Renal Tubular Pigmentation Associated with Senna-Related Metabolites

Figure 17, 18

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Renal Tubular Pigmentation Associated with Senna-Related Metabolites Gabrielle A. Willson1, David E. Malarkey2, Neil Allison1, Nancy Harris1, and Rodney A. Miller1

1Experimental Pathology Laboratories, Inc. P.O. Box 12766, RTP, NC 27709 2Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, P.O. Box 12233, RTP, NC 27709

Figure 5, 6

Introduction: Past oral administration of powdered senna to rats

has resulted in unidentified pigment deposits in renal tubules

(Mengs, et. al., 2004, Mitchell, et. al., 2006). Renal tubule brownish

pigment was seen in mice and rats dosed with emodin (an

anthraquinone metabolite of sennosides) and with anthraquinone in

NTP studies. Since some species of Cassia (Senna) cause skeletal

muscle degeneration and necrosis in domestic animals (but no

reported renal pigment), we wanted see if the renal pigment in

rodent studies was myoglobin or some other pigment(s)

(Vashishtha, et.al., 2009, Martin, et. al, 1981).

Methods: Slides from archived paraffin blocks from 4 rats and 4

mice with kidney pigment from the emodin and anthraquinone

chronic studies were stained with Schmorl's, PAS, Hall's Bile,

Prussian Blue stains and myoglobin by immunohistochemistry

(IHC).

Results: The IHC reaction for myoglobin resulted in positive

staining of small intracellular granules of purple pigment within the

proximal renal tubule epithelial cells in rat kidneys treated with

emodin and anthraquinone. The same reaction did not stain any of

the pigment as myoglobin in the mouse kidney sections.

The Schmorl’s reaction was variably positive in rats. However, when

positive, the reaction was associated with larger droplets and not

the small distinct granules.

The Prussian Blue reaction was variably positive in emodin rats and

mice and anthraquinone rats. Anthraquinone mice were strongly

positive for the iron stain.

The PAS reaction and Hall’s bile stain were all negative for staining

the small granules.

Conclusion: It appears that a portion of the renal cortical tubule

pigment in rats treated with emodin or anthraquinone may be

myoglobin, whereas, in mice it does not.

Lipofuscin was also a component of some of the brownish

pigmentation, but was associated with larger droplets, not smaller

distinct granule. This “wear and tear” pigment is likely related to the

renal pathology induced by anthraquinone in rats and emodin

treated rats and mice.

PAS and Halls’ Bile stains were negative.

In mice the only stain that was strongly positive was the Prussian

Blue (iron) correlating with hemosiderin (not myoglobin) related to

an induced anemia by anthraquinone in mice.

The low numbers of animals limited group or sex comparisons and

restricted evaluation to individual animal pigment staining results.

Myoglobin positive and negative controls are shown (Figures 1 and

2).

The brown pigment in emodin rats was mainly small

intracytoplasmic granules in proximal tubules (Figure 3). Some

brown pigment was seen interstitially and luminally, where it was

less granular and more amorphous and “dusty”. The brown

pigment in Emodin mice was less intracytoplasmic than in the rat

and more luminal and interstitial with coarser clumps (Figure 5). It

was less obvious than in the rat.

The brown pigment in anthraquinone rats was mostly

intracytoplasmic and present in small granules (similar to emodin

rats) with some larger amorphous and homogeneous hyaline

droplets which were also intracytoplasmic. In anthraquinone mice

the pigment was intracytoplasmic and formed small golden brown

granules (Figure 7). In the anthraquinone mice, the pigment was

more uniformly distributed throughout the cortex than the other

pigmented materials mentioned earlier.

Results of Special Stains

The immunohistochemical reaction for myoglobin resulted in

positive (reddish-purple) staining of an estimated 25-40% of the

distinct small intracellular granules of brown pigment seen only

within the proximal renal tubule epithelial cells of all the rat kidney

sections treated with emodin and anthraquinone (Figures 4 & 6).

The same immunohistochemical reaction did not successfully

identify any of the pigment as myoglobin in any of the mouse kidney

sections.

The Schmorl’s reaction (teal blue) for lipofuscin was somewhat

positive for emodin rats (Figure 8) and positive for anthraquinone

rats (Figure 10), whereas it was mostly negative for emodin mice

(Figure 9) and negative for anthraquinone mice. However, when

positive in rats, the positive reaction was associated with larger

droplets and luminal smudgy pigment and not the small distinct

granules.

