquid scintillation counter (Model 6892, Tracor Analytic, Elk Grove Village,

mEq/liter; plasma potassium, 4.9 0.1 mEq/liter; plasma 1 chloride, 104 1 mEq/liter; plasma osmolality, 308

mOsm/kg; plasma protein, 4.7 0.1 gldl; and plasma glucose, 7.6 0.3 mmollliter. The urine flow rate and inulin clearance were 5.2 0.7 .d/min/100 g body wt and 948 64 d/minh10O g body wt. Mean arterial pressure was 119 1 mm Hg. Calculated perfusion rate, length of the perfused segment of proximal Illinois, USA). In vivo microperfusion rate (PR) was calculated convoluted tubule, and perfusate chloride concentration and osmolality were not different between each experimental and as (in ni/mm): (1) corresponding control group (Table 1). The isoelectric point of PR = CR x [3HJ/[3H1 the kappa light chain used in the experiments was 7.7. where CR is the collectate volume divided by collection time; In proximal convoluted tubules (PCT's) perfused for 5 [3H], the [3H] activity of the collectate; and [3H19, the [3H1 minutes, absorptions of water (J), chloride (Jci), and glucose activity of the perfusate. (J0) did not differ between control (ATF5) and experimental Volume absorption (J) was calculated as (in nl/min/mm (BJP5) perfusions (Table 2). However, in those PCT's perfused tubule length): for 20 minutes with perfusate that contained BJP (BJP2O), 2,,, (PR — CR)/L (2) and J0 were less (P <0.025) than the corresponding values of J.. PCT's perfused for 20 minutes with ATF alone (ATF2O). The where L is the length of the perfused segment. decrease in Jci did not reach statistical significance. Tubules Chloride absorption (Jc1) was calculated as (in pEq/minlmm perfused for 20 minutes with albumin (ALB2O) in the perfusate tubule length):

Jci = {(P1 X PR) —

showed no changes in J,,,, Jci and J0 compared to control

(Cc1

X CR)}/L

(3)

where P1 is the perfusate chloride concentration and CCI is the collectate chloride concentration. Glucose absorption (JG) was calculated as (in pmol/minlmm tubule length): = {(['4CJ x PR) — (['4C] x CR)/(['4CJ

XPR)XL}xPRxPG (4) where [14CJ is ['4C] activity of the perfusate; [14C] is ['4C]

activity of the collectate; and P6 is the perfusate glucose concentration (in pmollnl).

(ATF2O) perfusions; this lack of effect of a high luminal concentration of albumin on J,,, agrees with Green, Windhager, and Giebisch [231. To summarize, perfusion with BJP produced time-dependent decreases in 2.,, and JG in the PCT in vivo; these decreases did not occur in tubules perfused for similar durations with albumin at the same concentration. Morphologic and immunocytochemical studies Inulin clearance averaged 844 71 td/min/lOO g body wt and the mean arterial pressure was 124 3 mm Hg in the 15 animals used in these studies. On light microscopic examination, the albumin-perfused tubules (N = 4) were normal in appearance,

Statistical analysis All values are given as mean SEM. Comparisons between except for rare epithelial cell vacuolation in one tubule. In two groups were analyzed for statistical significance by the contrast, none of the BJP-perfused tubules (N = 4) had normal unpaired t-test, with significance set at the 5% level. For morphology. All four nephrons had mild to moderate vacuolacomparisons among three groups, the Bonferróni modification tion of the epithelium with desquamation of tubule cells noted in of the I-test was used, with significance set at the 2.5% level. three tubules (Fig. 1). With the ABC technique, kappa light chains were identified in the tubule lumen and in discrete areas Results of the cytoplasm of the PCT cells (Fig. 2). In separate experiMicroperfusion experiments ments (N 2), immunofluorescence microscopy also showed Mean systemic variables of the 34 rats used in this portion of kappa staining in the lumen and within the cells of the PCT; the study were: hematocrit, 44 1%; plasma sodium, 150 1 lambda staining was negative.

