EXPERIMENTAL AND MOLECULAR PATHOLOGY 27, 377-391 ( 1977)

Effect of Chronic Renal Failure in Rats on Structure and Function of the Hepatic Endoplasmic Reticulum1

MARTIN BLACK,? LUIS BIEMPICA, SIDNEY GOLDFISCHER,

STANLEY GROSSMAN, AND IRWIN M. ARIAS 3

The Liver Research Center and Departments of Medicine and Pathology, Albert Einstein College of Medicine, New York, New York 10461

Received January 18, 1977, and in revised form March 30, 1977

Functional, histochemical, and ultrastructural studies of the liver were performed in rats rendered chronically azotemic following unilateral nephrectomy and segmental infarction of the remaining kidney. Zoxazolamine paralysis time and ketamine nar- cosis time were significantly prolonged; cytochrome P-450 content and benzpheta- mine and aminopyrine demethylase activities were decreased; and o-aminophenol UDP-glucuronyl transferase activity was unaltered. Histochemical examination of the liver by light microscopy revealed little abnormality except for lipid accumulation associated with increased serum cholesterol and triglyceride concentrations. Electron microscopy demonstrated alterations in endoplasmic reticulum and lysosomes. Di- chloroethane treatment of uremic rats prevented the ultrastructural changes and enhanced drug-metabolizing enzyme systems. These observations indicate that ex- perimental chronic renal failure influences the structure and function of the hepatic endoplasmic reticuhrm and support the hypothesis that some features of the uremic syndrome in man may result from altered hepatic subcellular function.

INTRODUCTION

The hepatic endoplasmic reticulum is a dynamic organelle which is responsible for the synthesis of many proteins, carbohydrates, and lipids and for biological transformation of endogenous and exogenous substrates including steroids, sterols, hormones, drugs, and chemicals (Ma et al., 1972; Moule, 1968; Camp- bell and Lawford, 1968, Orrenius et al., 1968). Its function is controlled by genetic ( Nebert and Bausserman, 1970), hormonal (Song and Kappas, 1970), dietary (Century, 1973), and other exogenous factors (Conney, 1967; Kuntz- man, 1969). Little information exists concerning the influence of renal disease upon its structure and function.

Several features associated with the uremic syndrome could theoreticahy result from changes in the function of this organelle. The uremic syndrome is manifested by aIterations in the metabolism of vitamins, hormones, proteins, lipids, steroids, and drugs. To determine whether any of these alterations result from changes in the function of the hepatic endoplasmic reticulum, studies were

1 Research supported by research grants from NIH (AM 02019, 05384, 17702, and 16281). a Present address: Temple University School of Medicine, 3420 North Broad Street,

Philadelphia, Pennsylvania 19140. 3 Author to whom reprint requests should be addressed.

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

37 7

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37s BLACK ET AL.

performed in rats rendered azotemic by unilateral nephrectomy and segmental infarction of the remaining kidney (Bricker et al., 1964; Lubowitz et al., 1971). This experimental model of chronic renal failure has been extensively investi- gated with respect to residual renal function (Bricker et al., 1964; Gonick et al., 1964; Hayslett et al., 1968, 1969; Lubowitz et al., 1971).

MATERIALS AND METHODS

Male Holtzman rats weighing 180-200 g were subjected to segmental in- farction of the left kidney and contralateral nephrectomy (Bricker et al., 1964; Lubowitz et al., 1971) resulting in the ablation of 60-75s of the original kid- ney mass. Control animals underwent sham operations consisting of bilateral flank incisions and manual manipulation of the kidneys. Following operation, both groups of rats were housed in general purpose cages and given a standard Purina Chow diet and water ad libitztm. NaCl (0.9% ) was added to the drink- ing water from the seventh postoperative day to avoid sodium deficiency (Lubowitz et al., 1971). B ecause cytochrome P-450-mediated drug metabolism is increased in the liver of normal rats treated with dichloroethane (DDT), a group of uremic and sham-operated rats received DDT dissolved in corn oil (25 mg/kg body weight) by intraperitoneal injection on 5 successive days from the fifteenth postoperative day. Other groups of uremic and sham-operated rats received similar volumes of corn oil.

