ContI. Nephrol., vol. 1, pp . 156-162 (Karger, Basel 1975)

Low Molecular Weight Proteinuria Y. Manuel! ,A. Colle, M. Leclercq and C. Tonnelle

First described in victims of chronic cadmium poisoning, low molecular weight (LMW) proteinuria has since been recognised as a sign of trauma to the renal tubules provoking a modification in the reabsorbtion and catabolism of urinary proteins. LMW proteinuria, often called tubular proteinuria (4), is characterised by the elimination of large amounts of protein of MW ~ 40,000 daltons (1.5- 3 S). These proteins comprise fragments of larger serum proteins (e.g. , albumin, immunoglobulin), monomeric and dimeric Ig light chains, and native microproteins such as ~-2-microglobulin , post-l'-globulins, a-2-microglobulins, carbonic anhydrase , lysozyme, and some other enzymes. Several of these proteins are at present the subject of considerable research.

Origins As shown in figure I, the serum proteins are filtered at the glomerular barrier, those larger than MW = 40,000 being largely excluded, but the smaller ones passing with relative ease; one molecule of MW = 10- 15,000 being passed for every two molecules of inulin at equal serum concentration. This differential filtration results in a glomerular filtrate containing almost equal amounts of proteins of MW > 40,000 and those of MW ~ 40,000 despite the enormous predominance of the former in the serum. Normal urine contains only little of this filtered protein, the rest, especially LMW protein, being reabsorbed , and partially catabolised in the tubules; this partial catabolism results in the relatively low levels of native serum proteins present in normal urine . Other protein components of normal urine are produced locally by the renal epithelial cells, or by the excretory passages (e.g.,

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lnstitut Pasteur, Lyon, France.

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proteinuria;

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Glomerular barrie r

proteinuria; proteinuria;

proteinuria; proteinuria;

proteinuria; proteinuria; proteinuria;

Norm a l u rine

Tub u la r urin e

Fig. 1. Schematic mechanism of LMW proteinuria; grey: protein > 40,000; black: LMW proteins.

Tamm-Horsfall glycoprotein, IgA). Bienenstock and Poortmans (3) have shown that the kidney possesses the capacity to synthesise many proteins, being, in this respect, comparable with the liver. If the reabsorbtion and catabolic functions of the renal tubule are diminished the composition of the urine will approximate that of the glomerular filtrate. This leads to the excretion of considerable amounts of LMW proteins; proteinurias of 5-7 g/24 h composed of 60-70 % LMW proteins are not uncommon. Metabolic studies on certain of these LMW proteins as described by Waldmann and Strober (16) , considered with clinical observations on myeloma patients, especially those with Bence-Jones proteinuria, and studies on the passage of ~-2-microglobulin into the urine seem to show that the proximal tubule is particularly involved in the reabsorbtion and, more especially , catabolism of the

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LMW proteins; this is particularly well demonstrated for the Ig light chains. It appears, however, that certain cells may be specialised in the treatment of particular proteins, This is of considerable importance as the relative concentration of different proteins in LMW proteinuria can vary quite remarkably.

Materials and Methods An overall appreciation of LMW proteinurIa is best obtained by electrophoresis in polyacrylamide/agarose gel - Uriel (15) - coupled with s eparate immunoelectrophoresis. The characteristic pattern ('tubular pattern ') observed, permits the identification of the proteinuria despite the variations in the amounts of the component proteins. Individual proteins can then be measured u sing qualitative methods. At present, il-2-mlcroglobulin and lysozyme can be measured routinely using either the techniques of Mancini et al. (7), or of Laurell (6), or better, in view of the low concentrations often involved, radioimmunoassay methods - Evrin et at. (5). It is sometimes also possible to measure fr ee Ig light chains using an antiserum reactive only to antigenic sites available on the fr ee light chain, but hidden when the chain is combined with a heavy chain in the intact Ig molecule.

I

Pre - a l b

::J

----i Alb o.- 2- micro

- i 1J-2 - micro

---i Post - y ---t Post-y

2

3

4

567

8

Fig. 2. Gel filtration of low molecular weight proteins:

1. Fraction No.5] 2. Fraction No.5' 3. Fraction No.6 4. Fraction No.6' 5. Fraction No.5] 6. Fraction No. 5' 7. Fraction No.6

from 25 ,000 to 10,000 MW. polyacrylamide agarose gel beads

from 25 ,000 to 10,000 MW : bioge\ P. 100

8. Fraction No. 6' Phosphate buffer 0.2M pH 7. Glass column 1,200 X 120 cm. Elution time : 80 ml/ h. Inclusion : 12 g proteins.

