Patterns of proteinuria in chronic pyelonephritis. The pseudo-glomerular pattern

CLINICA

CHIMICA ACTA

PATTERNS

OF PROTEINURIA

PSEUDO-GLOMERULAR

L. BONOMO

295

AND

IN CHRONIC

THE

F. P. SCHENA

Institute of Clinical Medicine *, II. Clinical Medicine, (Received

PYELONEPHRITIS.

PATTERN

Policlinic**,

Bari (Italy)

October 6, 1968)

SUMMARY

Proteinuria occurring in chronic pyelonephritis was investigated by agar gel and immunoelectrophoresis ; serum protein clearances were estimated as well. In chronic pyelonephritis, different patterns of proteinuria may occur due to selective proteinuria-with excretion of low molecular weight proteins-to unselective proteinuria-with proteins of low and high molecular weights. Serum protein clearances showed often a higher excretion of medium and high molecular weight proteins (IgG and IgM) than of low molecular weight proteins (albumin and transferrin). This pattern of proteinuria is a frequent characteristic of chronic pyelonephritis and its pseudo-glomerular nature is discussed.

Chronic pyelonephritis (C.P.) is a disease characterized by inflammation of the renal parenchyma; it is due to a definitive localization of bacteria in the interstitial tissue of the kidney with secondary effects on the glomerular, tubular and vascular structures. However it has been recently shown that active C.P. does not require the presence of demonstrable bacterial. Proteinuria is one of the signs which demonstrates the extent of kidney damage in C.P. although it does not exceed considerably the physiological level. Its persistence beyond the clinical evidence of the infectious episode indicates chronicity of the disease. According to Dogliotti et uZ.~, late glomerular involvement with the type of anatomical damage and the occlusion of the mainly involved nephrons would explain the low amount of proteinuria in C.P. On the other hand, compression by the interstitial infiltration on the renal structures would cause proteinuria to appear before gross parenchymal alterations, particularly at the glomerular level. Two patterns of proteinuria are currently distinguished, i.e. the glomerular pattern (due to plasma proteins filtered through damaged glomeruli) and the tubular pattern (resulting from tubular involvement by the disease process). * **

Dir. Prof. V. Chini. Dir. Prof. L. Bonomo.

Clin. Chim. Ada,

23 (1969) zgs-303

BONOMO,

296

fact,

SCHENA

Glomerular jwoteinuria can be different in relation to glomerular damage. In proteins of low molecular weight (albumin and transferrin) pass through the

glomeruli,

whose selective

function

in some cases may be lost to the point that also

proteins of larger diameter, like the IgG, a,-macroglobulin and even IgM pass through them. The amount of this proteinuria is increased due to absorption defect at the tubular level and probably also to other factors. Ttibular proteinuria has been studied in detail by several authors3-7. It is characterized by low molecular weight proteins (IOOOO to 30000) and probably produces the diffuse staining in the CI*, /I and post-y areas of the electrophoretic pattern. Trace amounts of these proteins are present in normal urine, while they are excreted more conspicuously in C.P. and in other congenital or acquired tubular diseases.

Antoine et a1.8 suggest that these low molecular weight proteins originate from damaged renal tubules, while othersg~1o are in favour of a defective tubular reabsorption of plasma protein fractions. In C.P., mixed proteinuria (glomerular and tubular) occurs of variable degree, according to the extent of the renal damage caused by the infectious inflammatory process. It seemed

interesting

to characterize

proteinuria

in C.P.

in relation

to its

glomerular components. Therefore, the degree of selectivity to serum proteins shown by the renal parenchyma following the damage produced by the chronic disease process was estimated by the serum protein clearances. A small group of cases with glomerulonephrosis tigated

and glomerulonephritis

and some normal

subjects

were inves-

as well.