Drug metabolism studies in uiuo were performed 20 days after operation, and the rats were decapitated 24 hr later. Blood was collected for determination of serum urea nitrogen, creatinine, cholesterol, and triglyceride concentrations. The liver and kidney were rapidly removed and weighed, and portions were taken for cytochemical and electron microscopic studies. Weighed aliquots were minced with ice-cold 250 ma1 sucrose in 10 mM phosphate buffer (pH 7.4), and 33% homogenates were prepared at 4°C using a motor-driven Teflon pestle homogenizer (A. H. Thomas Co., Philadelphia, Pa. ) . Liver and kidney homogenates were centrifuged at 13,OOOg for 20 min at 4°C in a Sorvall re- frigerated centrifuge. The supernatant fraction was centrifuged at 105,OOOg for 90 min at 4°C in a Model L Spinco ultracentrifuge. The microsomal pellets were washed and resuspended in 154 mill KC1 for cytochrome P-450 and o-aminophenol UDP-glucuronyl transferase determinations, and in 50 rnnl Tris (pH 7.5) for determination of aminopyrine and benzphetamine demethylase activities. Protein concentrations were measured (Lowry et al., 1951).

Intraperitoneal injection of zoxazolamine produces paralysis, the duration of which is inversely proportional to the rate at which the drug is metabolized by the hepatic cytochrome P-450 hydroxylating system (Trevor, 1971) , Zoxazolamine (80 mg/kg body weight) was administered intraperitoneally, following which the rats became paralyzed and were placed in a supine position. The time re- quired for resumption of an erect posture was recorded. Uremic and sham-oper- ated rats were decapitated at the time of recovery from zoxazolamine-induced paralysis; blood was promptly obtained by cardiac puncture, and serum zoxa- zoIamine concentrations were determined spectrophotometrically (Kunin et OZ., 1959). Ketamine hydrochloride (30 ing/lig body weight) was injected intra- pcritoneally, and the time take11 for tile rats to regain their normal posture \vas recorded ( Kuntzman, 1969).

UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 379

For conventional light microscopy, livers were rapidly fixed in cold 4% formaldehyde with 1% CaClz and incubated overnight at 4°C in 4% formalde- hyde with 1% CaC12. For cytochemical studies, incubation was in 2.5% glu- taraldehyde in cacodylate buffer, pH 7.2 (Sabatini et al., 1963), for 3 hr at 4°C. For ultrastructural studies, fixation was performed in 2% osmium tetroxide in phosphate buffer, pH 7.2 (Millonig, 1962), for 2 hr.

Light microscopy studies were performed on l- to 2-p-thick sections of Epon-embedded tissues stained with toluidine blue and on sections of paraffin- embedded tissues stained with hematoxylin-eosin and Mallory trichrome as well as for reticulin and iron. Frozen sections of 10-p thickness were stained for lipid with oil red 0 and were also incubated at 37°C for various enzymatic markers of cytoplasmic organelles: for nucleoside triphosphatase activity (plasma mem- brane and its specialized areas) using adenosine triphosphate as substrate ( Wachstein and Meisel, 1957)) pH 7.2, incubated for 15 and 30 min; for nucleo- side diphosphatase activity (endoplasmic reticulum) using inosine diphosphate as substrate (Novikoff and Goldfischer, 1961)) pH 7.2, incubated for 10 and 30 min; for thiamine pyrophosphatase activity (Golgi apparatus) using thiamine pyrophosphate as substrate (Gomori, 1952), pH 7.3, incubated for 20 and 45 min; for acid phosphatase activity (lysosomes) using p-glycerophosphate as substrate (Gomori, 1952), pH 5.0, for 15 and 30 min; for tetrazolium reductase activity (mitochondria) using nicotinamide adenine dinucleotide-tetranitro blue tetrazolium as substrate (Novikoff et al., 1961), pH 7.2, incubated for 10 and 20 min; for cytochrome oxidase (mitochondria) using 3,3’-diaminobenzidine (DAB), pH 6.0 ( Novikoff and Goldfischer, 1969), incubated for 10 and 60 min; and for catalase (peroxisomes) using DAB, pH 9.0 (Novikoff and Goldfischer, 1969), incubated for 60 to 90 min.

