K+-ATPase activity by FK506 involves calcineurin

Kidney International, Vol. 46 (1994), pp.

647—652

Evidence that the inhibition of Na7KtATPase activity by FK506 involves calcineurin JANIcE P. LEA, JEFF M. SANDS, STEVEN J. MCMAHON, and JAMES A. TUMLIN Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

Evidence that the inhibition of Na/K-ATPase activity by FK506 phorylation of the IL-2 specific nuclear promoter factors [10, 11] calcineurin. We reported that cyclosporin A (CsA) inhibits 1). Na'7K-ATPase activity in specific segments of the rat nephron. In this (Fig. Rapamycin is a new immunosuppressive drug which is structurstudy, we tested the hypothesis that cyclosporin A reduces NaVKATPase activity through inhibition of calcineurin. In T cells, cyclosporin A ally related to FK506 and binds to the same intracellular immuand FK506 bind to immunophilins and inhibit the phosphatase activity of nophilin but does not inhibit calcineurin activity [12]. Preliminary calcineurin; Rapamycin and SDZ 220-384 also bind to immunophilins but data indicate that rapamycin exerts considerably less nephrotoxinvolves

do not change calcineurin activity. Na47K-ATPase activity was measured in microdissected rat proximal tubule (S2 subsegment), medullary thick

ascending limb (mTAL), and cortical collecting duct (CCD). First we found that two inhibitors of calcineurin, pentafluorophenol (PFP, 100 mM) and peptide 412 (1 mM), significantly reduced Na/K-ATPase activity in

the CCD by 78% and 70%, respectively. In CCDs, FK506 inhibited NaVK-ATPase activity by 61 to 85% at concentrations of 1.5 to 6 ng/ml, but not at 0.5 ng/ml. FKSO6 (6 ng/ml) inhibited Na/K-ATPase activity in rnTALs by 56% but did not inhibit it in S2s or glomeruli. In contrast, Rapamycin (12.5 ng/ml) did not change Na/K-ATPase activity in CCD5 or mTALs, but at a concentration of 12.5 .rg/ml did block the inhibitory effect of FK506 (6 ng/ml) in both segments. SDZ 220-384 (600 ng/ml) did not change Na/K-ATPase activity in CCDs. Thus, in CCDs and mTAIs: (1) FK506, like cyclosporin A, inhibits Na4iK-ATPase activity; (2) Rapamycin and SDZ 220-384 do not inhibit Na/K-ATPase activity; and (3) Rapamycin prevents FK506-induced inhibition of Na/K-ATPase activity. These responses may be explained by a direct inhibition of calcineurin activity

yielding lower Na/K-ATPase activity in CCDs and mTAIs.

icity than cyclosporin A or FK506 [13]. SDZ 220-384 is a semi-synthetic derivative of cyclosporin A which binds to cyclophilin but does not inhibit calcineurin (personal communication, Dr. Römer, Sandoz Corp.). In addition to decreasing IL-2 transcription, calcineurin might

regulate Na/K-ATPase activity through dephosphorylation of its catalytic subunit. For example, Middleton et al showed that protein kinase C (PKC) was capable of phosphorylating the a subunit and inhibiting NaVK-transport activity in intact renal cells [14]. Bertorello et al reported that phosphorylation of the catalytic subunit of Na/K-ATPase by PKC inhibits the activity of the enzyme [15, 16]. Subsequently, Aperia et al demonstrated that stimulation of proximal tubular Na/K-ATPase by an a adrenergic agonist could be reversed by preincubation with either FK506 or a specific peptide inhibitor of calcineurin [17]. Thus, we

reasoned that if calcineurin regulates Na/K-ATPase activity via dephosphorylation, then drugs which inhibit calcineurin could decrease Nat'K-ATPase activity. First we examined the critical Cyclosporin A and FK506 are potent immunosuppressive drugs question, can inhibition of calcineurin reduce Na/K-ATPase which prolong renal allograft survival [1, 2] but also result in activity in unstimulated, isolated, intact nephron segments. For clinically significant nephrotoxicity [3, 4] and hyperkalemia [5, 6]. these experiments we used the specific inhibitors of calcineurin, We reported that cyclosporin A (CsA) causes a dose-dependent PFP [18] and peptide 412 [17]. Next, we examined the hypothesis

