Down-regulation of hepatic LDL receptor expression in experimental nephrosis

Kidney International, Vol. 50 (1996), pp.

88 7—893

Down-regulation of hepatic LDL receptor expression in experimental nephrosis NOSRATOLA D. VAZJRI and KAI Hui LIANG Division of Nephrology, Department of Medicine, University of California, Irvine, Irvine, California, USA

Down-regulation of hepatic LDL receptor expression in experimental nephrosis. Nephrotic syndrome (NS) is invariably associated with eleva-

tion of plasma total and LDL cholesterol concentrations. The present study was carried Out to test the hypothesis that nephrotic LDL hyper-

cholesterolemia is, in part, due to acquired LDL receptor (LDLR) deficiency. To this end, hepatic LDLR mRNA (Northern blot analysis) and protein mass (Western blot analysis) were measured longitudinally before and during the course of puromycin-induced NS. In addition, the rate of LDLR gene transcription by isolated hepatic nuclei was determined using nuclear run-on assay. Hepatic LDLR mRNA remained virtually unchanged during the 30-day course of the study period. However, after an insignificant rise on day 5, LDLR protein mass gradually declined to a level which was significantly below the baseline values (P < 0.05 ANOVA). This was accompanied by a normal rate of LDLR mRNA synthesis excluding impaired gene transcription as a cause. The fall in hepatic LDLR protein was associated with a marked rise in plasma total and LDL cholesterol concentrations but no rise in hepatic tissue choles-

LDL clearance is, in part, responsible for nephrotic hypercholes-

terolemia [9]. To our knowledge, the effect of NS on gene expression and production of LDLR has not been directly demonstrated. The present study was designed to test the hypothesis that NS results in down-regulation of LDLR expression which, in turn, contributes to the pathogenesis of hypercholesterolemia and dysregulation of cholesterol synthesis and catabolism. Methods Study groups

Male Sprague-Dawley rats (180 to 200 g) were purchased from Charles River Inc. (Wilmington, MA, USA) and maintained on

Purina Rat Chow (Purina Mills Inc., Brentwood, MO, USA). They were kept in a climate-controlled space with 12-hour light terol concentration. The latter observation is indicative of impaired hepatic cholesterol uptake and provides functional evidence for the

(500 lux) and 12-hour dark (5 lux) cycles. The rats were randomly

demonstrated acquired LDLR deficiency in the NS animals. Likewise, our findings elucidate the molecular basis of the previously reported impaired LDL clearance in NS. In conclusion, severe hypercholesterolemia in rats

assigned to the nephrotic (NS) and normal control (NL) groups.

with experimental NS is associated with and perhaps, in part, is due to down-regulation of LDL receptor expression.

Animals assigned to the NS group were given intraperitoneal (i.p.) injections of puromyein aminonucleoside (Sigma Inc., St. Louis, MO, USA), 130 mg/kg, on day 0 and 60 mg/kg on day 14. Animals

assigned to the control group were given placebo injections. All animals were provided free access to food and water. Subgroups of rats were studied at baseline (day 0) and on days 1, 5, 20, and Nephrotic syndrome (NS) is invariably accompanied by ele- 30 after the initial puromyein injection. Under general anesthesia vated plasma total and low-density lipoprotein (LDL) cholesterol (i.p. sodium pentobarbital, 50 mg/kg), the animals were sacrificed concentrations [1—4]. These abnormalities can potentially contrib- and liver was harvested for measurements of hepatie LDLR ute to cardiovascular complications and progressive renal insuffi- mRNA and protein mass. On each occasion, plasma concentraciency [5—10]. According to earlier studies, LDL synthesis is tions of albumin, total cholesterol, LDL cholesterol, triglycerides increased [4], and LDL clearance is decreased in NS [9]. Plasma and creatinine and 24-hour urinary excretion of protein and LDL concentration is a function of the rates of LDL synthesis and creatinine were determined using standard laboratory techniques. catabolism. Normally, 60 to 80% of LDL and 50% of VLDL are RNA preparation and Northern blot analysis cleared by the liver primarily via LDL receptor (LDLR) mediated The rats were killed between the hours of 8 to 10 a.m. The liver endocytosis. Thus, hepatic LDLR-mcdiated clearance plays a major role in LDL-cholesterol catabolism [11—14]. Primary LDLR was removed immediately, placed in liquid nitrogen and stored at deficiency in familial hypercholesterolemia is associated with —70°C until processed. Total RNA was prepared from I g of liver severe hypercholesterolemia [15]. In an earlier study, Warwick et by the guanidium thiocyanate extraction procedure as previously a! demonstrated a marked reduction in clearance of radiolaheled described [16]. RNA concentration was determined from the LDL in a group of patients with nephrotic syndrome [91. Based on absorbance at 260 nm using a spectrophotometer. Thirty microthis observation, they concluded that impaired receptor-mediated gram aliquots of total RNA were denatured in 2.2 M formaldehyde at 65°C for 15 minutes and run on 1.2% agarose/2.2 M formalde-