For ultrastructural studies, Epon-embedded tissues were sectioned at 0.5-l 1~ and stained with toluidine blue. The lobular area was identified in each case. Further thin-sectioning was performed using a diamond knife and an MTSB ultramicrotome. Sections were mounted on copper grids and stained with uranium ( Wachstein and Meiser, 1957) and lead (Reynolds, 1963), and coded specimens were blindly studied using a Siemens Elmiskop I or an RCA 3C electron microscope. All specimens for morphologic study were coded prior to examination, and the code was revealed after detailed pathologic description was recorded. Ultrastructural studies involved hepatocytes in comparable loca- tions within the hepatic lobule.

The cytochrome P-450 concentration in liver microsomal suspensions was determined by difference spectrophotometry using a Carey spectrophotometer as described by Omura and Sato (1964). M icrosomal suspensions were reduced with sodium dithionite and equally divided between two cuvettes, one of which was gassed with carbon monoxide. The reduced hemoprotein-carbon monoxide complex was detected by different spectroscopic scanning from 500 to 400 nm. An extinction coefficient of 91 for cytochrome P-450 was used.

Aminopyrine demethylase activity of microsomal suspension was measured by the method of Ernster and Orrenius (1965). The incubation mixture con- tained 10 mR1 aminopyrine, 5 mAf MgCIZ, 2 mR1 TPNH, and 1.5 mg of micro- somal protein/ml. The reaction was continued for 30 min and terminated by the addition of 20% trichloracetic acid. Formaldehyde production was mea-

380 BLACK ET AL.

sured by the Nash reaction (Nash, 1953). Benzphetamine demethylase activity was measured in a similar manner; the concentration of benzphetamine in the incubation mixture was 10 m&‘.

o-Aminophenol UDP-glycuronyl transferase activity of microsomal prepara- tions was determined by the method of Levvy and Storey (1949). The incuba- tion mixture contained 0.14 mA1 resublimed o-aminophenol, 1 rnnl uridine di- phosphoglucuronic acid, 500 mh1 Tris buffer, pH 7.4, 150 mJ1 MgCIz, and 1 mg/ml of microsomal protein. Incubation was for 20 min at 37°C.

Serum urea nitrogen, creatinine, cholesterol, and triglycerides were measured using standard Auto Analyzer procedures.

RESULTS

Following complete or sham operation, all animals lost weight for 48-72 hr (Fig. 1). Uremic rats in which the amount of infarcted left kidney was probably excessive continued to lose weight and died 4-7 days after operation. Surviving rats with renal failure gained weight during the subsequent 3-4 weeks at the same rate as sham-operated rats gained weight. Rats with a serum urea nitrogen concentration of 45 mg/lOO ml or greater were included in the experimental uremic group.

Rats with renal failure showed prolongation of zosazolamine paralysis time. Mean zoxazolamine paralysis time in 45 rats with renal failure was 6.6 2 0.3 (SE) hr compared with 4.2 * 0.2 h r in 27 sham-operated rats (P < 0.01). Mean blood zoxazolamine concentration on recovery from paralysis was 17.4 * 1.3 (SE) pg/ml in uremic rats and 18.1 +- 1.3 pg/ml in sham-operated rats indica- ing that both groups of rats recovered from paralysis at approximately the same plasma level of zoxazolamine. Pretreatment with DDT reduced zosazolamine paralysis in eight rats with renal failure (1.8 I- 0.2 hr, P < 0.01) and in eight sham-operated rats (2.3 +- 0.2 hr, P < 0.01).

Ketamine narcosis time in 34 rats with renal failure was 50.0 I+ 3.0 min com- pared with 40.2 * 4.3 min in 19 sham-operated rats (P > 0.01). Ketamine nar- cosis time was shortened in six uremic rats given DDT (38.5 * 14.3 min, P < 0.01) and in five sham-operated rats given DDT (27.3 * 2.3 min, P < 0.01).