inhibition of Na/K-ATPase activity in some but not all seg- that cyclosporin A inhibits Na/K-ATPase activity through ments of the rat nephron [7]. The mechanism by which cyclosponfl A specifically inhibits Nat/K-ATPase activity is unknown. Despite structural differences, cyclosporin A and FK506 inhibit interleukin-2 (IL-2) transcription in T lymphocytes through sim-

ilar signal transduction pathways [8, 9]. In these lymphocytes, cyclosporin A and FK506 bind to different cytosolic receptors (immunophilins) and form drug receptor complexes which inhibit the phosphatase activity of calcineurin, a calcium-dependent, calmodulin-stimulated protein phosphatase. Inhibition of calcineurin activity decreases IL-2 transcription by blocking dephos-

Received for publication October 22, 1993 and in revised form March 10, 1994 Accepted for publication April 7, 1994

© 1994 by the International Society of Nephrology

inhibition of calcineurin by comparing how FK506, Rapamycin, and SDZ 220-384 influence Na17K-ATPase activity in the rat nephron. These drugs were chosen because of their differential effects on calcineurin activity.

Methods Tissue preparation

Pathogen-free male Sprague-Dawley rats (75 to 100 g) were anesthetized with Nembutal (2 glkg body weight i.p., Abbott Laboratories, Chicago, Illinois, USA). The left kidney was selectively perfused with ice-cold dissection solution containing 1 mg/mI of collagenase (Type A, Boehringer Mannheim, Indianapolis, Indiana, USA) and albumin (BSA, bovine serum albumin, Sigma Chemical Co., St. Louis, Missouri, USA) and cut into cortical and outer medullary slices which were incubated at 37°C in the collagenase solution as described previously [19]. The

647

648

Lea et a!: Calcineurin involved in Na/K-ATPase inhibition

T cell receptor

Na

Na

L IL-2]


composition of the dissection solution was in (mM): NaCI 137, KC1

200

5, MgSO 0.8, Na2HPO4 0,33, KH2PO4 0.44, MgCl 1, Tris-HCI 10, and CaCI2 0.25. The following segments were isolated: gbmeruli, proximal tubules (S2 subsegment), medullary thick ascending limbs (mTALs), and cortical collecting ducts (CCDs).

150

Experimental protocol

100

Fig. 1. A diagram of calcineunn binding to immunosuppressant-immunophilin complexes in T cell activation and regulation of Na IKATPase. Binding of specific antigen to the T cell receptor begins a cascade of events that result in transcription of interleukin-2 (IL-2). Calcineurin causes a dephosphorylation of the nuclear factor of activated T cells (NF-AT) that binds to the IL-2 promotor region in T lymphocytes. When bound to their respective binding proteins, cyclosporin A (CsA) and FK506 complex with calcineurin and inhibit dephosphoiylation of NF-AT.

Microdissected tubules from each rat were divided in four groups of 5 to 10 mm each: groups 1 and 2 were used to measure total and ouabain-insensitive ATPase activity, respectively, while groups 3 and 4 were used to measure total and ouabain-insensitive ATPase activity when different concentrations of drug (FK506, Rapamycin, SDZ 220-384, PFP, and peptide 412) were added. Nat/K-ATPase activity was calculated by subtracting ouabaininsensitive ATPase activity from total ATPase activity [20]. Ten to

50

0 0

10

20

30

Number of glomeruli

30 glomeruli were microdissected and Na/K-ATPase activity Fig. 2. The relationship between number of glomeruli in an assay and was normalized per glomerulus. Preliminary experiments demon- Na /K-ATPase activity. NaIK-ATPase activity increased linearly (r = strated a linear relationship between the number of gbomeruli and 0.996) with number of glomeruli. Na/KtATPase activity (r = 0.996, Fig. 2). Measurement of ATPase activity

