Studies on the Causal Role ofHypoalbuminemia in Experimental NephroticHyperlipemia

Walter Heymann, … , Caroline Gilkey, Mildred Lewis

J Clin Invest. 1958;37(6):808-812. https://doi.org/10.1172/JCI103668.

Research Article

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STUDIES ONTHE CAUSALROLEOF HYPOALBUMINEMIAINEXPERIMENTALNEPHROTICHYPERLIPEMIA'

By WALTERHEYMANN,GERALDNASH, CAROLINEGILKEY, ANDMILDRED LEWIS

(From the Department of Pediatrics, Western Reserve University, School of Medicine,Cleveland, Ohio)

(Submitted for publication November 13, 1957; accepted February 6, 1958)

Previous work on the pathogenesis of the ne-phrotic hyperlipemia, in rats injected with anti-rat kidney sera obtained from rabbits (nephrotoxicserum, NTS), has indicated that the increase inplasma lipid concentration precedes the develop-ment of hypoproteinemia (1). They furthermorehave shown that the thyroid, pituitary, adrenals,ovaries, testes and pancreas glands are not essen-tial for its elicitation (2); that subtotal hepatec-tomy inhibits its development(3); and that incontradistinction to other fat depots, the hepaticlipid values are decreased (4). It was also shownthat the metabolic utilization of C'4 labeled tri-laurin and the clearance of this substance from theblood stream after intravenous injection proceededat normal rates (5). It was finally observed thatrenal tissue is essential for its elicitation, and thatthe severity of the hyperlipemia is related to theamount of present nephrotic kidney tissue (6).

Recently Rosenman, Friedman, and Byers havestated (7) that "the hyperlipemia observed in theexperimental nephrotic syndrome is initiated andmaintained by the external renal loss of plasmaalbumin."

The observations reported in the followingstudies were stimulated by the work of theseauthors. They are concerned with: a) relationof severity of hyperlipemia to severity of hypoal-buminemia; b) time relationship in the develop-ment of hyperlipemia and hypoalbuminemia; andc) the effect of saline, protein-free control urine,nephrotic urine, and dextran solutions on serumcholesterol and total lipid concentrations in ne-phrotic rats.

METHODSAND PROCEDURES

Rats of the Sprague-Dawley strain were used. Theywere fed Friskies and housed in single cages. Methodsused to produce the nephrotic syndrome in rats and to

1 Aided by grants of the Cleveland Area Heart Societyand the Northern Ohio Chapter of the National Nephro-sis Foundation.

determine the chemical data reported in this study havebeen described previously (8-12). Blood for analysiswas obtained after food had been withheld for 16 hours.

A first group of studies was designed to establish apossible relationship between severity of hypoalbuminemiaand hyperlipemia in the experimental nephrotic syn-drome. Serum protein, serum albumin, and total lipidvalues were obtained in 19 nephrotic rats of our colonyin whom nephrotic renal disease had persisted uninter-ruptedly for 1 to 11 months. These animals had beenstudied with weekly quantitative urinary protein de-terminations and monthly blood pressure readings. Theirfood intake was normal and their weight curves wereindistinguishable from control animals.

A second group of studies was planned to investigatethe time relationship in the development of hypoalbumine-mia and hyperlipemia within the first 24 hours afterinjection of NTS. Twenty-six rats of the same weightand sex were used for this purpose. They were givenNTS by intravenous injection. The potency of the twobatches of pooled sera used in these experiments had beenpreviously established by bio-assay. In amounts of 0.69and 1.38 ml. per 100 Gm. rat, they regularly inducednephrotic renal disease of equal severity, inducing pro-teinuria and hyperlipemia within the first 24 hours. Theanimals were killed 2, 4, 6, 8 and 24 hours after injec-tion. It was essential to obtain these values from the26 separate animals killed in the quoted intervals afterthe injection of NTS, because it had been noted that inrats the removal of 3 to 4 ml. of blood alone may de-crease blood lipid and cholesterol values appreciably for3 to 5 days.