hyde gel at 70 V for three hours. The separated RNA was Received for publication December 6, 1995 and in revised form March 22, 1996 Accepted for publication March 25, 1996

transferred to the nylon membrane (Zeta Probe; Bio-Rad Laboratories, Inc., Hercules, CA, USA) by capillary blotting method in 6 x SSC buffer overnight and immobilized by UV irradiation (Ultraviolet Crosslinker; Fisher Scientific, Pittsburgh, PA, USA).

© 1996 by the International Society of Nephrology

The membrane was incubated at 65°C in a solution containing 5 X 887

888

Vaziri and Liang: LDL receptor deficiency in nephrosis

SSPE, 5 x Derihard's, 1% SDS and 100 jig/mI salmon sperm DNA. The cDNA probe for human LDLR (1.9 Kb BamHI fragment of PLDLR2) [17] and human /3-actin cDNA (1.1 Kb EcoRI fragment) were obtained from American Type Culture Collection (Rockville, MD, USA) and labeled with [32P] dCTP

Medical Industries Inc., Los Angeles, CA, USA) and centrifuged

at 800 g for 10 minutes at 4°C. The pellet was resuspended in

buffer A containing 0.5% Triton-100 (Sigma Inc.) and centrifuged as before. The pellet was resuspended in 2.2 M Sucrose-Tris buffer containing 10 mivi Tris-HCI (pH 7.4) and 5 m'vi MgCl2 layered on (3000 Ci/mmol; New England Nuclear Inc., Boston, MA, USA) by 2.3 M ice-cold Sucrose-Tris buffer and centrifuged at 35,000 g for the random primer method (Prime-A-Gene System, Promega 60 minutes at 4°C. The nuclei were then collected and washed in Biotec Inc., Madison, WI, USA). Hybridization was carried out at 3 ml of buffer A and suspended in a storage buffer (25% glycerol,

60°C overnight in prehybridization solution containing 32P-la- 20 mvi Tris, 1.5 mrvi MgCl2, and 0.2 mi EDTA, pH 7.9). A beled probes. The blots were washed twice in 2 X SSPE/0.5% SDS proteinase inhibitor preparation (0.2 mt PMSF, 2.5 jig/mI of solution at room temperature, twice in 1 X SSPE/0.5% SDS aprotinin/leupeptin; Sigma Inc.), DTT and RNasin (20 jig/mI; solution at 37°C, twice in 0.1 X SSPE/0.5% SDS solution at 65°C, Promega Biotec Inc.) were added through all steps. The concen15 minutes each. The washed blots were exposed to the X-ray film (New England Nuclear) at —80°C for six hours for actin and two days for LDLR. The autoradiograms were scanned with a laser densitometer (PD 1211; Molecular Dynamics, Sunnyvale, CA, USA) to determine relative mRNA levels. The values obtained for actin were used as internal control. Western blot analysis

These measurements were carried out to determine the LDLR protein mass. To this end, hepatocyte plasma membranes were prepared as follows. Frozen rat liver was homogenized in 20 mrvi

tration of nuclei was measured by lyzing an aliquot of the resuspended nuclei in 0.1% SDS and determining the absorbance at 260 and 230 nm. The nuclei were then frozen at —70°C until used. Labeling and isolation of nascent RNA transcripts. Equal numbers of nuclei were used in transcription reaction at 26°C for 45 minutes in a 400 jil solution of 40 nM Tris-HC1 (pH 8.3), 150 mtvi NH4C1, 7.5 m'vi MgC12, 400 ji RNasin, 2.5 ms't DTT, 0.625 mM

