Renal Lesions in Baltic Grey Seals () and Ringed Seals ()

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INTRODUCTION

A severe reduction in the population size of seals in the Baltic was observed during the latter half of the 20th century. In the late 1960s, Jensen et al. (1) reported serious pollution of the Bal-

lesions. The lesions present both in the reproductive and nonreproductive organs of Baltic grey and ringed seals are believed to be part of a disease complex in which the adrenal changes (cortical hyperplasia) are suspected to be a crucial factor (6–8).

A report on the prevalence of lesions of the disease complex in Baltic grey seals during 1977–1996 showed improved gynaecological health, but there was an increased prevalence of colonic ulcers for the 10-year period, 1987–1996, compared to that of 1977 to 1986 (8).

Except for an earlier brief description of renal lesions in Bal- tic seals (6), no other report on renal lesions in grey and ringed seals has been published. In this paper, we present a detailed description of the pathology of the kidney in Baltic grey seals and ringed seals at the light microscopic and, in part, electron microscopic level. Correlations with other organ lesions in the same animals are also made.

MATERIAL AND METHODS Animals

Kidney tissue for light microscopy was obtained on necropsy of a total of 144 seals from 4 different sites: the Baltic, Swedish zoological gardens, Svalbard, and Scotland (Table 1).

The Baltic samples were from 76 grey seals (36 females and 40 males) and 29 ringed seals (17 females and 12 males). These animals were collected in the Baltic Sea and the Gulf of Bothnia during 1977–1996. Of the grey seals, 57 had drowned in fishing gear, 14 were found dead on the shore and 5 were shot due to disease. Of the ringed seals, 26 had drowned in fishing gear or seal nets, 2 were found dead on the shore and 1 was killed due to disease.

Five grey seals from Swedish zoological gardens were also investigated. These showed organ lesions similar to those common in Baltic grey seals. They had been fed fish from the Bal- tic for most of their lives (9).

For comparison, organs were studied from animals collected on hunting expeditions to Svalbard in 1981 (23 Arctic ringed seals, Phoca hispida hispida), and to Helmsdale in northern Scot- land 1988 (11 grey seals).

A severe reduction in the populations of grey and ringed seals in the Baltic occurred during the 1960s and 1970s.

Adult animals showed (and still show) a series of lesions inter alia in the female reproductive organs, intestines, integument, kidneys, adrenals, and skulls (the Baltic seal disease complex). The morphology and prevalence of light microscopic changes in the kidneys of 76 grey seals and 29 ringed seals collected in the Baltic proper and the Gulf of Bothnia during 1977–1996 are presented in this report.

Specific changes in the glomeruli were diffuse thickening of the capillary walls and the presence of large, rounded, hyaline bodies in the capillary or capsular walls. Specific changes in the distal convoluted tubules and the collecting ducts included focal replacement of the normal epithelium by multilayered cell proliferations. The prevalence and extent of the changes were age-related and thus correlated with the time of exposure to environmental toxicants. The lesions were more conspicuous in Baltic grey seals than in Baltic ringed seals. Similar findings were recorded in 5 grey seals from Swedish zoological gardens. These animals had been fed Baltic fish for most of their lives. Elec- tron microscopy was performed on 5 of the Baltic grey seals and on one of the grey seals from zoological gardens. Electron microscopy results mainly based on findings in one of the Baltic grey seals, included mesangial interposition in the glomerular capillary walls and the charac- teristics of intercalated cells in cell proliferations in the distal parts of the nephrons. Eleven grey seals from the Scottish coast and 23 ringed seals from Svalbard served as reference material. None of the reference seals showed the specific lesions described above. The authors propose that organochlorine pollution of the Baltic environment is a factor in the cause of these kidney changes.

Renal Lesions in Baltic Grey Seals (Halichoerus grypus) and Ringed Seals (Phoca hispida

botnica)

Anders Bergman, Anders Bergstrand and Anders Bignert

Report

Table 1. Number of animals providing kidney tissue. Ordered into species, sex and age.

Age Class, years < 1 1–3 4–10 11–25 > 25 Total

Baltic grey seal females 4 8 3 5 16 36

Baltic grey seal males 6 18* 11 5** 0 40**

Swedish zoo grey seal females 0 1 0 0 0 1

Swedish zoo grey seal males 0 0 0 2 2 4

Scottish grey seal females 3 3 0 0 0 6

Scottish grey seal males 0 5 0 0 0 5

Baltic ringed seal females 2 0 6 8 1* 17

Baltic ringed seal males 0 1 4 6 1 12

Arctic ringed seal females 0 1 1 5 1 8

Arctic ringed seal males 0 1 4 8 2 15

Total 144

*) Age estimated in one case. **) Includes a case with glomerular amyloidosis, not used in statistics.

tic by DDT and polychlorinated biphenyls (PCBs). Hook and Johnels (2) and Olsson et al. (3) suggested that this pollution was one factor responsible for the precarious situation for the seal populations. Helle et al. (4, 5) reported severe changes in female reproductive organs, in particular occlusions and stenoses of the uterus, presumably evi- dence of interrupted pregnancies. In addi- tion to these changes, Bergman and Olsson (6) reported a high prevalence of uterine leiomyomas in grey seals, and high prevalences of severe chronic lesions in nonreproductive organs in both sexes of grey seals and ringed seals (6, 7). The main lesions observed in the nonreproductive organs comprised claw deformations, regional intestinal (colonic) ulcers, adrenocortical hyperplasia, aortic wall changes and renal

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Electron microscopic investigations were performed on kidneys of 6 of the abovementioned grey seals (3 males and 3 females), in the following labelled individually. Of the males, 2, aged 3 years (Nos 1 and 2), were from the Baltic, and 1, aged 33 yrs (No. 3), was a captive seal. The females were all from the Baltic, 2 were aged 35 (Nos 4 and 5) and 1 was 40 years (No. 6). Animals Nos 1, 2, 4 and 5 had drowned in fishing gear and animals Nos 3 and 6 were shot due to serious illness.

Influence of autolytic changes? An important question about reports on microscopy in animals of fallen game is the impact of postmortem changes. For the reference seals, necropsied soon after death, these changes were negligible. The material of the Baltic grey- and ringed seals as well as that of the grey seals from Swedish zoological gardens presented here, was part of a large necropsy material from which cases with disturbing autolysis had been rejected. Slight autolytic changes were present in the kidneys of the Baltic seals and the seals from zoological gardens, but not to such an extent that they influenced adequate light microscopic observations.