855

a-

r

S

:0

1

s-

rn

"—p

a

:

'Ii

-.4

I.

w

q

I

Renal toxicity of Bence Jones protein

Fig. 1. Comparison of a BJP-perfused PCT (A) with an albumin-perfused PCT (B), both of which are identified by the India ink (dark particles)

in the lumen (L). Desquamation of epithelial cells (curved arrows) and mild vacuolation (arrows) of the lining PCT cells (E) are seen in the BJP.perfused tubule. In contrast, the albumin-perfused tubule (B), surrounded by arrows, is normal in appearance. (H&E stain, original magnification x 240).

Electron microscopy (N = 7) demonstrated prominent vacu-

Discussion

olation of the epithelium of the BJP-perfused PCT's and lysosomes that were more numerous and enlarged compared to the albumin-perfused tubules (N = 4) (Figs. 3 and 4A). Some of the lysosomes were markedly enlarged, distorted and atypical in appearance; structures resembling crystals were occasionally identified in the lysosomes (Figs. 4B and 5). In addition, cellular desquamation was seen in six of the seven BJP-perfused tubules and was absent in control tubules (Figs. 3 and 6A). Focal loss of

These data show that: (1) proximal convoluted tubules (PCT)

of the rat absorb human Bence Jones protein (BJP) by

endocytosis; (2) BJP is localized in endosomes and lysosomes of the PCT; (3) in proximal convoluted tubules (PCT's) perfused with BJP, Jv and JG decrease in time-dependent fashion; and (4) morphologic evidence of damage of the PCT is present after perfusion with BJP but not albumin. The kidney is an important site of uptake and catabolism of a the microvillus border of several cells of the PCT's was demonstrated only in the experimental perfusions (Figs. 4A and variety of low molecular-weight proteins that include immuno6B). Immunoelectron microscopy, using anti-kappa antibody globulin light chains (mol wt about 22,000), albumin, complethat was linked to colloidal gold, confirmed the presence of the ment protein D, lysozyme, insulin, growth hormone, parathyBJP in the lumen and in endosomes and lysosomes of the cells roid hormone, and glucagon [15, 23—31]. Proteins that are of the PCT (Figs. 5 and 6). The surrounding tubules, intersti- filtered by the glomerulus are absorbed primarily by the proxtium and mitochondria of these tubules (Fig. 6) and albumin- imal tubule [29, 31], although epithelia of other segments of the perfused nephrons did not show significant labeling with the nephron have been shown to absorb protein [32, 331. With the anti-kappa antibody and the BJP-perfused tubules did not stain exception of small peptides, such as bradykinin and angiotensin with the anti-lambda antibody. Thus, both light and electron II, that undergo hydrolysis along the luminal membrane of the microscopy techniques showed uptake of the kappa BJP into proximal tubule cells, proteins are absorbed by endocytosis and the epithelium of the PCT and alterations in morphology can be found initially in endosomes that fuse subsequently with compatible with acute epithelial cell-injury after 20 minutes of lysosomes, allowing hydrolysis of the absorbed proteins to perfusion in vivo; morphologic alterations of PCT damage were amino acids in an acidic environment [29, 31]. Our findings show that rat PCT cells absorb human BJP which was identified not seen in albumin-perfused tubules.

•

e

a

S

—

rn

a S

r

ii:.

ft

'4,,

—

—

r aS •

t St

•

S

a

w

S. U's

%

S

"4.

iv

et a!