ATP, 0.313 mivi CTP, 0.313 m GTP (Boehringer Mannheim Corp., Indianapolis, IN, USA) and 100 jiCi (cs-32P)UTP (3000 Tris-HC1 (pH 7.5) containing 2 m'vi MgCI2, 0.2 M sucrose, 5 mM Ci/mmol, New England Nuclear). To block elongation of tranphenylmethylsulfonyl fluoride, 5 jig/ml leupeptin, 10 jig/mI apro-

scription by RNA polymerase II, ci-amanitin (Boehringer Mann-

centrifuged at 3000 g for 10 minutes at 4°C. The supernatant was

reaction was terminated on ice, and RNase-free DNase RQ-1

tinin and 3 jig/mI pepstatin A [181. The crude extract was heim Corp.) was used at a final concentration of 2 jig/ml. The (Promega Biotec Inc.) was added to reach a concentration of 100 jig/mI. After an incubation period of 10 minutes at room temperfuged at 35,000 g for 45 minutes at 4°C. Protein concentration was ature, I ml RNAzo1 (Tel-Test Inc., Friendswood, TX, USA) and determined by using a Bio-Rad kit (Bio-Rad Laboratories). 200 jil chloroform were added. The solution was vortexed and One-hundred microgram protein aliquots were size fractionated placed on ice for 15 minutes. After centrifugation at 10,000 g for 10 minutes, the aqueous phase was removed and an equal volume on 4 to 12% Tris-glycine gel (Novex Inc., San Diego, CA, USA) at 120 V for two hours. After electrophoresis, proteins were trans- of isopropanol was added for RNA precipitation overnight at ferred to Hybond-ECL membrane (Amersham Life Science Inc., —70°C. The precipitate was then collected by centrifugation at Arlington Heights, IL, USA). The membrane was incubated for 14,000 g for 15 minutes and washed twice in 75% ethanol and one hour in buffer A (1 X PBS, 0.1% Tween-20 and 10% nonfat resuspended in DEPC water. The 32P-labeled RNA was run over milk) and then overnight in the same buffer containing 5% nonfat a G-25 Sephadex quick-spin RNase-free column (Boehringer

then centrifuged at 35,000 g for 30 minutes. The membrane preparation was then washed with the above buffer and centri-

milk plus I jig/mI mouse anti-bovine LDLR antibody (Cortex

Mannheim Corp.). A I jil aliquot of eluate was counted with a

Biochem Inc., Davis, CA, USA). Membrane was washed once for liquid scintillation counter (LS 9000; Beckman, Placentia, CA, 15 minutes then twice for five minutes in I X PBS, 0.1% Tween-20 USA). prior to a one hour incubation in buffer A plus diluted (1:1000) Hybridization. The 32P-labeled RNA solution was heated at horseradish peroxidase-linked sheep anti-mouse IgG (Amersham 95°C for five minutes, then quickly chilled on ice before hybridLife Science). The washes were repeated before the membranes ization to specific eDNA sequences. The linearized LDL-reeeptor were developed with chemiluminescent agents (ECL; Amersham (PLDLR7) eDNA and /3-actin eDNA were denatured in 0.2 M Life Science) and subjected to autoradiography for five minutes.

NaOH; 5 jig of eDNA was applied to the nylon membrane and baked at 80°C for two hours. Prehybridization of the filters was Nuclear run-on assay Nuclear run-on assay was carried out using the following steps performed for two hours at 55°C in hybridization buffer: 5 X SSC, 50% formamide, 5 X Denhard's reagent, 0.1% SDS and 200 as previously described [19]. Isolation of ral liver nuclei. Hepatie nuclei were purified for in jig/ml polyadenylic acid. An equal number of radioactive counts vitro transcription studies. To this end, rat liver (2 g) was of the 32P-RNA (1.5 >< 10) were added to freshly made hybridhomogenized in 10 volumes of buffer A (10 mrvi Tris-HCI [pH 7.I,

ization buffer, and the mixture was incubated for 48 hours at 55°C.