Necropses

Necropsy procedures included determinations of body and organ weights, body length and other body measurements, blubber thickness (nutritional status), tissue sampling for histology and environmental contaminant chemistry and, when appropriate, for parasitology, bacteriology and virology.

Many data are available from necropses on Baltic grey seals.

For this species correlations were made between age, prevalence and severity of 3 types of organ lesions: colonic ulcers, adrenocortical hyperplasia and aortic wall changes, and 3 types of kidney lesions: glomerular capillary wall thickening, glomerular capillary wall nodules, and tubular cell proliferations.

Severity (grades) of colonic ulcers were defined as 0 – no changes; 1 – minor ulcerations (3–10 mm in diam.); 2 – ulcerations of larger mucosal areas, often involving the full circum- ference of the mucous membrane; 3 – profound lesions, reach- ing the muscular tunic, and quite often the serosal tunic. In the present investigation, grade of adrenocortical hyperplasia was measured using the total weight of the 2 glands. Grades of changes of the aortic wall were defined as 1 – presence of focal

Figure 1. Glomerulus with no capillary wall thickening (Grade 0).

Basement membranes indicated by arrows. PAS x 360. Baltic ringed seal female, 5-yrs-old.

Figure 2. Glomerular capillary wall thickening, Grade 1 (slight).

Basement membranes indicated by arrows. PAS x 360. Baltic grey seal female, 33-yrs-old.

intimal fatty streaks; 2 – extensive intimal changes, occupying most of the distal part of the vessel; and 3 – deeper fatty changes with irregular form and loss of elasticity in the vessel wall.

Age Determination

Age was determined by examination of tooth sections according to Johnston and Watt (10). In 2 animals, teeth were lost during post-autopsy procedures so their age class was estimated: an old Baltic ringed seal female, around 30 yrs, and a male grey seal, 1 year. Criteria for age estimation were body size and weight, morphology of sex organs, tooth wear, etc. An important criterion was the degree of thymic involution; thymic tissue has not been observed grossly in Baltic seals older than 20 yrs (Bergman, unpubl. data).

The age and sex distribution for 5 age classes: < 1, 1–3, 4–

10, 11–25, and > 25-yrs-old are shown in Table 1. The lowest age limit for sexual maturity in grey seals was taken to be 4 yrs, i.e. the age of the youngest pregnant animals recorded in the Swedish investigation. The same age limit for sexual maturity in grey seal females is reported by Bonner (11), who also reported “a probably similar” age of sexual maturity in grey seal males. The corresponding lowest limits of sexual maturity reported for ringed seal females and males are 5 and 6 yrs, respectively (12). According to age determinations the oldest Baltic grey seal females were around 40, whereas the oldest male was 23-yrs-old. The oldest Baltic ringed seal was a male aged 30 yrs.

Of the reference animals (Table 1) all Scottish grey seals were immature ≤ 3 yrs), while the range of ages of the ringed seals from Svalbard was 3 to 32 yrs for females and 2 to 28 yrs for males.

Histology

Light microscopy (Figs 1-7): Small but representative pieces of organs were fixed in 10% neutral buffered formalin and embed- ded in paraffin. Sections, about 5 microns thick, were stained routinely with haematoxylin and eosin (H and E) and periodic acid Shiff (PAS) technique. When appropriate, other staining techniques were used including periodic acid silver methenamine (PASM), van Gieson, Masson’s trichrome, Ladewig’s modifi- cation of Mallory’s trichrome staining, Martius-Scarlet-Blue

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disposition by courtesy of Frank et al. (14) and Blomkvist et al.

(15), respectively. Detailed descriptions of the methods are found in these papers. Cadmium and lead were measured in the kidney cortex of 26 of our animals, 14 females aged between 7 and 41 yrs, and 12 males aged from yearlings to 20 yrs. Mercury was determined in the liver of 25 of the same animals. In the 26 Baltic grey seals, which were analyzed for cadmium and lead, concentrations of sPCB and sDDT were determined in extractable fat of blubber (15). Data on these organochlorines from 7 additional Baltic grey seals in our series were available at the Swedish Museum of Natural History and were added. Blomkvist et al. (15) also reported data on sPCB and sDDT concentrations in 7 of the reference young seals from Scotland. The sPCB concentrations were determined by comparison with a solution of the technical product Arochlor 1254 and the sDDT values were obtained by adding the concentrations of p,p’-DDT, p,p’-DDE and p,p’-DDD (15).

Statistics

Statistical tests were performed on results solely from Baltic grey seals. The nonparametric Kendall’s rank correlation test (16) was applied. This test is resistant to inappropriate leverage effects of extreme values. Males and females were treated separately.

Since repeated Kendall rank correlation tests were carried out on the same material, the α-level was adjusted according to

˘Sidák (38). The test was used to investigate:

–␣ The relationship between age and the 3 renal histological variables (CWT, CWN and TCP) graded above and some lesions present in other organs: colonic ulcers, aortic wall changes and adrenal weight (Table 2).

–␣ The relationship between grades of the 3 renal histological variables and grades of colonic ulcers, adrenal weight and aortic wall changes (Table 3).

–␣ The interrelationships between the 3 renal histological variables (Table 4).

–␣ To investigate whether a correlation existed between increasing concentrations of heavy metals (Cd, Pb and Hg) in kidney and liver tissues (Table 5) and on levels of sPCB and sDDT in the blubber (Table 6) and the degree of kidney lesions (CWT, CWN and TCP), colonic ulcers, aortic wall changes and adre-

Figure 4. Glomerular capillary wall thickening, Grade 3 (severe).

PAS x 360. Baltic grey seal female, 35-yrs-old.

Figure 3. Glomerular capillary wall thickening, Grade 2 (moderate).

PAS x 360. Baltic grey seal male, 23-yrs-old.

(MSB) method for fibrin, Verhoeff’s method for elastic fibers, van Kossa’s method for calcium and Congo Red for amyloid.

Frozen sections were stained for lipids with Sudan IV. For further information on the staining techniques see Bancroft and Stevens (13).

