Path. Res. Pract. 186, 619-624 (1990)

Morphological Findings in 70 Kidneys of Living Donors for Renal Transplant H. G. Rosenberg Escuela de Medicina, Departamento de Anatomia Patol6gica, Pontificia Universidad Cat6/ica de Chile, Santiago, Chile

P. S. Martinez, A. S. Vaccarezza and L. V. Martinez Escuela de Medicina, Departamento de Nefro-Urologfa, Pontificia Universidad Cat6/ica de Chile, Santiago, Chile

SUMMARY

Seventy donor kidneys for transplant were studied with light microscopy (LM), electron microscopy (EM) and immunofluorescence (1M) for C3, C 4, Clq, IgG, IgA, IgE, IgM, and antifibrin; the samples were taken just before transplanting the allograft kidney. Glomerular changes were found in 35.7% of apparently normal living donors: 9 cases showed relative glomerular ischemia with an irregular basal membrane (12.9%); 5 cases showed a diffusely widened basal membrane without antecedents of hyperglycemia (7.1%); in one case (1.4%) there was a lesion similar to type 1 mesangio-capillary glomerulonephritis with C3++, IgG++, IgA+, and IgM+; in another case (1.4%) there were scant isolated C3 glomerular, subepithelial deposits with indentation ofthe basement membrane of the immunocomplex type with a microhematuria which was demonstrated only after donation, and in 9 cases (among them two pairs ofsiblings) there were mesangial IgA and mesangial electron-dense deposits compatible with Berger's disease (12.9%). None of these glomerulopathies were evident under LM.

Introduction Morphological studies of renal biopsies made prior to performing the revascularization in living-related donor kidneys are scant. Most of them used samples taken one hour after the revascularization24,37,40 in search of transplant prognosis indices, such as the presence of more than two polymorphs per glomerulus, and not of morphological differences with the normal. Studies on acquired monorenal patients with long and short term follow-up are numerous; some show complete clinical norinality6, 10,26,30,34; others show hypertension and minimal proteinurias2,4,6,8, 12, 15, 17,22,31,39 and very few demonstrate glomerular changes 20,29,35,41. The pathogenesis of these lesions is under discussion since similar changes have ©

1990 by

Gustav Fischer Verlag, Stungart

been observed in resections of large parts of renal tissues11, 18. It has been a routine procedure in our hospital to obtain a sample of renal tissue from living-related donor kidneys prior to their revascularization. This material was analyzed from the morphological point of view, using light microscopy (LM), electron microscopy (EM), and immunofluorescence (1M). Material and Methods Seventy renal biopsies were obtained from living-related donors (LD) whose ages ranged from 18 to 58 years, during the period 1975-1987.

0344-0338/90/0186-0619$3.50/0

620 . Rosenberg, H. G. et al.

Fig. 1. Normal living donor, 35 years old, male: tissue recovered from Paraplast inclusion for LM. Three capillary loops (LC) with normal BM; mesangium not prominent. EM x 3000.

Fig. 2. Living donor, 26 years old, male; increased folds in the paramesangial BM (*). Slightly increased mesangial matrix. Irregular BM in free capillary loops (LC). Normal pedicles, focal and slightly fused. LC = capillary lumen. EM x 3000.

Forty-five were women (mean age 34 years) and twenty-five were men (mean age 28 years). All living-related donors were clinically normal and were nephrologically evaluated twice by the following work-up: full blood count, biochemical profile, creatinine clearance, 24 h proteinuria, urinalysis, plasma and urinary electrolytes, and urine cultures; an excretory urography and renal arteriography were also performed in every case. Donors were studied again when some morphological alteration was found in the renal biopsy. All were studied with LM, EM, and 1M using fluorescein-labeled antisera for C 3, C 4, Clq, IgG, IgA, IgE, IgM and antifibrin (Behring). The intensity of the fluorescent staining was graded as follows: negative (-), light (+), moderate (++) and marked (+++). The samples were obtained immediately before the administration of mannitol, perfusion with Collins solution and revascularization procedure. Tissue for LM was fixed in 10% buffered formalin (pH 7) and embedded in PARAPLAST PLUS@; serial sections 4 to 5 !-tm (microns) thick were stained with hematoxylin-eosin, periodic acid-Schiff, trichrome-Masson and silver methanamine. Tissues for 1M were snap-frozen, immersed in a small aluminum mold with OCT (Tissue-Tek II OCT®) with liquid nitrogen as soon as the biopsy was obtained and then stored in the deep freezer at -70°C or cut immediately in a cryostat at - 25°C at 2 to 4 !-tm (microns) in thickness. The specimen for EM was fixed in a 2.5% glutaraldehyde solution, 0.1 molar of cacodylate buffer at pH 7.4