1 mM EDTA, 0.25 M sucrose, 5 mM MgC12) using a polytron After hybridization, the strips were washed twice at 55°C for 15 homogenizer (Brinkmann Instruments Inc., Westbury, NY, USA) minutes each in 2 x SSC, 0.1% SDS and treated with RNase A at setting 5 for 15 seconds. The homogenate was then centrifuged (10 jig/mI; Boehringer Mannheini Corp.) at 37°C for 30 minutes, at 800 g for 10 minutes at 4°C. The pellet was resuspended in 4 ml then washed with 2 x SSC twice at 37°C. Radioactive RNA bound of buffer A and further homogenized using a Dounce homoge- to the various filters was quantitated by liquid scintillation countnizer (Fisher Scientific). The homogenate was then filtered ing. The values were corrected for nonspecific binding measured through 2 nylon filters (pore size, 210 jim and 53 jim; Spectrum in the blank filters.

889

Vaziri and Liang: LDL receptor deficiency in nephrosis

Table 1. Plasma concentrations of albumin and creatinine, 24-hour urinary protein excretion and creatinine clearance at baseline (day 0) and on

days 1, 5, 20 and 30 post-initial puromycin injections

Day I

Day 5

(N_=_6)

(N_=_6) 3.8 0.1 7.2 1.4 0.56 0.07 1.15 0.15

(N_=_6) 2.1 0.12 170 14.3 0.66 0.08 1.0 0.10

3.4

Plasma albumin mg/dl Urine protein mg/24 hrs Plasma creatinine mg/dl Creatinine clearance mI/mm

Day 20

Day 0 0.1

7.6 0.7

0.62 0.06 1.2

0.1

(N = 6) 2.0

P values

2.3 213 0.65 1.3

0.11

258 32 0.67 1.2

Day 30

(N = 6)

0.07 0.11

0.001

0.22

0.001

18

NS NS

0.08

0.16

Data are expressed as mean SEM. P 0.001 (one-way ANOVA and Student's t-test)

0

450

5

360

4.

I

ci)— 270

oE E

E 180

E 2' cci

cci

0

I.

(ci

(I)

90

U

cci

a-

0 DO

Dl

D5

D20

D3O

0

Fig. 1. Plasma concentrations of total cholesterol ([—J), LDL cholesterol —. ]) at baseline (DO) and on days J), and triglycerides (f-.([.

0

1, 5, 20 and 30 (Dl, D5, D20 and D30). A minimum of six rats were studied

at each point. Data are given as mean

SEM. *P < 0.001 (multiple

100

200

300

400

400

measure ANOVA).

- 300 Statistical analyses

Multiple measure analysis of variance (ANOVA), Students' t-test and regression analysis were used in statistical evaluation of the data which are expressed as mean SEM. P values equal to or less than 0.05 were considered significant.

Results

E 200

?5 100

General findings

Data are depicted in Table 1 and Figure 1. The NS group

0

exhibited marked proteinuria and hypoalbuminemia beyond day 5

400 200 300 100 0 of puromycin administration. This was accompanied by severe Plasma LDL cholesterol, mg/dI elevations of plasma total cholesterol and LDL-cholesterol concentrations and a modest rise in plasma triglyceride concentration Fig. 2. Correlations between plasma LDL cholesterol concentration (r = (Fig. 1). Plasma LDL cholesterol concentration directly correlated —0.89, P < 0.001) and urinary protein excretion (r = 0.85, P < 0.001) in the with urinary protein excretion (r = 0.85, P < 0.001) and negatively nephrotic ( and normal control (•) groups obtained dunng the study correlated with plasma albumin concentration (r = —0.89, P < period.

0.001; Fig. 2). No significant difference was found in hepatic tissue

cholesterol concentration between the NS and the normal control

groups (Fig. 3). There was no significant difference in serum creatinine concentration between the NS and the normal control group. Likewise, creatinine clearance values were comparable in the two groups.