Histological Grading: On average 4 sections from different sites of the kidneys in each animal were studied by light microscopy. Three kinds of lesions were graded: glomerular capillary wall thickening (CWT); glomerular capillary wall nodules (CWN); and tubular cell proliferations (TCP).

Classification of glomerular capillary wall thickening was based on our experience from domestic animals and man. The lesion was classified in 4 grades: 0 (no changes); grade 1 (slight); grade 2 (moderate); and grade 3 (severe). The classification was first performed by two of the authors independently. Full agreement was reached in about 90% of the cases. When there was disagreement the sections were re-examined together and discussed until agreement was reached. Examples of the different grades of glomerular capillary wall thickening are shown in Figures 1–4.

The prevalence of tubular cell proliferations was calculated using an indexed square in the microscope eyepiece. In sections of each case, 100 squares of 1 mm², strictly positioned edge to edge, were investigated at x100 magnification. Seventy such areas were investigated in the renal cortex and 30 in the medulla.

For each square, presence or absence of tubular-cell proliferations were recorded. The prevalence of glomerular capillary wall nodules was determined as the percentage of glomeruli with this change in the investigated cortical area (70 mm²). Grading of these two variables is presented in RESULTS, Histological grad- ing – age, sex and species differences.

Electron Microscopy Slices (Figs 8-12): of renal tissue 1 mm thick were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer for 48 hours. Post-fixation was performed in 1% osmium tetroxide in the same buffer for 2 hrs at 4°C followed by dehy- dration in alcohol-acetone and embedding in Lx 112. Ultrathin sections were stained with uranyl acetate and lead citrate.

Chemical Analyses

Data on tissue concentrations of heavy metals and the organochlorines PCB and DDT in Baltic grey seals were placed at our

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Table 2. Relationship between grades of 3 renal histological variables: glomerular capillary wall thickening (CWT), glomerular capillary wall nodules (CWN) and tubular cell proliferations (TCP), and grades of 3 variables in other organs: colonic ulcers (COLU), adrenal weight (ADW) and aortic wall changes (AWC), and age in Baltic grey seals.

τ = Kendall’s tau, p = probability to achieve the corresponding τ by chance, a p < 0.01 is considered significant which corresponds approximately to a true significant level of 5% after adjusting the level for repeated test according to ˘Sidák (38). NS = not significant.

AGE CWT CWN TCP COLU ADW AWC

Sex n τ p < τ p < τ p < τ p < τ p < τ p <

Males 27–33 .54 .001 .24 NS .39 .01 .22 NS .81 .001 .55 .001

Females 28–32 .71 .001 .72 .001 .49 .001 .29 NS .59 .001 .71 .001

Table 3. Relationship between grades of 3 renal histological variables: glomerular capillary wall thickening (CWT), glomerular capillary wall nodules (CWN) and tubular cell proliferations (TCP) and grades of 3 variables in other organs: adrenal weight (ADW), above, colonic ulcers (COLU), middle, and aortic wall changes (AWC), below, in Baltic grey seals.

τ = Kendall’s tau, p = probability to achieve the corresponding τ by chance, a p < 0.01 is considered significant which corresponds approximately to a true significant level of 5% after adjusting the level for repeated test according to ˘Sidák (38). NS = not significant.

ADW CWT CWN TCP COLU AWC

Sex n τ p < τ p < τ p < τ p < τ p <

Males 33 .52 .001 .29 NS .58 .001 .19 NS .46 .001

Females 31–33 .57 .001 .49 .001 .54 .001 .56 .001 .58 .001

COLU CWT CWN TCP ADW AWC

Males 33–38 .08 NS .31 .01 .35 .01 .19 NS .24 NS

Females 32–36 .43 .001 .32 .01 .37 .001 .56 .001 .34 .01

AWC CWT CWN TCP COLU ADW

Males 33–38 .60 .001 .45 .001 .44 .001 .24 NS .46 .001

Females 31–34 .79 .001 .67 .001 .57 .001 .34 .01 .58 .001

Table 5. Geometric mean concentrations (bold) and ranges, mg kg^–1, of cadmium and lead in kidney cortex wet weight and mercury in liver wet weight in Baltic grey seals of different sex and age groups. Number of individual specimens analyzed within brackets. Published by courtesy of Frank et al. (14).

Age, Cd Pb Hg

years

Baltic grey seal ≤ 3 0.68 (5) 0.13 (5) 18 (5)

males 0.25–1.3 0.085–0.18 11–35

Baltic grey seal ≤ 3 – – –

females

Baltic grey seal 4–25 1.5 (7) 0.13 (7) 34 (7)

males 0.84–2.7 0.093–0.24 23–92

Baltic grey seal 4–25 2.2 (6) 0.13 (6) 56 (6)

females 1.6–3.3 0.066–0.25 18–82

Baltic grey seal > 25 – – –

males

Baltic grey seal > 25 2.1 (8) 0.14 (8) 129 (7)

females 0.89–4.6 0.051–0.26 70–730

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Table 4. Interrelationship between grades of 3 renal histo- logical variables: glomerular capillary wall thickening (CWT), glomerular capillary wall nodules (CWN) and tubular cell proli- ferations (TCP) in Baltic grey seals. τ = Kendall’s tau, p = pro- bability to achieve the corresponding τ by chance, a p < 0.01 is considered significant which corresponds approximately to a true significant level of 5% after adjusting the level for repeated test according to ˘Sidák (38). NS = not significant.

CWN TCP

τ p < τ p < Sex

CWT .74 .001 .61 .001 Females

.45 .001 .38 .01 Males

TCP .57 .001 Females

.28 NS Males

Males, n = 39 Females, n = 36

nal weight.

–␣ Finally, to investigate the relationship between age and tissue concentrations of the chemical compounds under consideration.

RESULTS Baltic Seals

There were no qualitative differences between the species as regards renal lesions, so they are described together.

Macroscopic findings: Kidneys with se- vere lesions were pale, hard, and had lus- treless cut surfaces. In a few cases, the capsule was tightly adhered to the renal surface. There were no scars or change of shape in any of the cases. One male grey seal showed signs of embolic nephritis with several abscesses. Cysts of moderate size were found in 1 grey seal male. In 1 kidney of a male ringed seal a hard yellowish- white ramified calculus occupied the pelvic tree.