and 4°C and post-osmicated and embedded in epoxy resin. Thin sections stained with uranyl acetate and lead citrate were examined in a transmission electron microscope. Evaluation of the basal membrane thickness was performed on all photographs at 3000 to 20000 enlargements at free capillary loops, taking a measurement at every centimeter and obtaining an average for each case 13 ,19,23,28. Table 1. Morphological findings in 70 living-related kidneys (donors) Characteristics

Number of cases

Normal Relative glomerular ischemia Homogeneous BM thickening Mesangiocapillary GN Type I Scant subepithelial deposits IgA mesangial deposits

45 9 5 1 1 9

64.3% 12.9% 7.1% 1.4% 1.4% 12.9%

Total

70

100.0%

BM

= basal

membrane, GN

= glomerulonephritis.

Percentage

Donor's Glomerulopathies . 621

Results The following findings were recognized in the renal biopsies (Table 1): - Forty-five cases (64.3%) had a normal histology under LM, EM and 1M (Fig. 1). The morphology of the transplanted kidney corresponds to that already described elsewhere 28 • - Twenty-five donors (35.7%) showed preexisting glomerular lesions (Table 1) which can be grouped as follows: a) Nine donors (ages ranging from 25 to 49 years with a mean of 37 years) presented EM alterations consistent with relative glomerular ischemia, that is, an increase of the paramesangial basal membrane folds, a slight increase in the mesangial matrix and an irregularity in BM density, with focal thickenings of the lamina rarae interna (LRI) without dense deposits. Two women of this group, 43 and 44 years old, had 22% obsolete glomeruli under LM. In 5 of these nine donors, C 3 deposits in the arterioles were present and in one case there were also generalized mesangial IgM deposits in all the glomeruli; this case subsequently evidenced a proteinuria of 0.5 g to 0.8 g in 24 hours (Fig. 2).

Fig. 4. Living donor, 22 years old, female; (belongs to family III in Table 2). Glomerulus with diffuse mesangial IgA deposits. IgA x 200.

Fig. 3. Living donor, 33 years old, female; increased mesangium with mesangial cell interposition (M) in capillary loops (LC); some dense deposits are seen in the mesangial matrix (0). Endothelial cell (En). EM x 4000.

b) Five donors (4 men and 1 woman, with a mean age of 30 years) showed uniform and homogeneous BM thickening in free capillary loops as well as in the paramesangial zone,with a thickness varying between 4,500 Aand 6,650 A (average 5,168 ~), a higher value than our normal average of 3,510 A28 and that of others 19 ,23. Glomeruli were 1M negative in all these cases. A moderately positive C 3 was present in the arterioles of 3 donors, with normal LM; none of them had laboratory or clinical signs of diabetes mellitus. One patient, who ended in a severe acute rejection, showed the same BM thickening in three successive renal biopsies taken within 80 days of evolution. c) A 33 year-old female presented a lesion similar to type I mesangiocapillary glomerulonephritis with C 3++, IgC++, IgA + and IgM + deposits, mesangial immunetype deposits, mesangial circumferential interposition in capillary loops, and scant subepithelial dense deposits (Fig. 3). This nephropathy persisted in the recipient as was proven after 3 months by a renal biopsy; no clinical symptoms were found in the donor after a 3-year follow-up. d) A 25 year-old male showed scant granular C 3 deposits in capillary loops, subepithelial dense deposits with

622 . Rosenberg, H. G. et al.

involvement up to the dense layer of the BM, and an isolated complete segmentary interruption in the capillary wall (gap). Persistent microhematuria was observed in subsequent clinical controls. The lesions were interpreted as a scant immune complex disease with subepithelial dense deposits. e) A group of 9 donors (3 men and 6 women, with a mean age of 32 years) showed IgA or predominantly IgA mesangial deposits in all the glomeruli (Fig. 4). There were also IgM deposits in 4 cases, of C 3 in 4 cases, and of IgG in 2 cases. Electron microscopy showed mesangial dense deposits in all 9 cases: they were scant in 6 cases, moderate in 2 cases and abundant in the remaining case (Fig. 5). Irregular BM with thinning and segmentary splitting were found in 6 cases. Two cases showed type I collagen fibers in the mesangial matrix. Circumferential mesangial interposition in capillary loops was seen in one case, age 58, the oldest patient in the group; her biopsy (LM) contained 6 glomeruli, 3 of them being obsolete. In this group there were 2 couples of siblings who donated a kidney to a third brother (Families II and III, Table 2). In both cases the receptor's kidneys showed a severe renal retraction. In 2 families only (I and VII, Table 2) it was possible to

