Glomerulopathy with renal tubular oxalosis in Japanese rock ptarmigan (Lagopus mutus)

Abstract

Seven adult Japanese rock ptarmigan (Lagopus mutus) that died on a rearing farm and appeared to have suffered from glomerulopathy with renal tubular oxalosis were examined pathologically. Macroscopically, the kidneys showed enlargement of varying degrees. Histologically, the precipitation of a fibril-like substance in the mesangial area of the renal glomeruli was the most important and common lesion seen in the kidney. Calcification of glomeruli was observed in severe cases. In addition, oxalate precipitation in the lumen of the renal tubules was associated with glomerular lesions. The primary renal lesion may have been glomerulonephropathy, which was subsequently associated with tubular oxalosis as the secondary renal lesion.

Glomérulopathie avec oxalose des tubules rénaux chez des lagopèdes alpins japonais (Lagopus mutus)

Sept lagopèdes alpins japonais (Lagopus mutus) adultes qui sont morts dans un élevage et qui semblaient avoir soufferts d'une glomérulopathie avec oxalose des tubules rénaux ont été examinés. Sur le plan macroscopique, une hypertrophie plus ou moins importante des reins a été observée. Sur le plan histologique, la précipitation d'une substance ressemblant à de la fibrille dans la zone mésangiale des glomérules rénaux a été la lésion la plus commune et importante observée au niveau des reins. Une calcification des glomérules a été observée dans les cas graves. De plus, la précipitation d'oxalate dans la lumière des tubules rénaux a été associée à des lésions glomérulaires. La lésion rénale primaire a peut-être été la glomérulonéphropathie, qui a été par la suite associée à l'oxalose tubulaire comme lésion rénale secondaire.

Glomerulopathie mit renaler tubulärer Oxalosis bei japanischen Alpenschneehühnern (Lagopus mutus)

Sieben adulte japanische Alpenschneehühner (Lagopus mutus) die auf einer Aufzuchtfarm gestorben waren und die scheinbar an einer Glomerulopathie mit renaler tubulärer Oxalurie gelitten hatten, wurden pathomorphologisch untersucht. Makroskopisch waren die Nieren unterschiedlich stark geschwollen. Histologisch war die Ausfällung einer Fibrillen-ähnlichen Substanz im mesangialen Bereich der renalen Glomeruli die wichtigste und häufigste Läsion in den Nieren. In hochgradigen Fällen wurde eine Kalzifizierung der Glomeruli beobachtet. Die Oxalsäureausfällung in das Lumen der renalen Tubuli war zusätzlich mit Läsionen in den Glomeruli verbunden. Die primäre Läsion in den Nieren kann eine Glomerulonephropathie gewesen sein, die nachfolgend mit einer tubulären Oxalosis als sekundäre Nierenveränderung assoziiert war.

Glomerulopatía con oxalosis tubular renal en perdiz nival (Lagopus mutus)

Se realizó el examen patológico de siete perdices nivales (Lagopus mutus) adultas que murieron en una granja de crianza y que parecía que habían padecido glomerulopatía con oxalosis tubular renal. Macroscópicamente, los riñones mostraron incrementos de tamaño de diferente grado. Histológicamente, la precipitación de una sustancia similar a fibrilas en el área mesangial de los glomérulos renales fue la lesión más importante y común observada en el riñón. La calcificación de los glomérulos se observó en casos graves. Además, la precipitación de oxalatos en la luz de los túbulos renales se asoció con lesiones glomerulares. La lesión renal primaria puede haber sido la glomerulonefropatía, que se asoció con oxalosis tubular como lesión renal secundaria.

Introduction

Only a few cases on glomerular lesions have been reported in birds, although many have been seen in mammals (Brown, 1996). The lesions of avian glomeruli without visible cellular infiltrates are classified as glomerulopathy, even though they may be due to inflammatory processes. The lesions with infiltration of inflammatory cells, other than the macrophages of proliferative glomerulopathy, are classified as glomerulonephritis (Brown, 1996).

Generally, oxalate precipitation is caused by intoxication of engine antifreeze solutions containing ethylene glycol (EG), by ingestion of plants that contain toxic levels of oxalate, or by ingestion of plants infected with fungi that produce oxalates (Jones et al., 1996). A few cases of oxalosis in birds have been described. These include a field case of suspected EG poisoning in geese and an experimental EG poisoning in chickens (Riddell et al., 1967), tubular nephropathy (oxalosis) induced by EG toxicity in peafowl (Randall & Reece, 1996), and a field and experimental case of EG intoxication in ducks (Stowe et al., 1981). In all these cases, oxalate precipitation was usually seen in the renal tubule and rarely in walls of cerebral vessels. This paper describes the pathological changes of glomerulopathy associated with renal tubular oxalosis in rock ptarmigan (Lagopus mutus).