Rosenman, Friedman, and Byers (7) have shown thatwhen the loss of urinary proteins was prevented by liga-tion of the ureter on one side and lower vena cava-ureteranastomosis on the other side in rats injected with NTS,the development of hyperlipemia was inhibited. The in-travenous infusion of bovine albumin acted similarly anddiminished the degree of hyperlipemia in rats with estab-lished nephrotic renal disease. It seemed necessary tous to control these experiments by studying the effect ofnonprotein-containing rat urine, and also to investigatethe effect of dextran instead of albumin on the hyper-lipemia of nephrotic rats. For this reason, experimentswere planned to study the effect of bleeding, injections ofsaline, control and nephrotic urines, and dextran solu-tions 2 on the nephrotic hyperlipemia (Table I). Forty-

2 Plavolex®, Wyeth (6 per cent dextran in 0.9 per centNaCl solution).

808

ROLE OF HYPOALBUMINEMIAIN NEPHROTIC HYPERLIPEMIA

three nonedematous nephrotic rats were used. These hadbeen injected with antikidney serum one and one-half toseven months prior to the first bleeding. They had un-interrupted proteinuria (100 to 400 mg. per 24 hours), atewell, gained normally in weight, and all had marked hy-perlipemia with values ranging -for total lipids from1,050 to 2,300 mg. per cent and for cholesterol from160 to 420 mg. per cent. For reasons quoted above bleed-ing was spaced six to eight days apart Initial valueswere obtained three days prior to the onset of treatment,and 3.5 to 4 ml. of blood was obtained from the infra-orbital sinus (13) every six to eight days thereafter forthree to four weeks. All solutions were given by intra-peritoneal injections for 10 consecutive days in amountsof 4 ml. twice daily. Nephrotic and protein-free con-trol urines were collected after 10 mg. of streptomycinand 10 mg. of tetracycline (AchromycinO) per 20 ml. ofurine had been added to the sterilized recipient jar. Thesamples were then filtered through a No. 6 Seitz filterand urinary cultures were obtained to assure sterility.

RESULTS

Figure 1 represents serum protein and serumalbumin values (ordinate) related to the concen-tration of serum total lipids (abscissa) obtainedin 19 nephrotic rats with well-established diseaseof 1 to 11 months' duration. It is evident fromthese results that in these animals there is no cor-relation between severity of hypoproteinemia orhypoalbuminemia and severity of hyperlipemia.

Figure 2 represents serum total lipid, choles-terol, serum protein and albumin values obtainedin 26 rats 2, 4, 6, 8 and 24 hours after the intra-venous injection of antikidney sera obtained fromrabbits. From the 30 rats originally injected, 4rats were killed accidentally and were not availablefor bleeding after 8 and 24 hours. It can be seen(Figure 2) that total lipid and cholesterol valueswere increased beyond values ever obtained incontrol rats in some instances two hours, in mostinstances four hours, and in all instances eighthours after the injection of NTS. Serum albuminconcentration, however, remained within the rangeof normal without exception for six hours afterNTS injection. After eight hours, when hyper-lipemia was established in all instances, only oneof four albumin values was decreased below normalrange.

Table I summarizes the results obtained in 43nonedematous nephrotic rats which were bledevery six to eight days without being subjected toany intraperitoneal injections (eight rats, column

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1); rats which were bled in identical intervals andwere given 4 ml. of saline twice daily for 10 days(eight rats, column 2); rats which received 4 ml.of protein-free urine twice daily (eight rats, col-umn 3); protein-containing nephrotic urine (eightrats, column 4); and dextran solution (11 rats,column 5) by intraperitoneal injections. If serumprotein, blood urea nitrogen, cholesterol or totallipid values decreased to 30 to 50 per cent of theoriginal value obtained three days prior to the on-set of treatment, and if they returned to pretreat-ment levels within two weeks after treatment hadbeen discontinued, the result is listed in Table Ias +. If the decrease amounted to 50 to 70 percent, it is registered as + +, and in excess of 70per cent as + + +.

It can be seen (Table I, column 1) that the re-moval of 3 to 4 ml. of blood every six to eight daysdoes not alter the concentration of any of the se-rum constituents examined. Considering cho-lesterol and total lipid values, it can be noted(Table I, column 2) that total lipid values de-crease temporarily only twice out of eight timesduring the 10 day period of saline administration,whereas the cholesterol concentration was not af-fected. The intraperitoneal injection of 8 ml. ofprotein-free control urine for 10 days decreased