MATERIAL

AND MEiHODS

Our study was carried

out on

40

urine samples

obtained

from the following

group of patients : chronic pyelonephritis (25 cases), glomerulonephrosis glomerulonephritis (4 cases) and normal subjects (5 cases). The diagnosis of C.P. was established according findings previously described ll. Also for the diagnosis

(6 cases),

to the clinical and laboratory of the other nephropathies we

have followed the clinical and functional criteria generally accepted12. Urine of 24 h was collected, adding a small amount of sodium azide as conservative to avoid bacterial fermentation and protein denaturation; after completing collection the urine was filtered at 4’ through Whatman paper No. 12 and concentrated according to either of the following procedures: (a) the urine was put in Visking tubes and concentrated against polyethyleneglycol (Carbowax 6000) ; when the total amount was reduced to 20-30 ml it was dialysed for 48 h in buffered saline pH 7 ; (b) the urine was put in Visking tubes and concentrated by pervaporation with buffered saline, pH 7, while ventilated in a cold room at 4”. The concentrated samples were then lyophilized after estimation of their protein

content by the biuret method. Proteinuria was studied by agar gel electrophoresis, immunoelectrophoresis, and serum protein clearances were estimated with immunoplates (Behringwerke). For these procedures a small amount of lyophilized material was dissolved in pH 7 buffered saline to obtain a protein concentration of about 3-5 9%. Clin.

Chim.

Ada,

rzj (1969)

q-303

PROTEINURIA

IN

297

PYELONEPHRITIS

Agar gel electrophoresis was performed following the method described by Wieme13 and immunoelectrophoresis according to the micromethod of Scheidegger14; only urines from pyelonephritic patients were examined by these two methods. Serum protein clearances were tested according to the immunochemical method described by Soothill and modified by Cameron16 employing the Behringwerke anti-albumin, transferrin, IgG and IgM immunoplates. Estimations were performed on samples of the collected urine and of heparinized plasma taken soon after the 24-h urine collection had been completed. U-V For the calculation the formula p was applied, U and P being the protein concentration in the urine and respectively in the plasma, while V is the urine minute-volume. The selectivity of the kidney for the different serum proteins has been evaluated by a comparison between the clearance of such proteins with that of the albumin, the clearances being expressed as percentage of the albumin clearance with the following formula : UIgGPlgG

I/

PAlb UAlb-V

* IOOO/”

=

u UG -

P I@

PAlb . ~. UAlb

IOOO//O

RESULTS

In C.P. the quantity of uroprotein varies from 40 to 1500 mg/24 h (Tab. I), representing a moderate proteinuria if compared with that of patients with glomerulo-

Fig. I. Chronic pyelonephritis : glomerular proteinuria. Left: Agar gel electrophoretic patterns of the urine from cases with pyelonephritis: pool of normal human sera (above) and various degrees of proteinuria. Right: Immunoelectrophoretic patterns of the same samples developed with total anti-human antiserum: pool of normal human sera (above) and patterns from selective to non-selective proteinuria. Clin. Chim.

Acta,

23 (1969)

q5--303

‘d

7.95 7.80 6.75 6.60 7.95 6.00

Nephritis

Normal

M

9 6 45 40

28 2s 28 30 25

D.N. M.R. G.C. N.A. D.V.

36 37 38 39 40

E

M F M

Yf M

7.00 7.52

6.99

6.90 h.50 6.40 ‘1.75 5.45 7..50

7.36 6.15 8.88 6.57 8.88

5.10

M.A. F.G. D.M. C.A.

;