Na7K-ATPase activity was measured under Vm conditions

Calculations The amount of spontaneously hydrolyzed ATP and the specific described previously [7]. To measure total ATPase activity, tubules (groups 1 and 3) were incubated in a solution composed of radioactivity of 32P were measured as described previously [7]. (in mM): NaC1 50, KC1 5, MgCI2 10, EUTA 1, Tris-HC1 100, Enzyme activity for Na/KtATPase is reported in pmol ATP Na2ATP 10 (Grade II, Sigma). To measure ouabain-insensitive hydrolyzed per m tubule length (or per glomerulus) per minute ATPase activity (groups 2 and 4), the incubation solution was (pmol/mm/min or pmol/glom/min) and calculated from the folidentical to the above except NaCl and KC1 were replaced by lowing formula: Tris-HC1 and 2 mM ouabain (Sigma) was added [201. Different ATPase activity = MR concentrations of FK506 (0.5 to 6 ng/ml), a gift from Fujisawa — B/SRAjtubule length or number of glomeruli/min Corp., Rapamycin (12.5 ng/ml or 12.5 rg/ml), a gift from Wyeth Corp., SDZ 220-384 (600 nglml), a gift from Sandoz Corp., PFP (100 mM) (Sigma), and peptide 412 (1 mM) [17] (synthesized by where MR is measured radioactivity, B is the rate of spontaneous the microchemical facilities at Emory University) was added to ATP hydrolysis, and SRA is specific radioactivity. Spontaneous hydrolysis averaged 7 to 10% of measured radioactivity. groups 3 and 4.

using the 32P method of Doucet, Katz and Morel [20], as

649

Lea et a!: Calcineurin involved in Na/K-ATPase inhibition

Statistics

The results are presented as mean SE from paired experi-

20 E

ments. N indicates the number of rats studied. For comparisons between two groups, statistical significance was calculated using a

Student's t-test with a value of P < 0.05 being significant. For comparisons between 3 groups, a one-way analysis of variance (ANOVA) was used, followed by a multiple comparisons pro-

10

tected t-test to determine which groups were significantly different. Western analysis of calcineurin B protein expression

I

0

Microdissected tubules were emulsified in 50 .d of "High Test" P-412 (1.0 mM) PFP (100 mM) buffer containing various proteinase inhibitors. The composition of "High Test" buffer is as follows: 0.125 M Tris HCI, 20% glycerol Fig. 3. Calcineurin inhibitors and Na /K-A TPase activity in the CCD. PFP (100 mM) and peptide 412(1 mM) decreased NaIK-ATPase activity

(by vol), 6% SDS (by wt), leupeptin (10 mg/mi), chymostatin by 78% and 70%, respectively. Symbols are (U) Control; ()

drug.

(5 mg/mi), aprotinin (10 mg/mi), benzamidine (50 mM), PMSF (10

mg/mi), pepstatin (10 mg/mi) and sodium trypsin inhibitor (5 mg/mi). Total protein was measured spectrophotometrically at 595 nm using a BCA protein assay kit (Pierce Immunotechnology,

Rockford Illinois, USA) and size separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel (15%) electrophoresis. Protein from S2s and mTALs was added to each lane along with a positive

control from the rat brain. Following size separation, proteins were transferred to 0.45 j.tm nitrocellulose membranes (Gibco BRL) by electroblotting [21] and incubated for one hour with blocking buffer (TFBS-NFM). The composition of TTBS-NFM is as follows: Tn-buffered saline (TBS, 20 mrvi Tris HCI, 0.5 M NaCl, pH 7.5); Tween-20 (0.5% vol/vol), and non-fat dried milk (0.5% wt/vol) (Carnation Co., Los Angeles, California, USA). Excessive blocking buffer was removed and blots washed for 15 minutes with TBS for a minimum of two washes.

50 40

30

P.'zO.04

N=8

Q.

>

C)

a)

20

0) 0.. I—

z

10

0

P=NS N=18

P=NS

P<0.03 N=6

— N—5

I

j

______

:4:

]

mTAL CCD S2 Glomerulus Blots were then probed with monoclonal antibodies directed Fig. 4. Effect of FK506 (6 ng/ml) on Na/K-ATPase activity in various segments. FKSO6 significantly decreased Na/K-ATPase activity against calcineurin beta subunit (Upstate Biotechnology Incorpo- nephron in CCDs and mTALs. There was no effect on Na/K-ATPase activity in rated, Lake Placid, New York, USA). These primary antibodies either glomeruli or the S2 subsegment of the proximal tubule. Symbols

were diluted 1:800 in 15 ml of TTBS-NFM and the nitrocellulose blots were incubated for four hours. Blots were then washed three times for 15 minutes in TBS and then incubated with 20 ml of goat

are: (U) control; () FK506 6 ng/ml.