Comprehensive descriptions of macroscopic findings in other organs of the Bal- tic seals under consideration, and records on causes of death have been reported earlier by members of our group; in 1985 on grey and ringed seals (6) and 1999 (8) on grey seals, comprising the 20-yr period 1977–1996.

Glomerular Lesions

Light microscopy: The most striking feature was diffuse uniform thickening of the glomerular capillary basement membranes (Figs 2–4). Silver staining showed no de-

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posits, so called spikes or “tram track” structures, on either side of the membrane. No proliferation of endothelial or epithelial cells was observed at this level of resolution except occasional slight epithelial proliferations (crescents) on the inside of Bow- man’s capsule. Mesangial changes with increases of matrix or cell number were slight and were found also in young animals.

The basement membrane of Bowman’s capsule was less fre- quently thickened and laminated. Segmental sclerosis with ad- hesions between capillaries and capsule was observed in a few cases. Total global sclerosis was seen in a varying number of glomeruli in connection with vascular and interstitial lesions.

Small deposits of calcium were seen in the glomerular capillary walls in a few grey seals with severe changes.

A notable feature, observed in several cases, was the occurrence of large hyaline nodules (Fig. 5) in the capillary walls or in the basement membrane of Bowman’s capsule in some

Table 6. Geometric mean concentrations (bold) and ranges, mg kg^–1, of sDDT and sPCB in extractable fat of blubber in young Scottish grey seals and Baltic grey seals of different sex and age groups. Number of individual specimens analyzed within brackets. Published by courtesy of Blomkvist et al. (15).

Age, sPCB sDDT

years

Scottish grey ≤ 3 4.3 (7) 1.7 (7)

seal males and 2.7–7.2 1.2–3.1

females

Baltic grey seal ≤ 3 87 (9) 37 (9)

males 49–160 14–66

Baltic grey seal ≤ 3 110 (1) 98 (1)

females

Baltic grey seal 4–25 95 (7) 32 (7)

males 66–140 11–100

Baltic grey seal 4–25 240 (6) 84 (6)

females 57–770 13–180

Baltic grey seal > 25 – –

males

Baltic grey seal > 25 780 (10) 270 (10)

females 160–5300 87–1600

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Figure 7. Large tubular epithelial cell proliferations at the cortico- medullary border. H & E x 360. Baltic grey seal male,

23-yrs-old (the same as in Figure 3).

Figure 6. Glomerular deposits of amyloid in mesangial areas and peripheral capillary walls.

PAS x 360. Baltic grey seal male, 17-yrs-old.

Figure 5. Renal cortex showing large glomerular hyaline nodules (right glomerulus), capsular adhesion (left glomerulus) and islands of tubular epithelial cell proliferations (arrows). PAS x 180. Baltic grey seal female, 40-yrs-old.

glomeruli. They were structureless, stained pale red with H &

E and bright red with PAS, but were not stained by silver stains.

With the Martius-Scarlet-Blue (MSB) method for fibrin they were negative or stained weak rose to bright red. In a few grey seals, similar nodules were observed in the walls of the arterioli at the vascular pole.

Amyloid with Congo Red affinity and typical optical bi- refringence was present in the mesangial areas and the glomerular capillary walls of 1 animal, a 17-yr-old male grey seal (Fig.

6).

Staining for neutral fat showed small fat droplets, sometimes concentrated into large globules, in the capillary epithelial cells in the glomeruli of 15 grey seals (13 females and 2 males) in connection with fatty changes in the tubular epithelium.

Electron microscopy: Three glomeruli from each of the 6 grey seals were selected for the study. Autolytic changes were present

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in the capillary wall epithelial and endothelial cells of 5 animals (Nos 1–5). The basement membrane measured 400–500 nM in the 2 of the 3-yr-old males from the Baltic (Nos 1 and 2) and had a very narrow lamina rara externa and interna as seen in humans, but in contrast to what is seen in other animals, such as rats and rabbits. The basement membrane was considerably thicker, measuring 640–960 nM, in the 33-yr-old male from the zoological garden (No. 3) and in the two 35-yr-old Baltic females (Nos 4 and 5) as well as in the 40-yr-old Baltic female (No. 6), presumably due to old age. The basement membranes in the old animals (Figs 8–10) were occasionally duplicated or “split” with a thick outer, subepithelial, and a thin, subendothelial, layer. Be- tween them there was a fluffy layer containing fine fibrils (Fig.

9). Congo Red staining for amyloid was negative.

The material from the Baltic female No. 6, which was recov- ered immediately after it had been killed, was well preserved.

The endothelial cells were swollen and dislocated from the basement membrane. The basement membranes were duplicated as in the other old animals. Most striking were areas of cellular material, located in the basement membrane. The areas were bor- dered by a cell membrane and contained cell organelles, including mitochondriae, endoplasmic reticulum and peroxisomes (Fig.

10). These changes correspond to what is called “Mesangial in- terposition” (17, 18). The capillary wall epithelial cells were swollen with pale cytoplasm, few cell organelles and a moderate retraction (fusion) of foot processes. The cytoplasm of some epithelial cells contained a great number of vacuoles. There were

no electron-dense deposits in the capillary walls or mesangial areas.

Tubular Lesions

Light microscopy: A prominent feature was intraluminal prolif- erations of epithelial cells in the distal convoluted tubules and collecting ducts (Figs 5 and 7). The cells were monomorphic, large, pale, and polygonal. In some cases, they consisted of a monolayer on the tubular basement membrane, in others the tubules were distended and completely filled with solid islands of cells. They were always strictly located inside the basement membrane. Fat staining revealed fatty droplets in the cell cytoplasm mainly in the proximal convoluted tubules and in the medulla in connection with old age and severe glomerular and interstitial changes. This fatty change was focally distributed, usu- ally in the form of fine droplets. In one case only, with pro- nounced accumulation of lipids in the proximal convoluted tubules, lipids also occurred in proliferated cells as fine droplets, exclusively localized in the periphery of the cell clusters, close to the tubular basement membrane.

Proteinaceous casts were present above all in connection with proliferative changes in the tubular epithelium. Hyalinization of the tubular basement membrane was a common finding in connection with interstitial fibrosis. Small calcium deposits were observed, above all in the tubular basement membranes or epithelial cells.