~

Fig. 5. Living donor, 58 years old, female; (belongs to family IV in Table 2). Dense deposits in the mesangial matrix (0) especially around mesangial cells (M) with abundant type I collagen fibers in the II!~sangial matrix (*), between mesangial cells. LC = capillary lumen; P = podocyte. ME x 6000.

Table 2. Morphological characteristics of IgA nephropathy in nine living-related donors Family

Age at donation

Sex

23

F

Sister

IgA+ IgM+ C3+

+

28

F

Sister

IgA+ IgM+

+

28

F

Sister

IgA+ IgM+

+

22

F

Sister

IgA++ C3+

+++

29

M

Brother

IgA+ IgG+ C3+

+

29

M

Brother

+

V

35

Brother

++

VI

58

M F

IgA+ IgA+

Mother

IgA+

++

VII

37

F

Sister

19A++ C3+

+

II

III

IV

Relation

1M

Electron microscopy DMD

Collagen

Interposition

++

+ ++

1M = immunofluorescence, DMD = dense mesangial deposit; Interposition: mesangial circumferential interposition in free capillary loops; Collagen: type I collagen in mesangial matrix.

Donor's Glomerulopathies . 623 demonstrate an IgA nephropathy as the cause of their terminal renal insufficiency. Discussion A high percentage of family-related glomerular alterations have been found in LD, the most frequent being IgA glomerulopathy (12.9%). This percentage may be magnified in comparison to the normal population by a certain family tendency for this disease 36,38,59. In our material we have found 4 families in which two pairs showed IgA and electronmicroscopical glomerular deposits. Even though no common factor could be identified in the studies performed among family groups, a hereditary, environmental or exogenous factor, such as a virus capable of modifying-B-lymphocyte behaviour could be responsible for this 38 . Unknown factors have also been proposed 9. Only few cases of Berger's disease have been reponed!6 in living5,36 or cadaver donors. We think that this disease could be a much more frequent cause of renal destruction in our population than previously observed. The prognosis for a monorenal patient with Berger's disease is probably not as good as for a person who has two kidneys. Nevertheless, this remains to be demonstrated since controls over long periods of time do not exist. Our 9 cases were gathered from 1980 to 1987 and all were relatively young subjects. The two older ones, a man of 35 and a woman of 58, had the most intense lesions under EM, especially the latter, who evidenced an initial mesangiocapillary destructive lesion. There are two groups of donors with few glomerular changes which could represent a still not well known pathogenesis. In the first group (five cases) the basal membrane appears uniformly thickened in the free capillary loops as well as in the paramesangial zones, fully resembling the changes seen in diabetics. We have also found them in some cases of isolated idiopathic hematuria which have no apparent relation with diabetes mellitus 27 . Other authors have also found these changes in nondiabetic young LD32. These five cases were young with a mean age of 30, with negative 1M and normal LM. We cannot predict anything regarding the future of these monorenal patients. It must be taken into account that diabetic-type lesions have been shown to accelerate their course in monorenal patients. It is also important to emphasize that the BM thickening will not regress in the recipient! as we could demonstrate in one case in a post-transplant biopsy. The second group (9 cases) consists of those cases that we have called "relative glomerular ischemia with irregular basal membrane". This latter characteristic is stated in order to differentiate them from those with simple changes of the relative ischemia type such as the increase in paramesangial basal membrane folds. These donors had a mean age of 37, that is the oldest group of LD. Nevertheless, there are two cases, 25 and 26 years old, who already had this type of change, one of them with subsequently detected proteinuria. A greater intensity of the "relative ischemia" was seen in EM in older donors: 40 to 49 years

of age. The changes could represent precocious vascular damage as indicated by a strong positivity for C 3 in 5 cases with secondary glomerular changes which could worsen the prognosis for this group of "acquired" monorenal patients. This has also been observed by others 14,2!. We can conclude that approximately one third of living-related donors, considered to be clinically healthy, have glomerular lesions. Of these, the majority correspond to or are compatible with Berger's disease (lgA present, dense mesangial deposits in EM with or without collagen), more advanced lesions being found in older donors. There are few studies which show pre-existing glomerulonephritis in living donors 3,25. These lesions can only be found by using LM, 1M, and EM techniques. There are single publications which demonstrate immune deposits of C 3 and IgM, especially in living donors, but these findings were not correlated with the EM picture3. It would be of great interest to evaluate the future of monorenal donors, in relation with the morphological findings described in the donated kidney. Reports of this kind are practically non-existent in the current medical literature.