V

V

_

e

11

Sanders

*

856

Fig. 2. Using an avidin-biotin complex (ABC) immunostaining method, the presence of the kappa light chain in the tubule lumen (L) and in discrete areas of the cytoplasm (curved arrows) of a BJP-perfused PCT is shown. Mild cellular desquamation (arrow) is apparent; the desquamated cells also contained kappa light chain. E represents lining epithelial cells. (ABC stain, original magnification X240).

form of BJP [36]. Also, the use of renal cortical slices to study cellular transport and metabolism is controversial because of studies that have shown that the tubule lumens are collapsed or occluded [37] and oxygen delivery is inadequate to maintain metabolic processes in these preparations [38]. More recently, Batuman et al showed that BJP decreased glucose absorption has been shown to correlate with the degree of renal failure by proximal tubule, brush border vesicles in 30 seconds [39]. better than cast formation [5]. In the same study, patients with The osmolalities of the solutions that contained the BJP's were Bence Jones proteinuria and multiple myeloma had a higher not reported. incidence of renal failure and more defects in urine acidification Our in vivo microperfusion study showed that diminished and urine concentrating ability than those patients who had volume and glucose absorption by the PCT, the major site of the multiple myeloma but no Bence Jones proteinuria. Finally, BJP absorption, occurred in a time-dependent fashion after proximal tubule damage in the form of Fanconi syndrome has luminal exposure to the BJP in a concentration of 5 g/dl. In been recognized as a complication of Bence Jones proteinuria; preliminary studies, it was noted that the addition of BJP, which renal histology of these patients frequently showed destruction had been prepared by standard methods, increased the osmolaland electron-dense, needle-like crystals in the cytoplasm of the ity of ATF (D. W. Barfuss, personal communication). These osmotically active substances are usually electrolytes that are proximal tubule cells [34]. Experimental evidence of a direct cellular toxicity of BJP's acquired during the protein isolation techniques. Our preparahas been suggested by Preuss et al, who demonstrated that rat tion of the BJP's completely removed these substances since kidney slices incubated for four hours with crude extracts of the addition of dialyzed BJP, 5 g/dl, did not change perfusate BJP, 1.0 g/dl, had impaired cellular gluconeogenesis, ammonia- osmolality. Such small osmotic activities may profoundly alter genesis, and hippurate uptake compared to control kidney fluid movement in sensitive test systems, such as tissue slices slices incubated with protein extracts from the urine of patients and membrane vesicle preparations [40]. Thus, the discrepancy with nephrotic syndrome [35]. However, these results could not between our study and that of Batuman et al [391,regarding the be verified in a similar experiment that used a more purified time-dependent nature of the defect in JG, may be due to the within the endosomes and lysosomes of the PCT (Figs. 5 and 6) and are consistent with the mechanism of renal handling of low molecular-weight proteins. Clinical evidence suggests this process of BJP absorption and catabolism may also injure the kidney. In patients with multiple myeloma and myeloma kidney, atrophy of the proximal tubule

857

Renal toxicity of Bence Jones protein

.1;

Fig. 3. Electronmicrograph of a BJP-perfused tubule (A) and an albumin-perfused tubule (B). The BJP-perfused proximal tubule (A) has an activated lysosomal system (arrows), prominent apical vacuolation, and focal loss (curved arrows), of the microvillus border (my). By comparison, this albumin-perfused tubule (B) was normal in appearance. In this section, the microvillus (my) borders are in apposition. (Uranyl acetate and lead

citrate, original magnification x2,750).

presence of osmotically active impurities in their BJP preparation. The time-dependent decrease in solute transport is also important

majority of the BJP-perfused tubules had fragmentation and desquamation of cells (Figs. 1 and 2) or cytoplasmic compo-