anti-rabbit IgG antibody labeled with peroxidase (dilution

FK506 and Na/KtATPase activity 1:10,000). Excess secondary antibody was removed by washing with TBS and chemibioluminescence was performed according to FK506 (6 ng/ml) inhibited Na/K-ATPase activity in CCDs by the manufacturer's protocol (ECL Western Blot kit, Amersham 85% (P < 0.03) and mTALs by 56% (P < 0.04, Fig. 4). In contrast,

International) [22]. Bands detected on autoradiography were FK506 did not change Na/K-ATPase activity in glomeruli or S2s (Fig. 4). Three additional concentrations of FK506 were analyzed using laser densitometry [23]. Results

examined in CCDs: a therapeutic concentration (3 ng/ml); a low therapeutic concentration (1.5 ng/mI); and a subtherapeutic con-

Effect of calcineurin inhibitors on Na /K-A TPase activity

by 76% at 1.5 ng/mi (P < 0.01) and higher concentrations did not

centration (0.5 ng/ml). FK506 reduced Na/KtATPase activity

cause a further decrease (Fig. 5). However, FK506 did not

To assess the role of calcineurin in regulating Na/K-ATPase significantly affect NaJK-ATPase activity at 0.5 ng/mi (Fig. 5). activity, two specific inhibitors of calcineurin, PFP [18J and Rapamycin and FK506 on Nat'K-ATPase activity peptide 412 [17], were incubated with CCDs. PFP (100 mM) In contrast to FK506, Rapamycin (12.5 ngfml) did not inhibit significantly (P < 0.02; N = 6) reduced CCD Na17K-ATPase activity by 78%. Peptide 412 (1 mM), a sequence that overlaps the Na/K-ATPase activity in either CCDs (Fig. 6) or mTALs (Fig, autoinhibitory domain of caicineurin [17], decreased Na/Kt 7). FK506 and Rapamycin bind to the same immunophilin, FK ATPase activity by 70% in the CCD (P < 0.02; N = 6; Fig. 3). binding protein (FKBP), and high concentrations of Rapamycin These results provide strong support for the hypothesis that have been shown to displace FK506 from FKBP [24]. Therefore, calcineurin is involved in the regulation of Na/K-ATPase even if FK506 inhibits Na/K-ATPase activity because it blocks when activity has not been stimulated. calcineurin, pharmacologic concentrations of Rapamycin (12.5

650

I

Lea et al: Calcineurin involved in Na/K-ATPase inhibition

0 E

>, >

0

0 I-

z

NS

30 10

C)

20

a) Cl)

5

0C

0 0.5

1.5

6

3

Fig. 5. Dose-response of FK506 on Na/K-ATPase activiiy in CCDs.

FK506 inhibited Na/K-ATPase activity at concentrations of 1.5, 3, and 6 ng/ml. However, there was no significant effect on Na/K-ATPase activity at 0.5 nglml. Symbols are: (U) control; (iJ) FK506. Note: the data for 6 nglml is taken from Figure 4.

10 0

z

FK506 6.Ong/mI

Rapamycin Rapamycin 12.5 pg/mI + 12.5 ng/mI FK506 6.0 ng/ml

Fig. 7. Effect of FK5OO vs. Rapamycin on NaIK-ATPase activity in mTALs. FK506 (6 nglml) decreased Na/K-ATPase activity, while Rapamycin (12.5 ng/ml) had no effect. As in the CCD, Rapamycin (12.5 gIml) prevented FK506-induced inhibition of enzyme activity. Symbols

are (•) Control; (0) drug.

Calcineurin B protein ezpression in microdissected tubules To evaluate potential mechanisms for the sensitivity differences in nephron segments, we examined calcineurin 13 subunit protein

30 25

expression in S2s and mTALs. As shown in Figure 8, a 16 kDa

20

band corresponding to the known size of calcineurin B was

15

detected in the S2 subsegment and in the mTAL, Laser densitometry of the Western blot shown in Figure 8 demonstrated a relative

10

band density of 2.37 and 1.93 in two samples of S2s with 40 g total protein, and one of 1.32 in the mTAL at twice the amount of S2 protein. Control rat brain (100 g) showed a relative density of

C)

Ct a)

N=5

>

FK506 concentration, ng/mI

'-

N=8 NS

T

E

15

N=8

40

0

a)

CI)

50

I

20

Cl,

7.88,

Discussion FK506

Rapamycin Rapamycin 12.5 pg/mI

6ng/mI

12.5 ng/ml

FK5O66ng/ml

6. Effect of Rapamycin vs. FK506 on NaiK-ATPase activity in CCDs. In contrast to inhibition of Na'iK-ATPase activity by FK506 (6 Fig.

ng/ml), Rapamycin (12.5 ng/ml) did not change Na/K-ATPase activity. Rapamycin (12.5 ig/ml) prevented FK506-induced inhibition of Na/KATPase activity. Symbols are: (•) control; (0) drug. Note: the data for FK506 (6 ng/ml) are taken from Figure 4.