Materials and Methods

Case history

For preservation and breeding of Japanese rock ptarmigan (L. mutus), these birds are reared in bird houses at the foot of the mountains in Nagano, Japan (Omachi Alpine Museum, Omachi-City, Nagan, Japan). The birds are fed an artificial diet, apples and green vegetables. The artificial diet includes unhulled rice (13.0%), foxtail millet (16.0%), barnyard grass (16.0%), buckwheat (10.0%), wheat bran (16.0%), soybean flour (2.0%), Japanese oak powder (24.3%), and a vitamin complex (vitamins A, E, B₁, B₂, B₁₂, D₃, pantothenic acid, nicotinic acid, and folic acid) (1.7%). A diet supplemented with Rumex obtusifolius was fed to the birds from 1 to 30 days old. During the period from June 1999 to October 2000, seven adult birds (four males and three females) died in this rearing farm.

Histopathology

Following a postmortem examination, tissues from the liver, heart, lungs, spleen, kidneys, gizzard, proventriculus, pancreas, small intestine, large intestine, oviduct, ovary, peripheral nerves, and brain were removed and fixed in 10% buffered formalin. All tissue samples were then embedded in paraffin and 4-µm sections stained with haematoxylin and eosin were examined using a light microscope equipped with polarization filters.

Histochemistry

Alizarin red S stain was used for identification of calcium oxalate crystals (Proia & Brinn, 1986). Calcium oxalate is stained with alizarin red S at pH 7.0 but not at pH 4.2. Calcium phosphate and calcium carbonate are stained with alizarin red S stain at both pH 7.0 and 4.2. Calcium oxalate is soluble with 0.1 N hydrochloric acid but insoluble with 2 M acetic acid. Von Kossa silver stain was used for detecting calcium carbonate. Selected sections were stained by Azan, phosphotungstic acid haematoxylin.

Bacteriology and virology

We attempted to isolate bacteria from the liver, heart, spleen, lung, air sac, oviduct, and intestine using 5% sheep blood agar plates, DHL agar plates, and Sabouraud's agar plates. Virus isolation attempts were carried out from the trachea and kidney using chicken kidney cell culture or chicken embryonated eggs.

Results

Gross lesions

The kidneys of all the seven birds showed various degrees of enlargement. Deposition of a white substance was observed on the pericardium, hepatic capsule and renal capsule in bird 7. Four birds (birds 1 to 4) had yellowish discolouration of the liver. Other lesions included oviduct rapture (bird 1), gizzard erosion (bird 2), necrotic enteritis (bird 3), and aspergillus pneumonia (bird 5).

Histopathology

Renal lesions were observed in the glomerulus, tubules and interstitial regions (Table 1). Two pathologic conditions, glomerulopathy and tubular oxalosis were also observed.

Table 1.  Histological lesions of kidneys in Japanese rock ptarmigan^a

Part of kidney	Lesion	Bird 1	Bird 2	Bird 3	Bird 4	Bird 5	Bird 6	Bird 7
Glomerulus	Fibril-like substance	+	+ +	+ +	+	+	+ + +	+ + +
	Oxalate	–	–	–	–	–	–	+
Tubules	Oxalate	+	+	+	+	+	+ + +	+ + +
	Giant cell reaction	–	+	–	–	–	+	+
Interstitium	Fibrosis	–	–	–	–	–	+ +	+ +
^aLesion severity: –, no lesion; +, mild lesion; ++, moderate lesion; + + +, severe lesion.

Glomerulopathy

In mild cases, the deposition of fibril-like substances with some granules in the parts of the mesangial regions of glomeruli was scattered (Figure 1). In moderate cases, more fibril-like substance deposit was seen in the mesangial regions of glomeruli. In severe cases, deposition of the fibril-like substance was evident in the whole of the swollen glomeruli (Figure 2). The cellular elements decreased and the remaining cellular nuclei were atrophic. Calcification (calcium carbonate stained positive with Von Kossa silver stain) was seen in severely-affected glomeruli, tubules, and arterial walls. The fibril-like substance was stained alizarin red S stain at pH 7.0 but not at pH 4.2. It was soluble with 0.1 N hydrochloric acid but insoluble with 2 M acetic acid.