in the understanding of the mechanism of cell toxicity of BJP. nents (Fig. 6A) into their lumens. Therefore, the functional and We chose albumin as the control protein because its handling morphologic studies are complementary and show that a human by the PCT has been extensively studied [24—27], it does not Bence Jones protein can produce an acute injury to the epithealter water absorption of tubules in vivo [23], it is partially hum of the PCT of the rat in vivo. How BJP's produce cell damage is not certain. Clinically, not filterable, and it is absorbed and catabolized to amino acids in the PCT within six minutes [27]. Our data confirm the lack of all patients with Bence Jones proteinuria have renal failure; toxicity of intraluminal albumin since perfusion of nephrons some patients may excrete large amounts of BJP without with albumin, 5 gIdl, did not alter PCT function and morphol- evidence of renal damage [42]. It is possible that, because of ogy. In addition to albumin, we have also used the BJP as its their physiochemical properties, nontoxic BJP's are filtered but own control and showed that PCT function was not immedi- are poorly absorbed by the proximal tubule. Alternatively, ately altered by the perfusate containing the BJP. At this time, nontoxic BJP's are absorbed and readily catabolized by the however, we cannot state that this toxicity is specific for light proximal tubule while toxic BJP's or their intermediate catachains and in fact may occur with other low molecular-weight bolic products such as amyloid are not readily degraded in the lysosomes. For example, in an acid milieu (pH 4.5 to 5.0) proteins such as myoglobin and beta-lactoglobulin [8, 9]. Morphologic clues of early nephrotoxic cell damage—in- comparable to that of the lysosome [43], some, but not all, creased endocytic activity, vacuolation and focal loss of the BJP's have been shown to precipitate during incubation in vitro brush border [41]—were prominent findings in the proximal with the lysosomal fraction of homogenated human kidney tubules of those nephrons perfused with the BJP compared to tissue [44]; catabolism of these aggregated proteins may be those perfused with albumin. In the PCT's of the BJP-perfused impaired. Similarly, proteolytic cleavage of some BJP's has tubules, the lysosomes were enlarged, increased in number, and produced polymerization of the amino terminal fragments into several were distorted (Figs. 3, 4B, 5, and 6). Focal loss of the amyloid, which has also been found in lysosomes of macrobrush border of several cells of the epithelium of the PCT was phages and is thought to be the result of intralysosomal digesidentified (Figs. 3, 4A, and 6B). In addition to these findings, the tion [45]. Indirect evidence of precipitation of BJP or interme-

858

Sanders

--t-. _,i -*

et a!

—

-

- %,;-'_- -

—-•

It S -:

jt:t; B

:tt

1•.

Fig. 4. Electronmicrographs of 2 BJP-perfused PCT's. Figure 4A shows activation of the lysosomal system (curved arrows) and cytoplasmic protrusion on the luminal side of one cell where the microvillus border (my) is absent (arrows). (Uranyl acetate and lead citrate, original magnification x2,750). Figure 413 shows several markedly enlarged, distorted tysosomes (arrows) of a PCT cell and apical vacuolation (V). (Uranyl acetate and lead citrate, original magnification x2,750).

diate catabolic product is suggested by a study by Clyne et a! [46], who injected rats intraperitoneally with a human, kappa light chain and noted development of tubular proteinuria within 12 hours. Electron microscopy of the proximal tubules of these rats revealed many polygonal dense bodies, some of which had a crystalline appearance; several degenerative changes of the tubule cells were found [46]. The result of impaired degradation of BJP may be an overwhelming or "clogging" of the lysosomal system [47]. In turn, lysosomal overload could cause release of their digestive enzymes which may manifest as cytoplasmic vacuolatjon [47, 48]. Since some phagocytic cells internalize up to 186% of their surface area and 25% of their cell volume per hour [43, 49], this process could also diminish return of plasma

Another physicochemical property of BJP's—the isoelectric point (p1)—has been considered by some authors [12, 50] to be

atypical and appeared to contain crystals (Fig. SC). These

failure in human multiple myeloma, it is possible that this

changes occurred at a time when other morphologic alterations

toxicity of BJP's not only directly impairs nephron function but

an important factor conferring nephrotoxocity. Other studies [8, 10, 51—53] have shown no correlation between nephrotoxicity and the p1 of the BJP. Other proteins that include lysozyme (p1, 11.0) [54] and cationized albumin (p1 8.4) [25] have been

shown to damage renal tubules, but the p1's of these proteins are very high. By comparison, our BJP had a p1 of 7.7. At pH