Experimental renal failure resulted in increased serum concentrations of urea nitrogen, creatinine, cholesterol, and triglycerides (Table I). Pretreatment with DDT did not affect these results in rats either subjected to sham-operation or production of renal failure.

No significant difference between uremic and sham-operated rats was ob- served for liver weight/body weight ratio. Treatment with DDT increased the ratio (P < 0.05). In the uremic group, the kidney weight/body weight ratio was, as expected, reduced (P < 0.01) and was unaffected by DDT treatment. Mean liver microsomal protein concentration was unaffected by renal failure when compared with sham-operated rats but was increased in renal failure- DDT-treated rats (P < 0.05) and sham-operated-DDT-treated rats (P Y 0.01).

The mean hepatic microsomal cytochrome P-450 concentration was reduced in uremic rats as compared with sham-operated rats (P < 0.01) (Table II) and correlated in\.crsrly with zoxazolamiue pmdysis time ( r = 0.74) (Fig. 2). 111 12 operated rats with a serum urea nitrogen level less than 45 mg/lOO nil,

UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 381

TABLE I

Serum Urea Nitrogen, Creatinine, Cholesterol, and Triglyceride Concentrations in Experimental and Sham-Operated Rats with and without Pretreatment with DDTe

Group of rats Urea nitrogen Creatinine Cholesterol Triglyceride (mg/lOO ml) (mg/lOO ml) (mg/lOO ml) (mg/lOO ml)

Range Mean

Renal failure, no treatment

Sham-operated, no treatment

Renal failure, DDT treatment

Sham-operated, DDT treatment

45-212 72.6 f 3.3’ 1.4 f 0.16 104 f 8.YJ 113.5 i 10.4b (n = 74) (n = 44) cn = 27) (n = 28)

14- 35 23.7 f 0.66 0.8 f. 0.04 5.5 i 4.8 46.6 f 6.0 (n = 49) (n = 22) (n = 7) (n = 7)

47-235 73.6 f 14.1b 1.9 f 0.15* 108 & 14..Yib Not performed (n = 20) (n = 9) (n= 7)

12- 27 19.5 f. 1.3 0.6 i 0.05 63.3 & 4.2 Not performed (n = 16) (n = 9) (n = 6)

a DDT was administered on Days 15-19 following surgery. All subsequent, measurements were made on Day 21. See text for further details.

6 P < 6.01 as compared with resulk in sham-operated, no treat,ment rats.

hepatic microsomal cytochrome P-450 concentration was 0.398 -t- 0.022 nmoles/ mg of protein, which was not significantly different from the results in sham- operated rats. Treatment with DDT increased hepatic cytochrome P-450 con- centrations in uremic and sham-operated rats to levels found in control rats (Table II).

Aminopyrine and benzphetamine demethylase activities were significantly less in rats with renal failure than in sham-operated rats (P < 0.01) (Table II). DDT pretreatment increased enzyme activities in rats with renal failure and obliterated differences between results in untreated uremic and sham-operated rats. Hepatic o-aminophenol UDP-glucuronyl transferase activity was not sig- nificantly different in rats with renal failure and sham-operated rats; DDT treat- ment increased enzyme activity in both groups of rats (Table II).

Using conventional tissue stains for light microscopy examination, no differ- ences were found between livers of sham-operated and renal failure rats. Each contained isolated necrotic hepatocytes. Cytochemical methods for enzymatic staining of plasma membrane, mitochondria, peroxisomes, lysosomes, and endo- plasmic reticulum failed to identify major differences between the two groups. Several rats with renal failure showed loss of pericanalicular arrangement of lysosomes. Staining of lipids in rats with renal failure revealed an increased number of small lipid droplets without specific intralobular localization which was independent of serum urea nitrogen concentration. Most lipid droplets were 0.5 to 2 p in diameter and were localized at the sinusoidal aspect of hepatocytes.

Ultrastructural alterations in rats with renal failure consistently affected hepatocytes throughout the IobuIe (Figs. 3 and 4). Rough and smooth endo- plasmic reticulum showed several alterations. Changes in rough endoplasmic reticulum were more evident in centrolobular hepatocytes. Loss of parallel

382 BLACK ET AL.

140 -

120 UREMIC

100

A 80 INITIAL

WEIGHT

6.