Electron microscopy: Tubular cell proliferations could be stud-

Figure 8. Peripheral capillary loop of a glomerulus. The basement membrane is duplicated around the whole loop with a thick outer and a thin inner component. Between them is a fluffy layer. A slight “fusion”

of foot processes is seen on the outside. Below a mesangial area (M) and nucleus. Bar = 1 µm. Baltic grey seal female, 40-yrs-old. EM-case No. 6.

Figure 9. Part of glomerular capillary wall with irregularly oriented thin fibrils in the fluffy layer. Endothelial cell to the right. Bar = 0.5 µ^m.

EM-case No. 6.

M M

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D D P P

P P D D I I

D D

I I O M O M

I M I M

E N E N L L

E R E R

P P M M

ied only in the 40-yr-old Baltic grey seal female (No. 6); At high magnification (Fig. 11) individual cells of the proliferations were found to be cylindrical or polygonal with a large elongated nucleus. The chromatin was sparse and membrane-associated. The nucleoli were large. The cytoplasm contained, above all in the superficial parts of the cells, a great number of small vacuoles without content. There were shallow invaginations of the basal cell membrane. The intercellular spaces were narrow but fre- quently dilated containing interdigitating fingerlike protrusions (Fig. 12). Tight junctions were present at the surface and desmosomes were numerous. Cell organelles were few. There were very few mitochondriae and a sparse endoplasmic reticulum, but a great number of free ribosomes. In many cells, clusters of large, weakly stained osmiophilic bodies limited by a sin- gle membrane, were seen close to the nucleus.

Interstitial and Vascular Changes

These changes were only studied by light microscopy. The interstitium showed areas of chronic inflammation, mainly containing mononuclear cells and/or areas of fibrosis. Small foci of calcification were seen in some cases. Amyloid was present in the connective tissue of the interstitium as rather large deposits in 2 grey seal females, 35 and 40-yrs-old respectively, and as small deposits in a 17-yr-old grey seal male which, as mentioned above, also had amyloid deposits in the glomeruli.

Sclerotic changes in large renal arteries were found in ringed and grey seals above the age of 12 and 13 yrs, respectively. The

Figure 10. Mesangial interposition in glomerular capillary wall. Outer part of the basement membrane (OM). Endothelial cell with nucleus (EN). On its inside a thin wrinkled inner basement membrane (IM).

Between the 2 membranes part of the mesangial cell cytoplasm with mitochondriae (M), lysosomes (L), endoplasmic reticulum (ER) and a peroxisome (P).

Bar = 0.5 µm. EM-case No. 6.

Figure 12. Two tubular epithelial cells with small vacuoles in the cytoplasm. In the intercellular spaces are interdigitating fingerlike protrusions (P). Several desmosomes (D) are present. Bar = 1 µ^m.

EM-case No. 6.

Figure 11. Highly vacoulated part of tubular epithelial cell and adjacent cells. The cells are interdigitating (I). Desmosome (D). Bar = 0.5 µ^m.

EM-case No. 6.

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14A. Baltic grey seal females 4–25-yrs-old

(n = 8)

14B. Baltic grey seal males 4–25-yrs-old

(n = 15)

14C. Baltic grey seal females > 25-yrs-old

(n = 16)

15A. Baltic ringed seals,

≤ 30-yrs-old

females (n = 17), males (n = 12)

15B. Arctic ringed seals,

≤ 32-yrs-old females (n = 8), males (n = 15) Figure 14.

Figure 15.

13A. Baltic grey seal females ≤^3-yrs-old

(n = 12)

13B. Baltic grey seal males ≤^3-yrs-old

(n = 24)

13C. Scottish grey seals

≤^3-yrs-old females (n = 6), males (n = 5) Figure 13.

changes increased in frequency and severity with age. They included disruption and fragmenta- tion of the internal elastic lamina, proliferation of smooth muscle cells and increases in collagen and elastic fibers subintimally.

Changes of small arteries and arterioli were observed in older grey seals and included cases with hyalinosis and cases with prominent hyperplastic thickening of the vessel walls. Arteriolar changes were not observed in ringed seals.

Histological Grading, Chemical Analyses and Statistics

Histological grading—age, sex and species differences: A survey of the prevalence of specific renal changes in the different ma- terials according to age, sex, and species is presented in Figures 13–15. The scores for CWN (glomerular capillary wall nodules) and TCP (tubular cell proliferations) calculated as described above have been divided to obtain the same 4 grades of changes as for CWT (glomerular capillary wall thickening) as follows. For CWN: zero no changes; slight 1–20%; moderate 21–40; and severe > 40% (the highest score being 59%). For TCP: zero no changes; slight 1–

25; moderate 25–50; and severe

> 50 (the highest score being 78).

There were no males but 16 females older than 25 yrs among the Baltic grey seals (Table 1). In the age class 11–25 yrs the number of grey seals was rather small (6 females and 5 males). A comparison between the sexes in Baltic grey seals regarding frequency and grade of the histological variables was therefore performed using only 2 age classes: ≤ 3 (Figs 13A and B) and 4–25-yr-old animals (Figs 14A and B). Results in females > 25 years are shown in Figure 14C.

Results in Baltic and Arctic ringed seals, showing the same age distribution, are shown in Figs 15A and B.

In the ≤ 3-yr-old Baltic grey seals 2 of 12 females and 9 of 24 males showed slight TCP and 1 of the males showed slight CWN (Fig. 13A and B). Specific lesions were not observed in the 11 Scottish grey seals of corresponding age (Fig. 13C).

There was a higher prevalence

Figures 13–15. Prevalence of glomerular capillary wall thickening (CWT), glomerular capillary wall nodules (CWN) and tubular cell proliferations (TCP) in Baltic, Scottish and Arctic seals.

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of CWT and CWN in 4–25-yr-old Baltic grey seal females than in males, but the opposite relationship was present as regards TCP (Fig. 14A and B).

Most extensive and severe changes were seen in the 15 female Baltic grey seals above 25-yrs-old (Fig. 14C).

As seen in Figure 15A, slight specific changes of all 3 types were present in Baltic ringed seals, but they were less frequent and less severe than in the grey seals. These changes were not observed in the Arctic ringed seals of the same age class (Fig.

15B).

Interrelations between age and renal lesions as well as lesions of other organs: (Table 2). The grade of specific renal histologi- cal changes (CWT, CWN and TCP), adrenal weight (ADW) and aortic wall changes (AWC) were significantly correlated with age in females. In males, there was a significant correlation with age for these 5 variables except CWN. Colonic ulcers (COLU) were not correlated with age.