7.4 (the pH of our perfusate), this protein possesses a net

positive charge, which may facilitate absorption of the protein and therefore accelerate toxicity. Thus, the p1 of the BJP may be an important determinant of nephrotoxicity. In conclusion, we have shown that a human kappa light chain membrane to the surface of the cell. The combination of is absorbed by endocytosis and produces an acute toxic injury lysosomal dysfunction, release of digestive enzymes into the in cells of the rat proximal convoluted tubule. Since some BJP's cytoplasm, and decreased plasma membrane availability and are not nephrotoxic [5, 42], perfusion of the PCT with a variety turnover would cause reduced cell function and eventual cell of BJP's may allow in vivo characterization of the nephrotoxic death. The present study supports this hypothesis of lysosome potential of each BJP and subsequent correlation between overload by showing localization of BJP in an activated lyso- toxicity and the physicochemical property(s) of that protein. somal system of the PCT cells. Some of the lysosomes were Although these results cannot be extrapolated directly to renal

of the PCT cells were observed and volume and glucose also diminishes absorption of the BJP by the proximal tubule, absorption by the proximal convolution was impaired. Further study of this proposed mechanism is indicated.

allowing increased delivery of the protein to the distal nephron where the acid pH, lower tubule fluid flow rate, and Tamm-

r

'I.:,.,

S

a ... -

.-

I.

it;

I.

•

V.-

r

at.. ..j

I

_t .w

rT •

•

.1,1

B

7

-p

•0 ;j.: 4.

'I.

-

4

k

-• ..

I I ——

IS

—

I_n

it V.

-

k

I

L •

I II

I Fig. 5. Immunoelectron micrographs using an antikappa antibody stain, demonstrating several abnormal lysosomes of the BJP-perfused PCT's. Alteration of the lysosomal system was prominent in these tubules and was not seen in the albumin-perfused nephrons. A shows an enlarged lysosome (L) (Uranyl acetate and lead citrate, original magnification x 16,500). B shows evidence of autophagia of the cell with the presence of cytoplasmic organelles in a lysosome (center) (Uranyl acetate and lead citrate, original magnification x6,000). C shows a markedly enlarged, distorted lysosme (L) which contains structures resembling crystals and is surrounded by numerous smaller lysosomes, some of which are merging with it (Uranyl acetate and lead citrate, original magnification x7,700). The lysosomes in these figures stain with the anti-kappa antibody that is linked to colloidal gold (seen as small black dots).

860

:j 4

—C

—;.

.44

z—-

- -.

U—

-

—

'— — w

r

S

4i

!'

b;; .\

- q_

r

'

.4'

-. 2

Sanders et a!

Fig. 6. Immunoelectron micrographs of two other BJP-perfused PCT's, demonstrating kappa labeling of the endosomes and lysosomes (clear arrows) of the cells of the PCT. The mitochondria (curved allows) and other organelles of the cell are not labeled to a significant degree. In addition to activation of the lysosomal system with enlargement and atypical appearance of the lysosomes (L), vacuolation (V) and desquamation of cell fragments (solid arrows) into the lumen are noted (A) Uranyl acetate and lead citrate, original magnification x5,000). B also shows loss of the microvillus border (my) (Uranyl acetate and lead citrate, original magnification X6,000).

Horsfall glycoprotein facilitate BJP aggregation and allow cast formation which, in turn, further decreases nephron function. In addition, desquamation of proximal tubule cells and cell fragments may contribute to the tubular obstruction. Thus, dysfunction of the proximal tubule may be the first event to occur in the development of the tubulointerstitial nephropathy of multiple myeloma.