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8 Drinovec J, Malovrh M, Kandus A, Cizman B, Luzar S, Kosak M, Ravnik L, Kveder R, Ponikvar R, Mocivnik M, Benedik M, Varl J, Pavlovic SK (1987) Follow-up of donors in living related renal transplantion. Transplant Proc 19: 3645-3646 9 Egido J, Blasco RA, Sancho J, Hernando L (1985) Immunological abnormalities in healthy relatives of patients with IgA nephropathy. Am J Nephrol 5: 14-20 lO Fehrman I, Widstman V, Lundren G (1986) Long-term consequences of renal donation in humans. Transplant Proc 18:

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11 Fine L (1986) The biology of renal hypertrophy. Kidney Int 29: 619-634 12 Fotino S (1989) The solitary kidney: a model of chronic hyperfiltration in humans. Am J Kidney Dis 13: 88-98

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28 Rosenberg HG, Pulgar HA, Martinez LV (1983) El glomerulo normal en donantes vivos para transplante renal. Estudio con microscopia de luz, microscopia electronica e inmunofluorescencia. Rev Med Chile 111: 559-563 29 Rugiu C, Oldrizzi L, Lupo A, Valvo E, Loschiavo C, Tessitore N, Gamaro L, Ortalda V, Fabris A, Panzetta G, Maschio G, (1986) Clinical features of patients with solitary kidneys. Nephron 43: 10-15 30 Schmitz A, Christensen CK, Christensen T, Soiling K (1989) No microalbuminuria or other adverse effects of long-standing hyperfiltration in humans with one kidney. Am J Kidney Dis 13: 131-136 31 Stahl RA, Oberle G, Neumann HPA, Schollmeyer P (1986) Einzelniere-Risiko oder tolerabler Organverlust? Dt Med Wochenschr 111: 350-354 32 Steffes MW, Barbosa J, Basgen YM, Sutherland DER, Nanjarian JS, Mauer SM (1983) Quantitative glomerular morphology of the normal human kidney. Lab Invest 49: 82-86 33 Talseth T, Fauchaid P, Flatmark A (1986) The long-term prognosis of living kidney donors. Transplant Proc 18: 106-107 34 Thorner PS, Arbus GS, Celemajer DS, Baumal R (1984) Focal-segmental glomerulosclerosis and progressive renal failure associated with unilateral kidney. Pediatrics 73: 806-810 35 Tolkoff-Rubin NE, Cosimi B,Rubin RH, Colvin RB (1978) IgA nephropathy in HLA identical siblings. Transplantation 26: 430-433 36 Valenzuela R, Hamway SA, Deodhar SD, Braun WE, Banowsky LH, Magnusson MO, Osborne DG (1980) Histologic, ultrastructural, and immunomicroscopic findings in 96 one hour human renal allograft biopsy specimens. Immunologic and clinical significance. Hum Patholl1: 187-195 37 Waldo FB, Beischel L, West CD (1986) IgA synthesis by lymphocytes from patients with IgA nephropathy and their relatives. Kidney Int 29: 1229-1233 38 Weiland D, Sutherland DER, Chavers B, Simmons RL, Ascher J, Nanjarian J (1984) Information on 628 living related kidney donors at a single institution, with long-term follow-up in 472 cases. Transplant Proc 16: 5-7 39 Weymouth RJ, Seibel HR, Lee HM, Hume DM (1970) The glomerulus in man one hour after transplantation. An electron microscopy study. Am J Pathol58: 85-104 40 Zucchelli P, Caglioni L, Domini V, Pasquali S (1984) Focal glomerulosclerosis in patients with unilateral nephrectomy. Kidney Int 24: 649-655

Received August 4, 1989 . Accepted in revised form January 15, 1990

Key words: Donated kidneys - Glomerulopathies in donors - IgA donor's nephropathy - Transplantated kidney Prof. Dr. H. G. Rosenberg, Pontificia Universidad Catolica de Chile, Escuela de Medicina, Anatomia Patologica, Casilla 114-D, Santiago, Chile