(Gm) 40

20

0

-20

-40

0 4 8 12 16 20 24 28 32

DAY POST-Of?

FIG. 1. Weight gain in uremic and sham-operated rats following operation. Points represent mean and vertical lines represent standard error of the mean in 72 uremic rats and 49 sham- operated rats.

arrays of cisternae was common and associated with fragmentation, cisternal dilatation, and apparent increase in free ribosomes. Segmental loss of attached ribosomes was observed in the majority of hepatocytes (Figs. 4 and 5). Smooth endoplasmic reticulum revealed areas of hyperplasia in the majority of rats with renal failure. Glycogen was diminished and appeared only as individual rosettes through the cytoplasm (Fig. 4). Autophagic vacuole types of lysosomes which contained mitochondria, endoplasmic reticulum, or perosisomes in various stages of degradation (Fig. 4) were increased, whereas dense-body and multivesicular- body types of lysosomes were seen as frequently as in control rats. Abundant electron-dense lipid particles resembling very low density lipoproteins (VLDL) with a diameter of 60-100 nm were consistently observed in Golgi saccules and

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ZOXAZOLAMiNE PARALYSIS TIME (HiS) 9

FJ~. 2. Comparison of zokazolalllinc paralysis tiinc aucl Iiepatic cytochro~~lc I’-450 cc)lllc,llt ( L’ = 1 nanomolc/n~illigran~ of protein) in individual rats.

UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 383

Liver Microsomal Enzyme Activities in Experimental and Sham-Oper:tletl Rats with and wit,hout Pretreatment with DDTa

- Groups of rats Cytochrome Benzphetamine Aminopyrine 0-Aminophenol

P-450 demethylase demethylase UDPGT (nmole/mg (mg of formalde- (mg of formalde- (pg of O-amino- of protein) hyde/mg of hyde/mg of phenol glucuro-

protein/ protein/ nide/mg of pro- min) min) tein/min)

- Renal failure, 0.303 zt O.Olb 2.19 f 0.2b 4.73 f 0.32" 0.062 f 0.005

no treatment * (n = 74) (n = 40) (n = 47) (n = 20)

Sham-operated, 0.428 zk 0.01 3.21 z!z 0.48 5.79 f 0.39 0.058 f 0.006 no treatment (n = 41) (n = 18) (n = 26) (n = 15)

Renal failure, 1.004 f 0.06b 3.59 f 0.W 8.08 f 0.52* 0.075 f 0.009b DDT treatment (n = 20) (n= 9) (n = 11) (n = 7)

Sham-operated, 0.949 f 0.05b 3.39 * 0.39 7.31 xk 0.81b 0.092 f 0.006b DDT treatment (n = 16) (n = 9) (n = 10) (n = 14)

5 DDT was administered daily from Days 15 to 19. AI1 rats were killed by decapitation on Day 21. Numbers in parentheses indicate numbers of rats studied. See text for further det,ails.

b P < 0.01 as compared with results in sham-operated, no treatment rats.

secretory vacuoles (Song and Kappas, 1970; Trump et al., 1973) near the sinusoidal aspect of the plasma membrane and in the space of Disse (Fig. 6). Lipid particles were also present in intercellular spaces (Fig. 6). Mitochondria frequently showed bizarre forms (Fig. 4), and cristae were often laterally dis- placed. Peroxisomes were unchanged in population, morphology, or distribution. The plasma membrane of hepatocytes was morphologicaIIy unakered.

In DDT pretreated rats, no substantial cytochemical or ultrastructural differ- ences were present between rats with renal failure and controls. Proliferation of the smooth endoplasmic reticulum was pronounced in both groups treated with DDT, especially in centrolobular hepatocytes (Fig. 7).