50:29—40, 1975 2. ALEXANIAN R, BALCERZAK S, BONNET JD, GEHAN BA, HAUT A,

Acknowledgments

some aspects of clinical management. J Chron Dis 24:221—237, 1971 4. DEFRONZO RA, HUMPHREY RL, WRIGHT JR, COOKE CR: Acute

Portions of this work have been published in abstract form in Clin Res 34:205A, 1986, and Clin Res 34:607A, 1986. This work was supported by the Research Service of the Veterans Administration, the Alabama

References 1. KYLE RA: Multiple myeloma. Review of 869 cases. Mayo Clin Proc

HEWLETT iS, MONTO RW: Prognostic factors in multiple myeloma. Cancer 36:1192—1201, 1975 3. MARTINEZ-MALDONADO M, YIUM J, SUKI WN, EKN0YAN G:

Renal complications in multiple myeloma: pathophysiology and

renal failure in multiple myeloma. Medicine (Baltimore) 54:209— 223, 1975 5. DEFRONZO RA, COOKE CR, WRIGHT JR, HUMPHREY RL; Renal

Kidney Foundation, Birmingham District, and the National Kidney Foundation, Alabama Chapter. Dr. Sanders is a Research Associate of

the Veterans Administration. We thank Beverly B. Booker for her invaluable assistance, Drs. Robert G. Luke and Delon W. Barfuss for their advice on the project, T. Clint Mills for technical assistance, and the Media Services of the Veterans Administration and the University of Alabama at Birmingham for the illustrations.

Reprint requests to Paul W. Sanders, M.D., Nephrology Research and Training Center, Sixth Floor Zeigler Building, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

function in patients with multiple myeloma. Medicine (Baltimore) 57:151—166, 1978

6. OSSERMAN EF: Plasma-cell myeloma. II. Clinical aspects.

Med 261:952—1014, 7.

NEngI J

1959

BERGGARD I, PETERSON PA: Polymeric forms of free normal k and 1 chains of human immunoglobulin. J Biol Chem 244:4299—4307,

1969 8. WEiss JH, WILLIAMS RH, GALLA JH, GOTTSCHALL JL, REES ED, BHATHENA D, LUKE RG: Pathophysiology of acute Bence-Jones protein nephrotoxicity in the rat. Kidney

9. HOLLAND MD, GALLA

ill,

mt 20:198—210, 1981

SANDERS PW, LUKE RG: Effect of

861

Renal toxicity of Bence Jones protein

urinary pH and diatrizoate on Bence Jones protein nephrotoxicity in the rat. Kidney mt 27:46—50, 1985

33. Sritus W: Occurrence of phagosomes and phago-lysosomes in different segments of the nephron in relation to the reabsorption,

10. SMOLENS P, VENKATACHALAM M, STEIN JH: Myeloma kidney cast

transport, digestion, and extrusion of intravenously injected horseradish peroxidase. J Cell Biol 21:295—308, 1964

nephropathy in a rat model of multiple myeloma. Kidney mt 24:192—204, 1983

11. Koss MN, PIINI CL, OSSERMAN EF: Experimental Bence Jones cast nephropathy. Lab Invest 34:579—591, 1976 12. CLYNE DH, PESCE AJ, THOMPSON RE: Nephrotoxicity of Bence Jones proteins in the rat: Importance of protein isoelectric point.

34. MALDONADO JE, VELOSA JA, KYLE RA, WAGONER RD, HOLLEY

KE, SALASSA RM: Fanconi syndrome in adults. A manifestation of a latent form of myeloma. Am J Med 58:354—364, 1975 35. PREUSS HG, WEISS FR, IAMMARINO RM, HAMMACK Wi,

MURDAUGH HV: Effects of rat kidney slice function in vitro of proteins from the urines of patients with myelomatosis and nephro-