the elevated cholesterol values three times and thehyperlipemic total lipid values six times of eightsuch experiments. The magnitude of this effectwas the same as when protein-containing nephroticurine was used (Table I, column 4). The onlydifference was that in nephrotic rats treated withnephrotic urines, the concentration of total lipidsdecreased in each of the eight experiments and thecholesterol values seven out of eight times. Whendextran solutions were used, the temporary de-crease of lipid and cholesterol was as regularlyand markedly noted as in the studies using pro-tein-containing nephrotic urines. Because of theosmotic activity of dextran solutions, hematocritreadings were obtained at each bleeding in 3 of the11 rats. Even though an initial decrease from46, 37 and 43, respectively, to 30 was noted ineach instance, no correlation with total lipid orcholesterol concentration was noted. While thedecreased hematocrit readings did not return tonormal during the period of observation, the cho-lesterol and total lipid values increased to the pre-treatment levels within a week after the adminis-tration of dextran solutions had been discontinued.It should be re-emphasized that none of the ne-phrotic rats used in these studies was in theedematous phase of the disease.

NEPHROTIC RATS:TAL SERUM PROTEIN 8 ALBUMIN (ORDINATE),

TOTAL SERUM LIPIDS (ABSCISSA).

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FIG. 1. CORRELATIONOF SERUMPROTEIN AND SERUMALBUMIN CONCEN-TRATION (ORDINATE) WITH TOTAL LIPm VALUES (ABSCISSA) IN SERA OF

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DISCUSSION

If hypoalbuminemia were the only cause for thenephrotic hyperlipemia, an inverse relationshipbetween severity of plasma albumin depletion andseverity of the hyperlipemia could have been ex-pected. That this has not been established (Fig-ure 1) does not, however, eliminate the hypoal-buminemia as one of several factors that maypathogenetically be at play in the elicitation of thenephrotic hyperlipemia.

It has furthermore been shown (Figure 2) thathypoalbuminemia does not precede the develop-ment of hyperlipemia if studies are carried outwithin the first two to four hours after the injectionof NTS. It can, in fact, be seen (Figure 2) thathypoalbuminemia succeeds the development of thehyperlipemia in most instances. If hyperlipemiacan be noted within two hours after the adminis-tration of NTS it is almost inconceivable that hy-poalbuminemia could have developed prior to thatperiod of time.

The studies summarized in Table I show thatthe concentration of serum total lipids and cho-lesterol can be rather easily decreased by variousprocedures. The withdrawal of blood alone hasbeen mentioned before. If spaced six days apart,we feel reassured (Table I, column 1) that thisdoes not play a role in our studies. The injectionof saline alone (Table I, column 2) can, how-ever, occasionally decrease the total lipid concen-tration without affecting cholesterol values. Pro-tein-free urine (column 3) decreased total lipidconcentration markedly in six of eight experi-ments. In these six instances it would be difficultto distinguish the results from those reproduced incolumn 4 (Table I) where protein-containing ne-phrotic urine was used. It is thus probable thatin the studies of Rosenman, Friedman, and Byers(7) similar results, possibly differing in intensityonly in some of their experiments, would havebeen obtained if the nephrotic rats would havebeen infused with urine obtained from healthy con-trol animals instead of having been subjected toa vena cava-ureteral anastomosis. It also is evi-dent from column 5 (Table I) that dextran solu-tion would have had the same effect on blood lipidand cholesterol concentration as that observed bythe authors (7) when they infused nephrotic ratswith albumin solution. Wemay assume that this

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FIG. 2. TOTAL LiPm, CHOLESTEROL, SERUM PROTEINANDSERUMALBUMIN VALUESOBTAINED IN 26 RATS 2, 4,6, 8 AND 24 HOURS, RIEspEcTrvys, AFTER THE INTRA-VENOUSINJECTION OF NEPHROTOXICSERUM (NTS)

The shaded areas indicate the range of values obtainedin 12 to 21 healthy rats of the same age and weight.

effect of dextran solutions is not due to hemodi-lution in that only nonedematous animals wereused and hematocrit readings were unrelated tothe observed lipid and cholesterol values obtained.The mechanisms of the hyperlipemia-reducing ac-tion of dextran is unclear. It has also been de-scribed by Cohen and Tudhope (14) in non-ne-phrotic human patients with hypercholesterolemia.