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Low Molecular Weight Proteinuria

Gel filtration methods have been used to provide clinical information, but are mostly employed for the purification and isolation of the individual proteins (fig. 2). The concentrated urinary protein is passed through a column or Sephadex G 200, or G 100, Biogel P 100, or polyacrylamide/agarose gel beads. The fractions obtained are then further purified by chromatography on DEAE ~ephadex or by preparative electrophoresis either in liquid phase, or in polyacrylamide/gel.

Clinical Significance

There is now general agreement that LMW proteinuria reflects a functional disturbance in the proximal renal tubule. This disturbance may involve only proteins, and need not be associated with, for example, altered electrolyte excretion; indeed only one or two of the many microproteins may be excreted in quantity. This may reflect a lesion restricted to a few of the tubular epithelial cells specialised in the handling of these proteins. In all cases of LMW proteinuria we observe increased excretion of Ig light chains, but the relative amounts of all the other LMW components may vary remarkably (fig. 3). In some cases, i3-2-microglobulin may be almost completely absent, whereas in other cases (Wilson's disease) it may make up almost the total of the excreted LMW protein. Some patients excrete none, or only one, of the three o:-2-microglobulins. The amount of post-r-globulin or of lysozyme may vary similarly. With the exception of Wilson's disease none of these variations in the profile of the excreted microproteins appear to correlate with particular renal syndromes. Indeed, long-term LMW proteinuria can exist without any

-Alb

=:J

a.-2- micro

- - P-2-micro

---transf

- - Post-y -Post-y 2

3

4

5

6

7

8

5

Fig. 3. 1 to 8: sample of urine from burned patients at different evolutive states. S = normal serum.

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apparent clinical manifestation, as is the case in congenital asymptomatic tubulopathy. Only the observation of LMW proteinuria permits the identification of families carrying this trait. The above observations oblige us to consider that the measurement of a single microprotein cannot serve as a definite criterion of proximal renal tubule function, and that a technique, such as electrophoresis in polyacrylamide/ agarose gel, permitting an overall appreciation of the excreted microproteins must be employed. The increased excretion of Ig light chains may be an early sign of tubular dysfunction as, not only are these chains present in practically all frank LMW proteinuria, but they may also be observed as an isolated change in composition in an urine with a normal quantity of proteinuria. Such urines are characteristic of certain cases - Manuel and Greenland (8): (l) Patients who have suffered considerable tubular trauma (e.g., septicemia, severe burns, renal graft) may return to a normal proteinuria, but the lightchain-rich pattern will persist for a considerable time. (2) Victims of heavy metal poisoning - notably by cadmium or lead. Frank LMW proteinuria is frequent, especially in the cadmium victims; some 80 % of persons exposed to lead show a light-chain-rich proteinuria pattern. (3) Patients treated with gentamycin showed increased excretion of free light chains 2 days after the start of the treatment. This persisted, then disappeared a few days after the treatment was discontinued. (4) Children under 2 years old have similar light chain patterns in some 80 % of cases. Only some 10 % of normal adults show this pattern. (5) A typical LMW pattern may sometimes be observed without any obvious cause, for instance after prolonged decubitus, at the beginning of a tubulopathy, in endemic nephropathy, etc., but the quantity of protein excreted remains normal. We consider, then, that important clinical information can be obtained from the study of not only i3-2-microglobulin and lysozyme, but also the free Ig light chains. It is known that light chains are specifically reabsorbed and metabolised by the renal tubule, so that it is hardly surprising that they might be a very sensitive indicator of its functional state.

Biological Significance Little is known about the biological activity of most of the components of LMW proteinuria. Lysozyme and carbonic anhydrase, described by Poortmans et al. (12) as a 3 S 'Y-globulin, possess enzyme activity, and part of the a-2-microglobulin may be a retinol-binding protein although this had not been formally proved; i3-2-microglobulin (1), which has a MW of about 10,000 has been shown

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to have an amino acid sequence remarkably analogous to a single domain of immunoglobulin, being most similar to the third constant domain of the heavy chain of IgG - Peterson et al. (11). This protein was found in the supernatant from cultures of lymphocytes by Revillard et al. (13) then was found to be synthesised by, and present in the membrane of, lymphocytes by Bernier and Fanger (2). It has since been shown to be a membrane component of many nucleated cells, and can redistribute under the influence of specific antiserum. Some of the membrane i3-2-microglobulin has been shown to be associated with the HLA antigens - Neauport-Sautes et al. (10) and Nakamura et al. (9) suggests that it is in fact the constant microprotein of the HLA complex. Robert et al. (14) has demonstrated the presence of some HLA antigens in urine. Less is known about the biological properties of post-r-globulin. This protein undergoes changes both in its MW and its electrophoretic mobility on ageing, and appears to have a special affinity for certain peptides such as hydroxyproline-rich peptides perhaps derived from collagen, or fragments of immunoglobulin. Other microproteins are derived from catabolised serum proteins, notably albumin and IgG. This fact means that classical procedures for determining urinary clearances are not applicable to LMW proteinurias, as a considerable amount of protein having an antigenicity of say IgG may be present as fragments of much lower MW than the native protein. In order to present realistic clearances in cases of LMW proteinuria it will be necessary to identify serum proteins of suitable MW that are not degraded during their passage through the renal tubule. It is now known that the majority of the urinary microproteins are not derived from the kidney itself, but are concentrated from the serum where they are present in often very low concentrations by a sort of biological gel filtration. This allows the identification and eventually the purification of interesting serum microproteins. The possibility of induction of LMW proteinuria in experimental animals by chronic cadmium poisoning thus provides a valuable method for the isolation of such materials.