Kidney in! 16:345—352, 1979 13. MCINTIRE KR, POTTER M: Studies of thirty different Bence Jones

sis. C/in Sci Mol Med 46:283—294, 1974

protein-producing plasma cell neoplasms in an inbred strain of 36. FINE JD, REES ED: Renal cytotoxic effects of Bence-Jones protein. mouse. J Nat Cancer inst 33:631—648, 1964 14. PIINI CL, SILVA FG, APPEL GB: Tubulo-interstitial disease in multiple myeloma and other nonrenal neoplasias, in Tubulo-intersti-

37. WEDEEN RP, WEINER B: The distribution of p-aminohippuric acid

ha! Nephropathies, edited by COTRAN RS, BRENNER BM, STEIN JH, New York, Churchill Livingstone, 1983, pp. 287—334 15. SANDERS PW, VOLANAKIS JE, ROSTAND SG, GALLA JH: Human

38. BALABAN RS, SOLTOFF SP, STOREY JM, MANDEL U: Improved

complement protein D catabolism by the rat kidney. J C/in invest 77:1299—1304, 1986

(abstract) Lance! 1:150, 1977 in rat kidney slices. I. Tubular localization. Kidney mt 3:205—213, 1973

renal cortical tubule suspension: Spectrophotometric study of 02 delivery. Am J Phys 238:F50—F59, 1980 39. BATUMAN V, SASTRASINH M, SASTRASINH S: Light chain effects

16. SoloMoN A, FAHEY JL: Bence Jones proteinemia. Am J Med 37:206—222, 1964

17. NOORDEN SV, POLAK JM: Immunocytochemistry today: Techniques and practice, in immunocytochemistry. Practical applica(ion in Pathology and Biology edited by POLAK JM, NOORDEN SV, Bristol, Great Britain, John Wright and Sons, Ltd., 1983, pp. 16—17 18. VIALE 0, DELL'ORTO P, BRAIDOTTI P, C0GGI G: Ultrastructural

localization of intracellular immunoglobulins in epon-embedded human lymph nodes. An immunoelectron microscopic investigation using the immunogold staining (IGS) and the avidin-biotin-peroxidase complex (ABC) methods. J Histochem Cytochem 33:400—406, 1985

19. TIMMS BG: Post-embedding immunogold labeling for electron microscopy using "L. R. White" resin. Am J Anat 175:267—275, 1986

20. LAEMMLI UK: Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London) 227:680—685, 1970 21. FUHR J, KACZMARCZYK J, KRUTTGEN CD: Eine einfache colorim-

etrische Methode zur inulinbestimmung für Nieren-Clearanceuntersuchungen bei Stoffwechselgesunden und Diabetikern. K/in

Wochenschr 33:729—730, 1955 22. RAMSAY JA, BROWN RH, CROGHAN PC: Electrometric titration of chloride in small volumes. J Exp Bio/ 32:822—829, 1955 23. GREEN R, WINDHAGER EE, GIEBISCH G: Protein oncotic pressure

effects on proximal tubular fluid movement in the rat. Am J Physiol 226:265—276, 1974

24. MAUNSBACH AB: Absorption of I'25-labeled homologous albumin by rat kidney proximal tubule cells. J Ultrast Res 15:197—241, 1966 25. PARK CH, MAACK T: Albumin absorption and catabolism by

isolated perfused proximal convoluted tubules of the rabbit. J Clin invest

73:767—777, 1984

26. CARONE FA: Renal handling of proteins and peptides. Ann C/in Lab Sci 8:287—294, 1978

27. PARK CH: Time course of albumin absorption, accumulation, and metabolism in isolated perfused rabbit proximal convoluted tubules (PCT). (Abstract) Kidney In! 27:331, 1984 28. WOCHNER RD, STROBER W, WALDMANN TA: The role of the kidney in the catabolism of Bence Jones proteins and immunoglobulin fragments. J Exp Med 126:207—220, 1967 29. MAACK T, JOHNSON V, KAU ST. FIGUEIRED0 J, SIGULEM D: Renal

transport, and metabolism of low-molecular-weight proteins: A review. Kidney mt 16:251—270, 1979 30. RABKIN R, KITAJI J: Renal metabolism of peptide hormones. Miner Electrol Metab 9:212—226, 1983 31. CARONE FA, PETERSON DR. OPARIL 5, PULLMAN TN: Renal filtration,

tubular transport and catabolism of proteins and peptides. Kidney in!