Interrelations between lesions of other organs and renal le- sions: (Table 3). There was a significant correlation between ad- renal weight and the renal lesions in both sexes except for CWN in males. Colonic ulcers were significantly correlated with CWN and TCP in both sexes and with CWT in females. Aortic wall changes showed a significant correlation with the renal lesions.

Interrelations between renal histological variables: (Table 4).

The 3 renal histological variables investigated were significantly correlated with each other except for CWN and TCP in males.

Age and appearance of different lesions: The first signs of spe- cific renal lesions appeared in the Baltic grey seals already below 3 yrs of age: TCP at 1 yr in males and 2 yrs in females and CWN at 2 yrs in males. CWT first appeared at 4 yrs in males.

CWN and CWT first appeared at 7 yrs of age in females.

In Baltic ringed seals, TCP first appeared at the age of 7 in males and 10 in females. CWN first appeared at the age of 13 in males and 8 in females. CWT first appeared at the age of 21 in males and 13 in females.

In Baltic grey seals, slight colonic ulcers were found already in yearlings and the youngest animal fatally affected, due to per- foration of the intestinal wall, was 1-yr-old. Slight adrenocortical hyperplasia occurred in some 1-yr-old animals while slight aortic wall changes occurred in some animals aged 4 to 8 yrs.

In Baltic ringed seals, a slight colonic ulcer was found in a yearling, but although some severe cases were observed, none of them was fatal. In this species, slight degree of adrenocortical hyperplasia and aortic wall changes first appeared at the age of 5 and 8 yrs, respectively.

Heavy metals, DDT and PCB: The concentrations of heavy metals, according to Frank et al. (14): cadmium and lead in the kidney cortex and mercury in the liver of Baltic grey seals are presented in Table 5 as geometric means and range for different sex and age groups. There was no significant positive correlation between concentrations of cadmium, lead, and mercury and increasing age nor was there significant correlation between these heavy metal levels and grades of pathologic changes.

Concentrations of sDDT and sPCB, according to Blomkvist et al. (15) and additional analyses, in extractable fat from blubber of Baltic grey seals and the young grey seals from Scotland are presented in Table 6. The young grey seals from Scotland had much lower body burdens of these organochlorines than the grey seals of corresponding age from the Baltic. There was a significant positive correlation between concentrations of sDDT and sPCB and age in Baltic grey seal females, (p < 0.01 and p

< 0.001, respectively) but not in males. The females showed a significant positive correlation between increasing concentrations of sPCB and sDDT and 2 types of renal changes: CWT (p < 0.01 for sPCB and p < 0.001 for sDDT) and CWN (p < 0.01 and p <

0.01, respectively). Females also showed a significant positive correlation between increasing concentrations of sPCB and aortic wall changes (p < 0.01). In males, increasing concentrations of

sDDT showed a significant negative correlation with colonic ulcers (p < 0.001). There was no further significant correlation between the levels of these organochlorines and the grades of the pathologic changes.

Grey Seals from Zoological Gardens

Macroscopical changes of the kidneys; paleness and slightly increased consistency, were present only in the oldest of the 5 animals; 2 males, aged 32 and 33 yrs). Histologically, a 1-yr-old female showed no renal lesions, a 12-yr-old male showed slight tubular cell proliferations and the remaining 3 males, one aged 24 and the 2 males mentioned above, showed specific glomerular and tubular changes of slight to moderate degree. Renal vascular and interstitial changes were absent in the female and slight to severe in the males.

The 1-yr-old female showed no lesions, while the 4 males showed lesions in the nonrenal organs of the same character and about the same prevalence as those occurring in adult Baltic grey seals.

Reference Seals

There were no macroscopical changes in the kidneys of the reference seals and the specific changes (glomerular capillary wall nodules, glomerular capillary wall thickenings, and tubular cell proliferations), which were present in the Baltic seals were not observed. Other minor changes were found. As observed in young Baltic grey seals an increase in mesangial width also occurred in the young grey seals from Scotland. Four of the 11 animals showed slight changes of this kind. Three showed small accumulations of mononuclear cells in the pelvic interstitium.

Of the 23 Arctic ringed seals, 4 showed focal minor changes of the interstitium (mononuclear cell accumulations and fibrosis) and 1 showed a slight increase in mesangial width. Proteinaceous casts and focal calcifications were common in the tubules of the papillary regions. Renal vascular changes of the same kind as in Baltic ringed seals were found in older animals while changes in arterioles were not observed.

Compared to the findings in the Baltic ringed seals few lesions of significance were observed in “nonrenal” organs in the reference ringed seals. The lesions were restricted to the adrenals.

Out of the 23 reference ringed seals investigated there were 2 individuals (9%) with adrenocortical hyperplasia. The prevalence of this change was much higher in Baltic ringed seals—among the 29 Baltic ringed seals records on macroscopic adrenal morphology were available for 25 of the individuals, and 14 of these (56%) showed adrenocortical hyperplasia. No lesions of significance were observed in “nonrenal” organs of the young grey seals from Scotland.

DISCUSSION

Light Microscopic Methods

The quantitative calculations of the prevalence of glomerular capillary wall nodules (CWN) and tubular cell proliferations (TCP) described in Material and Methods comprise a small area of the kidneys. The distribution of the lesions in the sections was random and we concluded that the quantitative records could be used in the statistical analyses with reasonable certainty.

Glomerular Lesions

Diffuse thickening of the glomerular capillary basement membranes with little or no mesangial changes is seen in several domestic animal (17) and in human (18) diseases such as membranous glomerulonephritis, and amyloidosis.

Membranous glomerulonephritis is characterized by immune deposits, called “spikes” on the epithelial side of the glomerular capillary basement membrane. Similar changes have been

(10)

observed in connection with drug abuse or intoxication with heavy metals (19). The latter observation is interesting considering the possibility of heavy metal poisoning of the Baltic seals.

“Spikes” were not observed in sections stained with silver methenamine, however, and electron microscopy showed no electron dense deposits in the capillary walls of the 6 animals investigated.

Mesangial interposition is a nonspecific lesion seen in several glomerular diseases. It may be caused by a stimulation of the mesangial cells by immunological mechanisms (17, 18). How- ever, we did not performed immunohistological investigations for immune complexes in our animals.