Our major findings are: (1) inhibition of calcineurin reduces Na7K-ATPase activity even in the basal state; (2) FK506, a drug

which binds to FKBP and inhibits calcineurin in T cells [12], inhibits Na/K-ATPase activity in the same nephron segments as cyclosporin A [7]; (3) Rapamycin and SDZ 220-384, drugs which bind to immunophilins but do not change calcineurin activity, do not inhibit Na/K-ATPase activity; (4) the segmentspecific effects of FK506 may be linked to the differential expression of calcineurin protein in the nephron. Role of calcineurin in regulating Na *IK±A TPase activity

In T cells, cyclosporin A and FK506 bind to distinct intracelluPase activity by displacing FK506 from FKBP. When CCDs and lar proteins (immunophilins), cyclophilin and FKBP, respectively mTA.Ls were incubated with both FK506 (6 ng/ml) and Rapamy- [24]. In vitro, the drug-receptor complexes, cyclosporin A-cycto-

gIml) could prevent FKSO6-induced inhibition of NaIK-AT-

cm

(12.5 Wml), the inhibitory influence of FK506 on Na7K-

ATPase activity was not observed (Figs. 6 and 7). Effect of SDZ 220-384 on Na/K4-ATPase activity

SDZ 220-384 binds to cyclophilin, the same immunophilin which binds to cyclosporin A, but has no effect on calcineurin activity (personal communication, Dr. Romer, Sandoz Corp.). SDZ 220-384 (600 ng/ml) did not change Na/K-ATPase in the 4 CCD (control: 10 2 pmol/mm/min; SDZ 220-384: 11 pmol/mm/min, N = 6).

philin and FK506-FKBP, bind to and inhibit the activity of

calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase [11]. Studies in T lymphocytes show that these drug-receptor complexes inhibit calcineurin activity at concentrations which decrease IL-2 transcription [12]. In fact, the importance of inimunophilin binding to FK506-induced inhibition of calcineurin was demonstrated by Fruman et at who noted that a pharmacologic concentration of Rapamycin (1 .rm) reversed the inhibition of IL-2 transcription by FK.506 [121. Presumably, this was due to displacement of FK506 from FKBP [12]. Lane et al showed that calcineurin's phosphatase activity in peripheral blood mononuclear cells (PBM) was inhibited by 1 M FK506-FKBP or

Lea eta!: Calcineurin involved in Na/K-ATPase inhibition

651

Calcineurin B expression in rat nephron

16 kDA

4

'4 4#c

9

Fig. 8. Ca!cineurin B protein expression in rat nephron. A 16 kDa band, consistent with the size of Calcineurin B, was detected in the S2

I

(40 g protein/lanes 3 and 4) from two experiments compared to a less dense band in the mTAL (80 g protein/lane 2). Rat brain

PS2 Exp-B

(100 j.tg protein/lane 1) was used as a positive control.

I Brain

mTAL

PS2 Exp-A

cyclosporin A-cyclophilin, but not by either drug or immunophulin

separately. However, up to 10 M Rapamycin-FKBP did not change PBM calcineurin activity. They proposed that cyclosporin

rat nephron. Because tissues with increased expression of calcineurin are less sensitive to FK506 [26, 27], we reasoned that different segments of the rat nephron might express varying