DISCUSSION

The metabolism of several endogenous and exogenous substrates of enzymes primarily located in the hepatic endoplasmic reticulum is impaired in chronic renal failure. These include vitamin D (Avioli et af., 1968; DeLuca, 1971) and cortisol (Englert et al., 1958). Abnormalities in the half-lives of several drugs metabolized by liver microsomal enzymes occur in uremic subjects including sulfasoxazole (Anton and Corey, 1971), chloramphenicol (Kunin et al., 1959), and Pentothal (Richards et al., 1953). Antipyrine metabolism was normal in a group of uremic subjects (Lichter et al., 1973); however, since many of these patients were receiving other medications which influence drug metabolism, an underlying defect could not be excluded. Possible involvement of the hepatic endoplasmic reticulum in uremia is also suggested by analysis of plasma lipids and albumin synthesis in patients with chronic renal failure (Bagdade, 1970; Coles et al., 1970).

384 BLACK EY’ AL.

The rats with experimental chronic renal failure used in this study gained weight at a normal rate postoperatively and appeared healthy. Cytochemical and morphological studies of liver revealed morphologic alterations, particu- larly affecting the endoplasmic reticulum, characterized by vesiculation and loss of organized cisternal arrangement primarily in centrolobular hepatocytes. Similar morphologic alterations occur in other experimental and pathological conditions (Schmukler and Arcasoy, 1969; Trump et al., 1973). Changes in cytoplasmic lipid were frequent in rats with renal failure. Large lipid droplets, considered to be triglyceride-rich “storage” fat ( Roheim et al., 1971), were numerous in livers from rats with renal failure, which also revealed 60 to lOO-nm electron-dense particles in intercellular spaces and the space of Disse. These particles are probably VLDL carriers for triglycerides (Hamilton et al., 1967) and are associated with enhanced lipid turnover (Hamilton et al., 1967) and hyperlipidemia in uremic humans (Bagdade, 1970) and rats (Biempica et al.,

FIG. 3. Control, sham-operated rat. Hepatocyte, centrolobular area. Cytoplasmic organelles have a normal appearance. Rough endoplasmic reticulum (RER) is abundant and organized in parallel arrays. The smooth endoplasmic reticulum (SER) is associated with glycogen rosettes (G) and prevails near the plasma membrane. Mitochondria (M) display transverse cristae, tend to be round, and are uniformly distributed. Peroxisomes (P) show frequent nucleoids. Lysosomes (L), an inconspicuous Golgi apparatus (GA), and bile canaliculus (BC) are also identified. x9450.

UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 385

FIG. 4. Rat with renal failure. Hepatocyte, centrolobular area. The endoplasmic reticulum is altered. RER cisternal arrays are absent, and Inany cistemae are dilated (arrows). Other areas of the ER show vesiculation (arrowheads), and SER areas are enlarged. Autophagic vacuoles (AV) are increased, and the Golgi apparatus (GA) displays numerous VLDL particles within saccules and vacuoles. X11,200.

1971). Autophagic vacuoles containing partially altered organelles were in- creased in rats with renal failure reflecting rapid turnover of cytoplasmic com- ponents, usually associated with subcellular damage (DeDuve and Wattiaux, 1966). The alterations in hepatic mitochondria observed in rats with chronic renal failure differ from abnormalities reported by Heitz et al. (1971) which include increased mitochondrial size and mass and reduced volume of lyso- somes, Golgi apparatus, lipid droplets, and glycogen per hepatocyte.

Zoxazolamine and ketamine require the hepatic endoplasmic reticulum for metabolism. The pharmacologic effect of both drugs was prolonged in rats with chronic renal failure. Test and control animals recovered from the pharma- cologic effect at virtually identical plasma levels of zoxazolamine.

Studies of enzyme systems located principally in the hepatic endoplasmic reticulum revealed reduced cytochrome P-450 concentration and benzphetamine demethylase and aminopyrine demethylase activities. o-Aminophenol UDP- glucuronyl transferase activity was not decreased in uremic liver, thereby simu- lating the situation in cholestasis where cytochrome P-450 concentration is

3kxi BLACK L;‘1’ AL.

decreased ( IIutterctr El al., 1X2) and ghicuronidaliug enz)‘mc acli\,ity is uormtil ( Black and Rilling, 1969). Cytochemical preparations for nucleoside phosphn- tase activity showed no difference between sham and renal failure rats. This enzyme parallels the tissue distribution of UDP-glucuronyl transferase in nu- merous cell types (Novikoff et al., 1962).