16:271—278, 1979

32. MADSEN KM, HARRIS RH, TISHER CC: Uptake and intracellular distribution of ferritin in the rat distal convoluted tubule. Kidney mt 21:354—361, 1982

on glucose

and alanine uptake up renal brush

Kidney mt 30:662—665, 1986 40. SCHAFER JA: Mechanisms coupling

water in

border membranes.

the absorption of solutes and

mt 25:708—716, 1984 41. FLAMENBAUM W, KAUFMAN J, CHOPRA S, MCNEILJS: Membrane the proximal nephron. Kidney

alterations

in acute renal failure, in Cellular Pathobiology of

Human Disease, edited by TRUMP BF, LAUFER A, JONES RT, New York, Gustav Fisher, Inc., 1983, pp. 321—349 42. WOODRUFF R, SWEET B: Multiple myeloma with massive Bence

Jones proteinuria and preservation of renal function. Aust NZ J Med 7:60—62, 1977 43. STEINMAN RM, MELLMAN IS, MULLER WA, COHN ZA: Endocytosis and the recycling of plasma membrane. J Cel! Bio! 96:1—27, 1983 44. TAN M, EPSTEIN W: Polymer formation during the degradation of

human light chain and Bence-Jones proteins by an extract of the lysosomal fraction of normal human kidney. Immunochemistry 9:9—16, 1972 45. GLENNER 0G. EANES ED, BLADEN HA, TERRY W, PAGE DL:

Creation of "amyloid" fibrils from Bence Jones proteins in vitro. Science 174:712—714, 1971

46.

CLYNE DH, BRENDSTRUP L, FIRST MR, PESCE AJ, FINKEL PN,

CL: Renal effects of intraperitoneal kappa injection. Induction of crystals in renal tubular cells. Lab

POLLAK YE, PIRANI chain

Invest 31:131—142, 1974 47. DEDUVE C, WATTIAUX R: Functions of lysosomes. Ann Rev Physiol 28:435—492, 1966 48. STRAUS W: Altered reabsorption of protein by the renal cortex in

rats treated with hypertonic saline or mannitol. J Histochem Cytochem

23:707—721,

1975

49. STEINMAN RM, BRODIE SE, COHN ZA: Membrane flow during

pinocytosis. A stereologic analysis. J Cell Biol 68:665—687, 1976 50. COWARD RA, DELAMORE 1W, MALLICK NP, ROBINSON EL: The

importance of urinary immunoglobulin light chain isoelectric point (p1) in nephrotoxicity in multiple myeloma. C/in Sci 66:229—232, 1984 51. JOHNS EA, TURNER R, COOPER EH, MACLENNAN 1CM: Isoelectric

points of urinary light chains in myelomatosis: Analysis in relation to nephrotoxicity. J C/in Patho! 39:833—837, 1986 52. SMOLENS P. BARNES JL, STEIN JH: Effect of chronic administra-

tion of different Bence Jones proteins on rat kidney. Kidney Int 30:874—882, 1986

S. MOUGENOT B, BAUDOUIN B, DE MEYER-BRASSEUR M, LEMAITRE V. MICHEL C, MIGNON F, RONDEAU E, VANHILLE P, VERROUST P, RONCO P: Multiple myeloma and severe renal failure:

53. ROTA

A clinicopathologic study of outcome and prognosis in 34 patients. Medicine (Baltimore) 66:126-137, 1987 54. COJOCEL C, D0CIU N, BAUMANN K: Early nephrotoxicity at high plasma concentrations of lysozyme in the rat. Lab invest 46:149— 157, 1982