Duplicating or “splitting” of the capillary basement membrane is observed in many glomerular diseases, but nothing certain is known about the cause or importance of this change.

Amyloid was demonstrated in the glomeruli of a grey seal male. These depositions had probably developed secondary to purulent processes. The animal had been killed illegally by shot- gun and showed multiple abscesses in soft tissues of the skull, cervical and neck region with similar changes in the kidneys, the latter as a result of embolic spread. Bacteriological investi- gation revealed septic Escherichia coli infection.

The size and frequency of the hyaline globules is remarkable.

Similar, strongly PAS-positive, “fibrinoid”, globules with locali- zation in the capillary walls or the capsular wall are present in the exudative form of diabetic glomerulopathy in domestic animals (17) and in man (18, 19). Their connection with diabetes mellitus is unclear and they are not considered to be specific for diabetes (19). We have not been able to obtain fresh blood from the majority of our animals and have therefore no possibility to ascertain if the hyaline globules in the glomeruli were associated with an elevated blood sugar or any other endocrine disor- der. Hyaline nodules are also seen in the glomeruli in para- proteinemias such as light chain disease or Waldenström’s macroglobulinemia, but they are located in the mesangial areas only and have no resemblance to the changes in the glomeruli of the seals (19).

Tubular Lesions

Proliferative changes: Hyperplasia of epithelial cells of the kid- ney was described by Bruckner et al. (20) after PCB (Aroclor 1242) administration to rats, however, it was restricted to the papillary epithelium.

The epithelial cell proliferations present in the Baltic seals were localized mainly in the collecting ducts. In the mamma- lian kidney, the epithelial cells in the collecting tubules are of 2 main types; principal or light cells and intercalated cells. The principal cells are pale with few and widely spaced, short mi- crovilli on the cell surface. The basilar plasma membrane shows few, short, interdigitating processes and infoldings (21).

The intercalated cells are rounded and contain numerous mitochondriae with closely packed christae. The apical cytoplasm contains a number of small vesicles coated with studs.

Free ribosomes are numerous in the cytoplasm. Three subtypes have been described, but the morphological differences between them are difficult to evaluate. It has been suggested that they are transition forms of the same cell modifying its appearance according to functional demands (22). It has also been proposed that the principal and intercalated cells may have a common pro- genitor. Both cell types are active in the ion transport in the collecting tubules (23, 24).

The morphology of the the proliferating cells in the collecting tubules of the grey seals corresponds well to the description of the intercalated cells. The most characteristic features of these cells, the fingerlike and interdigitating foldings, the great number of free ribosomes and the numerous vacuoles in the cytoplasm of the apical parts of the cells are apparent (Figs 11 and 12).

Hypertrophy of intercalated cells has been observed by some

authors (25) in potassium-depleted rats in association with an increased H+/K+ ATP-ase activity. This is a matter of dispute, however. The question has been surveyed by Stranton et al. (26).

Proliferative tubular changes, but of metaplastic type, will be discussed below in connection with hyperoestrogenism.

Fatty changes: Sudanophilic lipid is normally present in the tubular epithelium, regularly and in large amounts in cats, occasionally in pigs, and rarely in horses. There was no tubular fat in young seals, but it is improbable that the fatty changes were an effect of old age alone, since the kidneys of many old animals contained no fat.

Fatty change in the tubular epithelium is a common sign in intoxications with a number of substances including PCBs (20, 27). It is therefore possible that an intoxication with external substances, as will be discussed in more detail below, is the cause of the fatty changes. Parenchymal cells of the kidneys, the myo- cardium and, especially, the liver are known to develop fatty changes (28, 29). Besides impacts of external toxic substances, fatty changes occur in starvation or malnutrition (28, 29), and due to bacterial toxins (28) and metabolic disorders such as diabetes mellitus (29). Besides effects of external toxic substances, effects derived from bacterially infected colonic ulcers, as well as the not unusual states of starvation or malnutrition must be considered when discussing the background for the fatty changes observed in the Baltic seals.

Renal vascular lesions: The changes in renal arteries seem to be very similar to arteriosclerosis in other animals and man and are correlated with aging. This change was also found in the reference ringed seals. The occurrence of arteriolosclerotic lesions in the Baltic grey seals, especially those of hyperplastic type, deserves further investigation.

Interstitial changes: Interstitial fibrosis and/or inflammatory cell accumulations were more common in kidneys of older animals. It can not be decided if these changes are due to old age and circulatory changes alone or if external etiologic factors are also involved since we had no age-matched controls.

The most probable background for the amyloid depositions present in the renal interstitium of 2 aged grey seal females was chronic ulceration of the intestine.

Are the Electron Microscopic Findings Representative?

The light microscopic examination showed a complete uniformity of glomerular and tubular changes with variation in inten- sity only. The EM structures of the proliferating cells in the collecting tubules could be studied only in 1 animal. The indica- tion that these cells in this case were intercalated cells is strong.

Considering the uniformity of the light microscopic changes we think we are justified in regarding this observation as representative for the material as a whole.

Mesangial interposition in the glomeruli was also demonstrated convincingly only in 1 animal, but this observation was sustained by the findings in 2 other animals though with less certainty due to autolysis.

Renal Lesions Reported in Other Marine Mammals

There are few reports on the histology of renal lesions in marine mammals. Interstitial nephritis due to leptospirosis is re- ported in northern fur seals (Callorhinus ursinus) (30), Califor- nia sea lions (Zalophus californianus) (31) and Pacific harbor seals (Phoca vitulina richardsii) (32). A case with nephrolitiasis in a harbor seal (33) and a case with a renal fibrosarcoma in a northern fur seal (34) have also been reported. Renal amyloidosis as we observed in a few cases has also been reported in bottlenose dolphins (Tursiops truncatus) stranded along the Texas Gulf coast (35). There are as far as we know, no reports from other marine animals of tubular cell proliferations and the glomerular changes which are prominent and common in the Baltic seals.

(11)

Is there a Common Etiologic Factor Behind Renal and Other Organ Lesions in the Baltic Seal Disease Complex?