These studies provide evidence that hepatic subcellular structure and function are altered in experimental chronic renal failure. The morphologic changes were not detected using light microscopic or enzyme cytochemical examination of sections of liver. Although some enzyme systems showed altered activity while others remained unchanged, the major role of cytochrome P-450 in hydroxyln- tion reactions magnifies the potential significance of its quantitative reduction. The extent to which these changes may contribute to pathophysiologic features of chronic renal failure in man require further study.

Pretreatment of chronically azotemic rats with DDT, an enzyme-inducing agent which is slowly eliminated by normal rats (Ortega et al., 1956), prevented most of the ultrastructural changes and enhanced microsomal enzyme activity in these rats as well as in control rats. These observations suggest that the hepatic endoplasmic reticulum in rats with chronic renal failure is capable of response to drugs and chemicals which result in endoplasmic reticulum prolifera- tion and increased activity of many of its substituent enzymes in normal rats. The effect of such agents on hepatic mechanisms of drug metabolism in humans with chronic uremia has been partially explored (Lichter et al., 1973).

FIG. 5. Rat with renal failure. Centrolobular area. Several RER cisternae (ER) are dilated and show irregularly implanted ribosomes with denuded segments of the ER membranes. %39,200.

UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 387

FIG. 6. Rat with renal failure. Centrolobular area. Electron dense VLDL particles are clustered within Golgi apparatus structures (GA). These particles are also seen in the inter- cellular space between two adjacent hepatocytes (arrows). X20,000.

Several potential mechanisms for the observed alterations in structure and function of the hepatic endoplasmic reticulum in experimental chronic renal failure were considered:

(i) Increased lipid peroxidation is suggested by the multiplicity of effects seen on various organelle membranes; however, this mechanism seems unlikely because the membrane lesions were morphologically and biochemically re- stricted and not diffuse. In addition, preliminary studies reveal no increase in diene conjugation, malondialdehyde production, or cellular respiration in livers from rats with chronic renal failure.

(ii) Chronic renal failure may alter the formation, degradation, or regulation of the turnover of endoplasmic reticulum proteins. This possibility has not been experimentally studied; however, DDT administration produced changes in endoplasmic reticulum structure and function in rats with renal failure similar to those in control rats.

(iii) Are the observed abnormalities due to nutritional depletion, electrolyte abnormalities, or hypertension in uremic rats ? Nutritional depletion is unlikely since all rats gained weight at a similar rate (Fig. 1). Recent studies by Shafritz et aI. (1976) reveal comparable results in pair-fed rats with chronic renal failure. Decreased formation of active complexes between albumin-synthesizing polysomes and endoplasmic reticulum membranes with concomitant release of albumin into the cytosol was also demonstrated (Shafritz et al., 1976).

3% BLACK E2’ AL.

FIG. 7. Rat with renal failure and DDT pretreatment. Centrolobu!ar area. Large areas of cytoplasm show hyperplastic smooth endoplasmic reticulum. ( SER) . ~12,800.

Following the operative procedure, nephron adaptation is successful with respect to sodium, potassium, and bicarbonate balance (Lubowitz et nl., 1971). The concentration of serum electrolytes was not measured in the present study; however, serum electrolyte concentrations were repeatedly normal in other studies using the same animal model of chronic renal failure (Lubowitz et al., 1971). Arterial blood pressure was not measured in the present study; however, other rats treated similarly have normal or mildly elevated arterial pressure (Lubowitz et al., 1971). Hyptertension resulting from any etiology has not been studied with respect to its role in altering hepatic endoplasmic reticulum structure or function.

The mechanism whereby chronic renal failure produces the observed hepatic effects is unknown. Altered endoplasmic reticulum composition, fluidity, and/or regulation are possible but have not been studied in detail. The dynamic nature of the hepatic endoplasmic reticulum and the effect of many substrates and metabolites on its regulation suggest that chronic renal failure with its profound metabolic consequences influences the function of this essential organelle.

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UREMIA AND HEPATIC ENDOPLASMIC RETICULUM 389

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