E F P F

FM

: M

32 33 34 35

7.62 7.61 7.50 6.45 8.70 6.76 6.42

7.75 8.21 7.74 7.63 7.20 7.71

8.60 7.71 6.30 6.96 7.99 7.51

Nephrosis

~~~~-~

Chronic pyelrmephritis

F F M F M M

23 24 25

22

27 I8 58 76 73 75 72 56 58 73 75

56

F

: i:

z M

; bl F F

F

g%

P.T.

Blood

F.M. D.M. C.M. SM. F.L. D.M.

60

2:

60 69 50 59

-

Diagnosis

26 27 28 29 30 31

L.G. R.V. C.F. C.L. T.R. G.F.

C.M. L.F. SM. L.G. L.M. S.F.

-

METIIOD

D.S. D.C. D.D. C.F. FM. P.M. A.M. D.R. D.L. D.C. P.M. A.M. D.R.

-

BIURET

____--.

THE -

13 14 15 16 17 18 19 20 21

iY

3500 3450

~too

3200

4900

4500 3450 4300

5200

1350

1250

3600 3010 3200 1450

950 *go0 2550 29ocl 3850 2850 4050 4900 6400

2150

4x50 3.500 3500

3850 4x50 3450

2150

5650 4900

1870 2700 41.50 3850 2850 5400

Alb.

180

365 316 182 190

247 240 117 305

145 130

220

137

220 182

200 97 182 1875 105 215 235 1575 1875 115 190 230 217

245 2735 190 235 1875 ‘90

235 125 21.5 1875 235 1875

Transf.

50

900

820

rg8o

300 1.500

2goo

2850

1500 1300

rho0

lODO

1600

2020

2680 3360 3000

470

500

460

IgOO

500 500 800 1500

-

-

110 -

2360 5600 6000

3360 4400 4400 860 360 I700

3360 6400 3040 5040

2040

340 -

-

-

1700 I400 1000 650 *go0 700 600 700 1300 350 1300 650 1300

-

2680 3040 6400 1840 -

500

2520 700

2000

1400 1000 1500

1500 1085

1100

1550

1800

I22

-.~

IJrine

ml/24 h

4800

137 -

170 -_

170 .-

-

-

IgM

280

2280 2180 4080 2080

740 3900 II20

10x2

7200 1680

%

IgG

61 55 70 50 45

355 500 82 190

771 3497 595 3646 2745 616

418 446 44 917 239

II0

%E 513

1% 460 242 193

8:; 1495 601 276

142

971 195 323 657 156 1405

P.T. W/24 h

0.30 0.20

0.28

0.74 2.2

2

40 3.3

51

IO2

515

6;:

211

200

24 1.8 6.4 98.5 0.3 52 3.2

I.2

i? 2.7 I.5

0.17

16 0.6 7.4 30 45 20

14 I.5 28

$3

30

Alb.

0.01

0.24 0.65 0.2 I 0.01

82:: 3.4 0.14

3.5 6.1 0.76 29 21 4.8

2.5

0.38 I.1 1.7 0.29 5 0.9

0.21 2

0.22 I I

0.17

0.52

I

0.3 I

0.1

0.4

0.9

0.33 3.9

0.52

0.25

2.4

Transf. “b

_-._-..-. ~

2

0.05

0.13 0.10

z

k!

:

0.57

-8

3

o*

0.2

0.32

-

-

-

-

0.1

-

0.13

3

38 I1 I

1.7

z.1

0.1

2.5 53

40 0.36 II 5.6

0.5

0.28

i

13

-

0.14 0.28 0.1

0.3 0.08 8.6 9 3.6 17 0.41 0.50 15.2

0.47 2.58

0.09 -

IgM

0.5 46

IO

35 3.1 I

I&i

TABLE

II

19 20 *I 22

13 14 15 16 17 18

No.

0.00385 0.0002g O.OOI49 o.oo8zg 0.00006 0.00477 0.00045

D.S. D.C. D.D. C.F. F.M. P.M.

A.M. D.R. D.L. D.C. P.M. A.M. D.R.

&c. N:A.. D.V.

0.00072 o.000a4 0.00128 0.00005 0.00005

0.00008 0.00006 0.00002 0.00003 0.00001

Z.? D.ki. C.A.

0.00016 0.00028 0.00060 0.00008 0.00005

O.OOOI5 0.00036 0.00027 0.00006

0.0032g 0.02146 o.oI5gI 0.00047

0.00333 0.03022 0.00052 0.00048

KY. SM. F.L. D.M.

D.N.

0.00461 0.01252 0.00002 0.00075 0.00160 0.00715

O.OOIZ4 0.00016 0.00022 0.00285 0.00005 0,00162 0.00100

0.00018 0.00018 0.00163 0.00048 0.00283 0.0000g

0.000I2 0.00002 o.oo*Ig 00.0597 0.00068 0.00120

0.01027 0.000I4 0.03080

o.ooorg o.oorog

o.oogor

IgG

o.oog86 0.03476 0.00724 0.04086 0.04924 0.01181

0.00436 o.oooog 0.00052 0.00354 0.000I3 0.00978 0.00372

0.00100 0.00218 0.00378 0.000*4 0.00262 0.00446

0.00158 0.00027 0.00164 0.00587 0.000*5 o.ooog3

0.00633 0.0002I 0.00302 0.00036 0.00146 0.00156

Tram/.

mllmin

0.03444 0.072go 0.00573 0.13682 O.I4000 0.02417

F.M.

0.00048 0.00083 O.OOII8 0.00056 0.00206 0.00060

:.z C.L. T.R. G.F.

0.00234 0.00027 0.00054 0.0038X

0.0007g

0.00994

Alb.

Clearances

0.0027.1 0.00008 0.03579 0.01075 0.00052 0.001g7

L.G.

C.M. L.F. SM. L.G. L.M. S.F.

Cases

\‘alues in y. of albumin and transferrin clearances.

,~ENALCLEARANCBS OG ALBUMIN,TRANSFERRIN,IgG AND TgM

0.00698 -

0.00038

0.00?210 0.01138

0.00070

IgM

450 300 213 62 IO0

4I 3000 97

98

47 126 20 3; 48

28

II0 3I 34 42 216 205 826

200 260 320 42 127 740

57 337 4 54 48 47

63 26 I29 I33 270 40

J@

Alb.

283 ZI 138 85 I37 48

4 2.5 6 551 I33 60

30 24 5; 1800 25 793

Cl.

18

24

92

2