LDLR mRNA and protein Data are shown in Figures 4, 5 and 6. No significant difference was observed in hepatic LDLR mRNA levels between the normal control group and the subgroups of the NS animals studied at days

890

Vaziri and Liang: LDL receptor deficiency in nephrosis

I>

A

I

4

w

I I

I

0

N)

C.)

a 0

N)

0

-4 C)

0

3

III. N)

0

-&

LDL R/acttn niRNA ratio

C.)

N)

Fig. 4. A. A representative autoradiograph of the Northern blot of hepatic LDL receptor (LDLR) and the cotresponding actin in the normal control (NL) (lane 1) and the nephrotic rats (NS) at days 5 (lane 2), 20 (lane 3), and 30 (lane 4) in the course of induction of nephrosis with two puromycinaminonucleoside injections given on days 0 and 14. Total RNA (30 g/!ane)

was isolated from liver, as described under the Methods section, and

0

Liver cholesterol concentration myig wet tissue

2

•,-28S

0

Plasma cholesterol mgld/

a C 0

I

separated by electrophoresis on 1.2 agarose/2.2 M formaldehyde gel. RNA was transferred to a nylon filter and hybridized with the 32P-labeled 1.9 Kb

Fig. 3. Comparison of plasma and hepatic tissue cholesterol concentrations in the nephrotic (NS) animals (day 30) and the normal control group (NL). Data are given as mean SE.M (N = 6 in each group). *P < 0.00!.

1, 5, 20 or 30. Thus, LDLR mRNA levels remained virtually unchanged during the induction (days 1 to 20) and chronic phase (day 30) of puromycin-induced NS. In contrast, after an insignificant rise at day 5, hepatic LDLR protein measured by Western blot gradually declined during the observation period reaching its

eDNA to human LDLR mRNA and 1.1 Kb eDNA to human -actin mRNA used to control for possible variations in the loading dose. B. Longitudinal measurements of LDLR mRNA (normalized against corresponding actin mRNA) in the norma! control (NL, N = 6) and subgroups of ncphrotic (NS, N 6 in each subgroup) animals studied at the above time points. Data are given as mean SEM.

1

LDLR —

23 4

5

e—

—200 kDa

lowest point on day 30 (P < 0.05 ANOVA). There was no significant change in either hepatic LDLR mRNA or protein 24 hours after puromycin injection (Figs. 4 and 7). This observation tends to exclude the possible acute effect of puromycin on LDLR protein production in the study animals. Plasma LDL cholesterol concentration was inversely related to hepatic LDLR level (r = 0.80, P < 0.01; Fig. 8). LDLR gene transcription rate

In an attempt to explore the cause of depressed LDLR protein expression in the NS animals, we measured the rate of LDLR gene transcription using isolated liver nuclei in a nuclear run-on

Fig. 5. A representative autoradiograph of the Western blot of hepatic LDL receptor (LDLR) in the normal control (NL) (lane 1) and the subgroups of nephrotic rats studied at days I (lane 2), 5 (lane 3), 20 (lane 4), and 30 (lane 5) in the course of induction of nephrosis with two puromycin-aminonucleoside injections given on days 0 and 14. Hepatocyte plasma membrane (100 jig/lane) was isolated from liver as described under the Methods Section, and separated by eleetrophoresis on 4 to 12% Tgris-g!ycine gel. Protein was transferred to Hybond-ECL membrane and hybridized with I jig/mI mouse anti-bovine LDLR.

Discussion assay. The results are depicted in Table 2. As can be seen, the rate The occurrence of proteinuria and hypoalbuminemia in rats of LDLR gene transcription in the NS group was similar to that found in the normal control group. This finding excludes impaired assigned to the NS group was accompanied by an expected rise in gene transcription as a cause of the LDLR deficiency in the NS plasma total cholesterol and LDL-cholesterol concentrations. The elevation of total and LDL-cholestcrol levels in the study animals animals.