The interrelations between grades of 3 lesions in other organs (adrenal weight, colonic ulcers and aortic wall changes) and grades of the renal lesions (glomerular capillary wall thickening, glomerular capillary wall nodules and tubular cell proliferations) were calculated in Baltic grey seals (Table 3). Adre- nal weight correlated well with the renal lesions except for CWN in males. Colonic ulcers were well correlated with the renal lesions except for CWT in males. Aortic wall changes correlated well with the renal lesions. These findings indicate a common etiology.

Old Age or Pollutants?

Old age indicates a long exposure time for an eventual pollut- ant, but is not a measure of body burden. Concentrations of PCBs in Baltic herring and the fish predator, guillemot (Uria aalge) have shown a gradual decline from the mid-1970s (36). Old animals collected during the beginning of the collection period have therefore been exposed to higher amounts of pollutants than animals of equivalent age collected at the end of the period.

The renal changes were closely related to age. This raises the question: are they due to old age or to pollutants alone or both?

The ringed seals from the Baltic and Svalbard showed a com- parable age distribution (Table 1). None of the Arctic animals (Fig. 15B) showed the specific renal lesions (glomerular capillary wall thickening, glomerular nodules and tubular cell proliferations) present in Baltic ringed seals (Fig. 15A). It is therefore improbable that these lesions in the Baltic ringed seals are an effect of old age.

A similar comparison between the Baltic and the Scottish grey seals was only possible for the age group ≤ 3 yrs. Glomerular nodules were found in one and tubular cell proliferations in 11 of the 36 Baltic seals in this age class (Fig. 13A-B), while none of the 11 Scottish seals showed these lesions (Fig. 13C).

Since the specific and identical lesions were present in the 2 closely related Baltic seal species and were not observed in the reference seals it seems probable that these lesions are due to environmental factors.

Pollutants and Pathogenesis

For part of the seals individual data were available from investigations on heavy metal tissue concentrations in the Baltic grey seals (14) and on tissue concentrations of PCB and DDT in the Baltic grey seals and in the young Scottish grey seals (15).

Heavy metals: Intoxication with cadmium (Cd), lead (Pb) and mercury (Hg) is well known to cause renal lesions. The levels of the tissue concentrations of Cd and Pb in the seals presented here do not indicate a high exposure of these elements in Baltic seals (14). Furthermore, Frank et al. (14) found no difference between heavy metal concentrations in seal kidney and liver from various parts of the Swedish waters, including the Swedish West Coast, and those reported in seals from other areas of the world.

High concentrations of Hg, up to 730 mg kg^–1, were found in some aged Baltic grey seal females. These high concentrations are ascribed to a highly age dependent accumulation of Hg and Se (14). Since the concentrations of Se, known to protect animals from toxic effects of Hg (37), paralleled those of mercury in the seals, influence by this metal on renal histology was im- p r o b a b l e .

There was no correlation between heavy metal tissue concentrations and renal changes in the Baltic grey seals. We conclude that there are no indications that intoxication with heavy metals is a cause of the renal lesions observed. Furthermore, the char- acteristics of these lesions were not consistent with those present in heavy metal intoxication in domestic animals and man.

Organohalogens: The Baltic environment is known to be highly contaminated by organohalogens, especially PCB and

DDT substances (1, 36, 39). Long-term time trend monitoring of DDT and PCB concentrations in herring collected in the Baltic and at the Swedish West Coast since the end of the 1970s, have shown decreasing concentrations of both DDT and PCB. How- ever, during the entire study period (1967–1995), the PCB concentrations were 2–3 times and the DDT concentrations 3–5 times higher in the Baltic than at the Swedish West Coast (40).

Higher concentrations of polybrominated diphenylethers (PBDE) were found in harbor seals from the Baltic compared with harbor seals from the Swedish West Coast (41). Another study (42) has shown higher concentrations of polychlorinated dibenzofurans (PCDFs) in Baltic than in Swedish West Coast herring, but low concentrations of both PCDFs and 2, 3, 7, 8- substituted polychlorinated dibenzo-para-dioxins (PCDDs) were found in seals from Swedish waters (43).

In the material presented here, the young grey seals from Scot- land had substantially lower body burdens of organochlorines than the corresponding young grey seals from the Baltic (Table 6). Similarly, it is known that PCB and DDT levels are considerably lower in Arctic ringed seals than in ringed seals from more polluted areas such as the Baltic (44).

There was a wide range of the concentrations of sDDT and sPCB in extractable fat from blubber in the Baltic grey seals (Ta- ble 6). One reason for this wide range may be that the fat de- pots are much reduced in diseased or emaciated animals without a parallel reduction in the amount of these fat-soluble con- taminants. The concentrations in the blubber at the time of death may therefore not be an appropriate measure of the exposure over time. The metabolic turnover of these organochlorines is insufficiently known.

In Baltic grey seal females, a significant positive correlation was found between increasing concentrations of both sPCB and sDDT and grades of 2 renal lesions, CWT and CWN. In Baltic grey seal males, increasing concentrations of sDDT were significantly negative correlated with colonic ulcers. We cannot find a causal explanation for this negative correlation. Considering the previous reservations these results must be interpreted with great caution.

After the regulations and bans in the countries around the Bal- tic in the 1970s, the concentrations of PCBs and several other organochlorines such as DDT, hexachlorocyclohexans (HCHs) and hexachlorobenzene (HCB) have decreased in biota (15, 36, 39, 40). A simultaneous, slow recovery in the population size of grey seals has been observed (45). These circumstances are supported by the findings from autopsies of Baltic grey seals, which show that gynaecological health has improved (8) and also that the occurrence of severe skull bone lesions tends to decline (Bergman et al., unpubl. data). We conclude that there are indications that the decline in the Baltic grey seal population and the development of the seal disease complex are associated with impacts of PCBs and related substances.

Experimental Effects of PCBs on the Kidney

Although Brandt et al. (46), performing autoradiography on mice, found target cells for a polychlorinated biphenyl metabolite in lung and proximal convoluted tubules of the kidney, there are few renal lesions reported in experimental animals after administration of PCBs, besides the papillary epithelial hyperplasia in rats, mentioned above (20). Sudanophilic vacuolation of the epithelial cells in the proximal convoluted tubules has been observed in rats treated with Aroclor 1242 (20, 27).

Endocrine Imbalance

A hormonal imbalance has been suggested to be a factor in the etiology of the Baltic seal disease complex (6). Adrenocortical hyperplasia is a prominent finding in this complex and the present investigation has shown that it is statistically related to the severity of renal lesions.