The coexistence of hypoalbuminemia and hyper-lipemia is well-established in all forms of the ne-phrotic syndrome. We believe, however, that acausal relationship between them has not beendemonstrated, in that: a) There is no relationshipbetween severity of hypoalbuminemia and hyper-lipemia; b) hyperlipemia does not succeed, butusually precedes the decrease in serum albuminconcentration; and c) protein-free urine and dex-tran instead of albumin solutions decrease the

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WALTERHEYMANN,GERALDNASH, CAROLINE GILKEY, AND MILDRED LEWIS

nephrotic hyperlipemia markedly. Clinical ob-servation has furthermore taught us that hypoal-buminemia of comparable severity due to insuffi-cient protein intake, as in kwashiorkor (15), oras noted in cases of hypocupremia and idiopathichypoproteinemia (15), or as occasionally seen inchronic nephritis (16), is not necessarily associ-ated with increased cholesterol or total lipid con-centration. We therefore believe that a causalrelationship between hypoalbuminemia and hy-perlipemia has not been shown to exist as yet.

'SUMMARY

1. No correlation between severity of hypoal-buminemia and hyperlipemia was noted in 19 ne-phrotic rats with renal disease of 1 to 11 months'duration.

2. The development of hypoalbuminemia andhyperlipemia was studied in 26 rats 2, 4, 6, 8 and24 hours after the injection of anti-rat kidney sera.Hyperlipemia preceded, rather than succeeded, thehypoalbuminemia.

3. In nephrotic rats the intraperitoneal adminis-tration of nonprotein-containing rat urine de-creased the hyperlipemia almost as regularly andmarkedly as when protein-containing nephroticurines were used. The effect of dextran solutionson the experimental hyperlipemia was markedand regularly noted.

4. It is concluded that a causal relationshipbetween hypoalbuminemia and hyperlipemia in theexperimental renal disease of rats has as yet notbeen established.

REFERENCES1. Heymann, W., and Hackel, D. B. The early develop-

ment of anatomic and blood chemistry changes inthe nephrotic syndrome in rats. J. Lab. clin. Med.1952, 39, 429.

2. Heymann, W., Hackel, D. B., Gilkey, C., and Salehar,M. Relation of pituitary and adrenal glands to thenephrotic syndrome in rats. Lab. Invest. 1953, 2,423.

3. Heymann, W., and Hackel, D. B. Role of liver inpathogenesis of experimental nephrotic hyper-lipemia. Metabolism 1955, 4, 258.

4. Heymann, W., and Hackel, D. B. Hepatic and ex-trahepatic depot lipids in rats with experimentalnephrotic hyperlipemia. Metabolism 1957, 6, 169.

5. Heymann, W., Matthews, L. W., Lemm, J., Olynyk,P., Salehar, M., and Gilkey, C. Fat metabolismin nephrotic hyperlipemia. Metabolism 1954, 3, 27.

6. Heymann, W., and Hackel, D. B. Role of kidney inpathogenesis of experimental nephrotic hyper-lipemia in rats. Proc. Soc. exp. Biol. (N. Y.)1955, 89, 329.

7. Rosenman, R H., Friedman, M., and Byers, S. 0.The causal role of plasma albumin deficiency inexperimental nephrotic hyperlipemia and hyper-cholesteremia. J. clin. Invest. 1956, 35, 522.

8. Heymann, W., and Lund, H. Z. Nephrotic syndromein rats. Pediatrics 1951, 7, 691.

9. Weichselbaum, T. E. Accurate and rapid methodfor determination of proteins in small amounts ofblood serum and plasma. Amer. J. clin. Path.Tech. Sect. 1946, 10, 40.

10. Wilson, W. R., and Hanner, J. P. Changes of totallipid and iodine number of blood fat in alimentarylipemia. J. biol. Chem. 1934, 106, 323.

11. Schoenheimer, R., and Sperry, W. M. A micro-method for the determination of free and com-bined cholesterol. J. biol. Chem. 1934, 106, 745.

12. Archibald, R. M. Colorimetric determination ofurea. J. biol. Chem. 1945, 157, 507.

13. Stone, S. H. Method for obtaining venous bloodfrom the orbital sinus of the rat or mouse. Science1954, 119, 100.

14. Cohen, H., and Tudhope, G. R. Dextran sulphate:Use as an anticoagulant, and action in loweringserum cholesterol. Brit. med. J. 1956, 2, 1023.

15. Cooke, R., Goodman, H., and Rapoport, M. Discus-sion in Proceedings of the Eighth Annual Con-ference on the Nephrotic Syndrome, J. Metcoff,Ed. The National Nephrosis Foundation, Inc.,1956, p. 98.

16. Heymann, W. Unpublished data.

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