Summary Low molecular weight (LMW) protein urias vary widely in their microprotein composition. In general, there is little correlation between a given microprotein composition and a defined clinical disease (with the exception of the predominant iJ-2-microglobulin in Wilson's disease). Free immunoglobulin light chains are a practically invariable component of, and may be the only detectable LMW protein in, 'tubular' proteinuria. The origins and significance of some frequently occurring urinary LMW proteins are discussed.

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References 2 Berggard, I. and Beam, A.G.: Isolation and properties of a low molecular weight {l-2-microglobulin in human biological fluids. J. bioI. Chern. 243: 4095 (1968). 2 Bernier, G.M. and Fanger, M. W.: Synthesis of {l-2-microglobulin by stimulated lymphocytes. J. Immun. 109: 407 (1972). 3 Bienenstock, J. and Poortmans, J.: Synthesis and assembly of IgA and other urinary proteins. Commumcation 21st Colloquium, Brugge 1973, Elsevier, Amsterdam. 4 Butler, EA. and Flynn, F. V.: The proteinuria of renal tubular disorders. Lancet ii: 978 (1958). 5 Evrin, P.E.; Peterson, P.A.; Wide, L., and Berggard, I.: Radioimmunoassay of {l-2-microglobulin in human biological fluids. Scand. J. clin. Lab. Invest. 28: 439 (1971). 6 Laurell, C.B.: Electroimmunoassay. Scand. J. clin. Lab. Invest. 29: Supp!. 124, No. 21 (1972). 7 Mancini, G.; Carbonara, A.O., and Heremans, J.F.: Immunochemical quantitation of antigens by using radial immunodiffusion. Immunochemistry 2: 235 (1965). 8 Manuel, A. and Greenland, T.B.: Immunoelectrophoresis: a convenient method of studying 'minimal change' in proteinuria; in Protides of the biological fluids, pp. 393-400. Proc. 21st Colloquium, Brugge 1973, Elsevier, Amsterdam. 9 Nakamuro, K.: Tanigaki, N., and Pressman, D.: Multiple common properties of human {l-2-microglobulin and the common portion fragment derived from HL-A antigen molecules. Proc. natn. Acad. Sci. Wash. 70: 2863 (1973). 10 Neauport-Sautes, c.; Bismuth, A.; Kouri/sky, F., and Manuel, Y.: Relationship between HL-A antigens and {l-2-microglobulin as studied by immunofluorescence on the lymphocyte membrane. J. expo Med.139: 957 (1974). 11 Peterson, PA.; Cunningham, B.A.; Berggard, I., and Edelman, G.M.: {l-2-mlcroglobulin. A free immunoglobulin domain. Proc. Natn. Acad. Sci. Wash. 69: 1967 (1972). 12 Poortmans, J.R.; Jeanloz, R.W., and Schmid, K.: 3S -y-l-globulin levels of normal human serum and urine. Biochim. biophys. Acta 133: 363 (1967). 13 Revillard, J.P.: Vincent, c.; Leclercq, M., and Manuel, A.: Human migration inhibition factor: a preliminary report. Transplant. Proc. 4: 243 (1972). 14 Robert, M.; Vincent, c., and Revillard, J.P.: Presence of HL-A antigens and {l-2-microglobulin in tubular proteinuria. Transplantation (in press, 1975). 15 Uriel, J.: Methode d'electrophorese dans des gels d'acrylamide-agarose. Bull. Soc. Chim. bioI. 48: 969 (1966). 16 Waldmann, T.A. and Strober, w,: Renal regulation of serum protein metabolism; in Protides of the biological fluids. pp. 419-422. Proc. 21st Colloquium, Brugge 1973, Elsevier, Amsterdam.

Dr. Y. Manuel, Institut Pasteur, Lyon (France)

Low molecular weight proteinuria.

Low molecular weight (LMW) proteinurias vary widely in their microprotein composition. In general, there is little correlation between a given micropr...
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