293

777

-

Alb.

Ighf

88

cl.

II 7 I.5 60 20

4 2.8 1.6 I2

46 36 0.2 0.5 3.2 60

42 80 38 16 2

28 I77

I33 IO0 272 129 I36 8 43 200 108 6

7 7

2850 9 I970

47 90 42

64

-

I97

-

I4

I92 51 -

-

729

583

333 -

BONOMO, SCHENA

3oo

nephrosis; also patients with glomerulonephritis show similar amounts of proteinuria. The electrophoretic pattern on agar gel (Fig. I) of pyelonephritic proteinuria changes usually in agreement with its amount. Actually, there are cases with a prevalent excretion of prealbumin and albumin and, at the other extreme, others with a uroprotein pattern quite similar to the serum protein patterns, as well as various intermediate forms. Similarly, also immunoelectrophoresis (Fig. I) shows different patterns, from cases in which only some arcs (prealbumin and ~bumin) appear to others in which a,-trypsin, haptoglobin, ceruloplasmin, transferrin and y-globulins (I&, IgA, IgM) are found. Furthemrore, arcs very near the application trough and parallel to those of the described fractions were often detected. This is usually ascribedl’$l* to protein fragments, present not only in pathologic proteinurias but also in normal subjects as demonstrated by immuno-diffusion studies with antisera for the il and x type of the light chains. By the serum protein clearances described by Blainey et ~2.19in various renal diseases, we evaluated glomerular permeability to proteins of different molecular size and the extent of renal damage.

IOOj

lJ----

T.R.

67,000 A

80.000 150,000 t -.-, Tk IgG

Molecular

lQQQcOOO

l&i--

kveijht

Fig. 2. Glomerular permeability to proteins in chronic pyelonephritis. Ordinate: Albumin (A), transferrin (Tr), IgG and IgM clearances are expressed as percentage of albumin clearance (IOO%). Abscissa: molecular weight of proteins. Values are reported on logarithmic paper. or. N.A. = normal subject o T.R.

l C.IM. Chronic pyelonephritis l C.F. I

Clilz. Chim. Acta,

23

with various degrees of selectivity.

(1969) 295-303

301

PROTEINURIA IN PYELONEPHRITIS

According to this method, sdective ~~ote~ff~~~us are those with a low clearance, usually less than I?$, of the large a,-macroglobulin and IgM molecules in comparison to that of the medium (IgG) and small (albumin and transferrin) molecules. Nonselective proteiwrias are those with a high clearance of the large molecules, including proteins with medium and low molecular weight. Intermediate patterns occur between these two extremes. Our investigation on the serum protein clearance in C.P. (Tab. II) did not show high values of the albumin and transferrin clearance, in contrast with findings in glomerulonephrosis, while the clearances of the higher molecular weight proteins (IgG and IgM) were greater, and in some cases exceeded those of albumin and transferrin (Fig. 2, Tab. II). This is partly in accordance with the recent results of Goddard and Hobb90. The analysis of our results shows different degrees of permeability, from high to low selectivity in the different cases (Figs. I, 2; Tab. II). In some of them transferrin clearance exceeded that of the albumin. Actually, proteins with similar molecular weight can behave differently as far as clearance is concerned. The formation by the albumin and other serum proteins of high molecular weight complexesz11a8can explain this behaviour. Albumin and transferrin could also aggregate in polymeral forms23-25. DISCUSSION

Proteinuria in C.P. is generally considered of minor importance, due to its limited amount, although its persistence reveals the chronic course of the disease%*. Our results show that by electrophoretic and immunoelectrophoretic analysis, several degrees of proteinuria can be distinguished, which is useful for its chnical interpretation (Table III). TABLE

III

DEGREES OF PROTEINURIA -_--. Agar ---

gel

SELECTIVITY IN

CHRONZC

ACCORDING

TO AGAR

GEL

AND

IMMUNOELECTROPHORET~C

ANALYSIS

OF

PYELONEPHRITIS

ebectrophoresis