A and FK506-induced immunosuppression results from drug- amounts of calcineurin. To address this hypothesis, we performed immunophilin complexes blocking the phosphatase activity of Western blot analysis using a monoclonal antibody to Calcineurin calcineurin [25]. 13 in FK506-sensitive (mTAL) and FK506-insensitive (S2) Others have demonstrated that phosphorylation of the Nat' nephron segments. We found greater Calcineurin f3 expression in K -ATPase catalytic subunit decreases enzyme activity in vitro the S2 than in the mTAL (Fig. 8). Preliminary data demonstrating [14—16]. Bertorello and Aperia noted that stimulation of protein that calcineurin enzyme activity in S2s is 20-fold greater than in kinase C activity by phorbol ester or diaglycerol significantly mTALs or CCDs [28] is also consistent with the hypothesis that reduced Na/KtATPase activity in the rat proximal tubule [16]. sensitivity to cyclosporin A or FK506 may be related to the In related studies, Aperia et al found that inhibition of calcineurin amount of calcineurin present. A second possibility is that there is phosphatase activity by FK506 reversed a adrenergic mediated differential expression of isoforms of calcineurin or immunophilstimulation of proximal tubule Nat'K-ATPase [17]. They em- ins in different nephron segments. For example, one isoform of phasized that changes in phosphatase activity can modify enzyme cyclophilin, cyclophilin C, appears to be absent from the liver and activity. Our studies extend these results in the following way by T cells of mice but is particularly abundant in the kidney [29]. A showing that the inhibitory influence of FK506 can be seen even third possibility is that cyclosporin A and FK506 interact to a when Nat'K-ATPase activity is not stimulated at least in the lesser or greater extent with various isoforms of Nat'K-ATPase. mTAL and CCD. Moreover, we found that FK506 does not affect However, we and others find no difference in a or J3 isoform Na/K-ATPase in the S2 segment possibly because there is subunit expression in microdissected CCDs, mTALs, and S2s increased expression of calcineurin. Thirdly, differences in the using RT-PCR and Western analysis [30, 31]. influence of other drugs in this category indicate that simply binding to immunophilins (that is, FKBP or cyclophilins) is Possible relationship between nephrotoxicity, inhibition of Na / insufficient to inhibit Nat'K-ATPase. K -A TPase activity, and immunosuppressive mechanisms Nephron segment-specific inhibition of Na t/KtA TPase

Initial clinical experience with FK506 suggested that it was less

It is intriguing that both FK506 and cyclosporin A [7] cause nephrotoxic than cyclosporin A [32]. However, more recent segment-specific inhibition of Nat'K-ATPase activity along the studies indicate equivalent degrees of nephrotoxicity between the

652

Lea et al: Calcineurin involved in Na/Kt-ATPase inhibition

two drugs [33, 34]. Our data indicate a greater degree of Na/

14. MIDDLETON JP, KFIAN WA, COLLIN5WORTH G, HANNUN YA, MED-

the same biochemical event, namely inhibition of caleineurin

16. BERTORELLO A, APERLA A: Na/K-ATPase is an effector protein for

FORD RM: Heterogeneity of protein kinase C-mediated rapid regulaK-ATPase inhibition in the CCD (Fig. 4) which is consistent with tion of Na/K-ATPase in kidney epithelial cells. J Biol Chem 268: the 100-fold greater potency of FK506 to inhibit IL-2 transcription 15958—15964, 1993 in T cells [9]. Moreover, the present results, along with our earlier 15. BERTORELLO A, APERIA A, WALAAS SI, NAIM AC, GREENGARD P: study [7], raise the possibility that both the immunosuppressive Phosphorylation of the catalytic subunit of Na7lC-ATPase activity in and nephrotoxic effects of cyclosporin A and FK506 result from renal tubule cells. Proc Nail Acad Sci USA 88:11359—11362, 1991

protein kinase C in renal proximal tubule cells. Am J Physiol 256:

activity.

Acknowledgments

This work was supported by National Institutes of Health grants R29-DK41707 and R01-DK45688. JPL performed this work during the tenure of an Institutional National Research Service Award from the National Institutes of Health (grant T32-DK07656). JMS performed

this work during the tenure of an Established Investigatorship from the American Heart Association. Portions of this manuscript were presented at the Annual Meetings of the American Federation for Clinical Research, Washington, DC, 1993, and the American Society of Nephrology, Boston, MA, 1993; and published in abstract form (Clin Res 41:326A, 1993 and JAm Soc Nephrol 4:871, 1993). The authors thank Dr. William F. Mitch for his critical reading of this manuscript.

Reprint requests to Dr. Janice P. Lea, Emoty University School of Medicine, Renal Division, 1364 Clifton Roa4 NE, Atlanta, Georgia 30322, USA.

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