Figure 1.  Mild glomerulopathy. Focal deposition of fibril-like substance with granules in the glomerulus (arrows). Haematoxylin and eosin, bar = 50 µm.

Figure 2.  Severe glomerulopathy. Fibril-like substances deposit the whole glomerulus (arrows). Atrophic and destructed nuclei are scattered in the glomerulus. The blood vessels disappear in the glomerulus. Haematoxylin and eosin, bar = 50 µm.

Tabular oxalosis

Numerous transparent crystals were seen in the lumen of the renal tubules, especially the distal tubules. Under polarized light, the crystals were observed as birefringent rosettes (Figure 3). Crystals were stained with alizarin red S stain at pH 7.0 but not at pH 4.2 (Figure 4). Crystals were soluble with 0.1 N hydrochloric acid but insoluble with 2 M acetic acid. Occasionally, the formation of giant cells was seen around the crystals in the tubules (Figure 5). Interstitial fibrosis was observed in severe cases (birds 6 and 7) (Figure 5). Rarely, gouty nodules were seen in the kidney (bird 7).

Figure 3.  Many birefringent oxalate crystals in tubules and rarely in the glomeruli (arrow) of the kidney of a bird affected with severe glomerulopathy. Under polarization light. Haematoxylin and eosin, bar = 100 µm.

Figure 4.  Alizarin red S staining of tubular crystals and glomerulopathy at (4a) pH 7.0 and (4b) pH 4.2. Tubular crystals and glomerular calcium substances are stained positively with alizarin red S at pH 7.0 but not at pH 4.2. Bar = 100 µm.

Figure 5.  Giant cell reaction around the oxalate crystals in the kidney of a bird affected with severe glomerulopathy. Fibrosis is also seen in the interstitium of the kidney. Haematoxylin and eosin, bar = 100 µm.

Lesions in other organs

Lesion in other organs included fatty degeneration of hepatocytes in birds 1 to 4, salpingitis (bird 4), gizzard ulcer (bird 2), and egg peritonitis (birds 1, 2, and 4). An amorphous substance, possibly urate, was deposited in the epicardium and hepatic capsule of bird 7. No oxalate crystals were evident in the brain or other organs.

Bacteriology and virology

Aspergillus fumigatus was isolated from the lung, liver, heart, spleen, kidney, and air sac of bird 5. No viral agents were isolated from the kidney and trachea of any of the birds.

Discussion

The exact pathogenesis of the accumulation of fibril-like substance in the glomeruli is not known. Fibril-like substances as well as tubular crystals were stained with alizarin red S stain at pH 7.0 but not at pH 4.2. They were further confirmed to be oxalate by their insolubility in 2 M acetic acid. In one severe case (bird 7), a small amount of oxalate crystal was found within the fibril-like substance. The fibril-like substance, possibly the precursor of oxalate crystals, was deposited predominantly in the mesangial region. As no inflammatory cells were present either within the glomeruli or in the periglomerular interstitium, the condition was better designated “glomerulopathy” than “glomerulonephritis”.

Histological findings, polarization observation, and histochemical staining proved that the crystals in the tubular lumen are probably oxalate. Generally, oxalosis is caused by ingestion of natural oxalate-containing plants, or from feed contaminated with aspergillus (Jones et al., 1996). A diet supplemented with the oxalate-bearing plant R. obtusifolius had been given to the birds from 1 to 30 days old. No glomerular lesions were reported in cases of EG toxicity in birds (Riddell et al., 1967; Stowe et al., 1981). The present case revealed no information of a possible EG intoxication. The glomerular lesion caused an increased permeability of blood vessels, resulting in the excretion of the blood oxalate into the tubular lumen. These facts indicated that the primary renal lesion may have been a precipitation of the fibril-like substance in the glomeruli (glomerulonephropathy), and then associated with tubular oxalosis as the secondary renal lesion.

Translations of the abstract in French, Germany and Spanish are available on the Avian Pathology website.

The authors would like to thank Dr Y. Ando and Mr T. Fujisawa for providing photographs, Mr M. Kobayashi and Miss Megumi Shimada for histological and histochemical assistance, and the Omachi Alpine Museum (Omachi-City, Nagano, Japan) for the supply of Japanese rock ptarmigan.