ImmwnoeleGtrop~oresis -

Albumin in small amount; amounts of other proteins.

First degree absence or trace Arc of albumin: traces of other arcs: presence of parallel arcs very near the application spot.

Second degree Prealbumin, albumin, post-albumin; more Albumin; traces of ai-trypsin, haptoglobin evident traces of xi-, tc,-, & and y-globulins. and transferrin; IgG and protein fragments in small amounts.

Albumin: amounts

:

a,-, tlg- and &globulin traces of y-globulins.

Third degree in greater Prealbumin, albumin: more evident arcs of ai-trypsin, haptoglobin and transferrin; IgG and protein fragments.

Albumin, czi-, K-, p- and y-globulins amounts.

Fourth degree in clear Albumin, a,-trypsin, haptoglobin, ceruloplasmin, transfer-k, IgG, IgA, IgM and protein fragments clearly evident. C&m. Chim. ACta, 23

(1969)

295-303

BONOMO,

302

SCHENA

Also other autllorsas~27 emphasize the interest of the qualitative analysis of proteinuria in the diagnosis of C.P., although they are mainly concerned with tubular proteinuria. Recent ultramicroscopic studies show that in physiologic proteinuria the large size protein molecules do not filter through the glomeruli2*, while in pathologic proteinuria the major part of proteins may pass through the cytoplasm of the epithelial cells tightly stuck to the basal membrane 29p30.Hence, the existence should be admitted of pores31+ that may be damaged by the inflammatory process and allow the leakage of conspicuous amounts of albumin and high molecular weight proteins as well. Actually, it seems

established

that

glomerular

proteinuria

results

from the

proportion of glomerular pore size, altered by the pathological process, to the molecular size of proteins. In C.P., our investigations showed a marked increase of medium and high molecular weight protein clearances with the pattern of unselective proteinuria, and therefore of marked renal damage, although the clearance of low molecular weight proteins (albumin and transferrin) was not high. This pattern usually results from definite kidney damage particularly at the glomerular level, although various interpretations Actually,

may be put forward. an inflammatory infectious

process in the renal parenchyma

and the

urinary tract might explain the high rate of IgG and, in some patients, also of IgM excretion in the urines, according to the recent hypothesis of Goddard and HobbsZo. In addition, the excretion of y-globulin fragments can yield higher estimates of the immunoglobulins in the immunochemical methods with immunoplates; in fact, these fragments have the same immunological reactivity as whole immunoglobulin molecules. Finally, a defective tubular reabsorption, as occurring in C.P., also a tubular diseaseZ8, might favour the urinary excretion of y-globulins and their fragments. In conclusion, the qualitative analysis of proteinuria appears useful for the diagnosis

and prognosis

kidney structures of JJ-globulins into The mixed occurring in C.P.

of pyelonephritis

as an index

of the involvement

of the

(glomeruli, tubules and capillaries). Furthermore, the chronic loss the urines may be relevant for the course of the disease. pattern (glomerular and tubular) is characteristic of proteinuria It differs from that of glomerulonephritis, that like C.P., shows

slight proteinuria, but of glomerular type alone, and from acquired tubular diseases, e.g. cadmium poisoning, in which the pattern is purely of the tubular type. It seems reasonable to suggest that proteinuria of C.P. should be more properly defined as “pseudo-glomerular” proteinuria, in view of the extra-glomerular factors involved in its determination. ACKNOWLEDGEMENT

This work was supported in part by Consiglio Immunologia) Rome, Italy.

Clin. Chim. .4cta, 23 (1969) 295-303

Nazionale

delle Ricerche

(Gruppo

PROTEINURIA

IN PYELONEPHRITIS

303

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Clin. Chim. Acta, 23 (1969) 295-303