891

Vaziri and Liang: LDL receptor deficiency in nephrosis

3,

A

Dl

F

C

NL

—28S

za:

LDL R

.. E

_; —======*===

a: -J

0-j

Actin

S

B

lii

0'

.. E

I

2

a: -J

3OO

0-J

0—

o

U_jC

M

wtwai mI*4A

za:

400

H

3

Dl

NL

Fig. 7. A. A representative Northern blot of hepatic LDL receptor (LDLR) and the corresponding actin in the normal control (lane 1, NL) and at 24 hours after puromycin (130 mg/kg) injection (Dl). B. Comparison of hepatic

200

LDLR/actin mRNA obtained before and 24 hours after puromycin

IIII

100

01

5

injection (N = I

6

in each group). Data are given as mean

SEM.

I

20 10 Time, day

500

30

Fig. 6. Longitudinal measurements of LDLR protein in the normal control (•, N = 6) and subgroups of nephrotic (U) rats studied at baseline (day 0)

and on days 1, 5, 20 and 30 (N = 6 at each time point). Data are given as mean SEM. p < 0.05 (multiple measure ANOVA).

was associated with a normal hepatic tissue cholesterol concentration. The normality of hepatic tissue cholesterol concentration despite severe hypercholesterolemia suggests that hepatic uptake of LDL particles may be defective in the NS animals. If true,

impaired receptor-mediated hepatic uptake, which is a major pathway of LDL catabolism, may, iii part, contribute to the associated hypercholesterolemia. In addition, such a defect may play a role in dysregulation of hepatic cholesterol biosynthesis [20,

21] and catabolism [22] in this model. In this regard, we have found an inappropriately elevated HMG-CoA reductase, an inappropriately depressed hepatic cholesterol 7a-hydroxylase, and a normal hepatic tissue cholesterol concentration in animals with NS as compared to the control animals with equally severe dietary

400 q) 31)

(/)jj

$

.

U

a: (j) 200 _J

U.

100

0 0

I

100

200

300

400

Plasma LDL cholesterol, mg/dI

Fig. 8. Correlation between plasma LDL cholesterol concentration and hypercholesterolemia. In contrast to the NS animals, the latter hepatic LDL receptor (LDLR) obtained in the nephrotic (U) (day 30) and animals exhibited a markedly increased hepatic cholesterol con- the normal control (•) groups (r = 0.80, P < 0.01).

centration, an appropriate down-regulation of HMG-CoA reduc-

tase and upregulation of cholesterol 7a-hydroxylasc [21, 221. Dysregulations of HMG-CoA reductase and cholesterol 7a-hy- extracellular cholesterol modulates gene expressions of these key droxylase in the NS animals was thought to be, in part, related to enzymes in cholesterol metabolism to maintain cholesterol hothe disproportionately low intracellular-to-plasma cholesterol meostasis [23—25]. Thus, if present, impaired hepatic LDL uptake concentration ratio. This is because intracellular rather than precludes appropriate down-regulation of endogenous production

892

Vaziri and Liang: LDL receptor deficiency in nephrosis

Table 2. Relative transcription rates of the hepatic LDL receptor gene and -actin gene in normal control (NL) and nephrotic (NS) rats Counts/minute hybridized to PLDLR2 eDNA ______ NL NS

Counts/minute hybridized to

-actin eDNA _______

per 10 cpm total RNA

720 87 856 26

772 109 947 33

down regulation of hepatic LDLR production. The demonstrated acquired LDLR deficiency in the NS animals may contribute to the associated hypercholesterolemia in a qualitatively analogous manner to that of primary LDLR deficiency in familial hypercholesterolemia.

PLDLR2/-actin cDNA (ratio) 0.95 0.91

0.02 0.04

Acknowledgment

The authors are grateful to Dr. Fadia Haddad for her technical assistance with the nuclear run-on assay.

Counts hybridized to specific eDNA sequences were corrected by subtracting background counts hybridized to filter containing the vector DNA (PcD). The results are expressed as mean SEM (N = 6, P> 0.1).

Reprint requests to N.D. Vaziri, M.D., Division of Nephrology, Department of Medicine, UCI Medical Center, 101 The City Drive, Orange, California 92668, USA.

(via inhibition of HMG-CoA reductase) and up-regulation of catabolism (via stimulation of cholesterol 7a-hydroxylase) of cholesterol, steps which are necessary for mitigating the associ-

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