The PAS reaction (red) was mostly positive for larger droplets and

negative for the small granules in emodin rats and anthraquinone

rats (Figure 11 & 13). PAS stains were negative for mice (Figure 12

& 14).

The Prussian Blue reaction (blue) in rats was mostly negative in

emodin rats (Figure 15) and mice. Anthraquinone mice were

strongly positive for the iron stain (Figure 18) and anthraquinone

rats had mixed results, one mostly positive (Figure 16) and one

mostly negative (Figure 17).

Hall’s Bile reaction (green) was negative for all rats and mice.

Figure 3, 4 Results

Reddish discoloration of the urine has been described as one of the

side effects of senna laxative use in humans. In rats, oral

administration of powdered senna for 13 weeks or for 104 weeks

resulted in dark discoloration of the kidneys macroscopically and

unidentified pigment deposits in renal tubules at the microscopic

level (Mengs, et al. 2004; Mitchell, et al. 2006). No pigment was

observed in the renal tubular epithelium of mice in a National

Toxicology Program (NTP) study of Senna in the 39-Week Study of

Senna in Heterozygous F1 p53 (+/-) Transgenic Mice. Renal tubule

brownish pigmentation (unidentified) was reported in mice and rats

dosed with emodin (an anthraquinone metabolite of sennosides) in

a series of NTP studies ranging from 14 days to 2 years (NTP,

2001). Studies of anthraquinone have also reported increased

incidences or severity of unidentified pigment deposits in the kidney

of both rats and mice (NTP, 2005). Because Cassia (Senna) can

produce skeletal muscle degeneration and necrosis in domestic

animals, it was deemed interesting and useful to determine whether

the renal pigment recorded in rodent studies is myoglobin or some

other pigment(s).

Introduction

Figure 1, 2

Figure 7, 8

In this limited sampling of tissues, it appears that a portion

of the renal cortical tubule pigment in rats treated with

emodin or anthraquinone may be myoglobin, whereas,

in mice it does not appear to be myoglobin.

Lipofuscin also seemed to be a component (the less

granular and more amorphous and “dusty”component) of

some of the brownish pigmentation. This “wear and tear”

pigment is likely related in some manner to the

nephropathy induced by anthraquinone in rats and related

to kidney changes of hyaline droplets (rats) and

nephropathy (mice) in emodin treated rats and mice.

PAS and Halls’ Bile stains did not contribute to definitive

identification of the granular brown pigment. PAS-positive

renal epithelial droplets were related to the anthraquinone

induced renal lesions in rats and renal hyaline droplets

induced by emodin in rats.

In mice the only stain that was strongly positive was the

Prussian Blue (iron) which indicated that the pigment in

the renal cortical tubules was hemosiderin (not

myoglobin) related to an induced anemia by anthra-

quinone. The variable presence of Prussian Blue positivity

in emodin-exposed rats and mice and anthraquinone

exposed rats was likely related to renal pathology.

The results of this probe into the origin of the renal tubule

pigment indicate that the pigment may be the result of a

myopathy in rats but not in mice.

Discussion

References

To begin to address this issue we retrieved archived paraffin blocks

and prepared kidney sections stained with Schmorl's, PAS, Hall's

Bile, Prussian Blue stains and myoglobin by IHC (Novusbio, Lot #

YF051410R) from the following rats and mice from the respective

chronic studies:

Methods

Abstract Figure 11, 12

Figure 9, 10

Figure 13, 14

Emodin High dose male rat

Emodin High dose female rat

Emodin Mid dose male mouse

Emodin High dose female mouse

Anthraquinone High dose male rat

Anthraquinone High dose female rat

Anthraquinone Two high dose male mice

Myoglobin IHC

(red-purple)

Schmorl’s

(teal blue) PAS (red)

Prussian Blue

(blue)

Hall’s Bile

Stain (green)

HM181 Emodin

Rat Positive 50% Positive

Negative granules

Positive droplets Negative Negative

HF473 Emodin

Rat Positive Equivocal

Negative granules


MM185 Emodin

Mouse Negative Negative Negative Negative Negative

HF477 Emodin

Mouse Negative Negative Negative Negative Negative

HM239

Anthraquinone

Rat

Positive Positive Negative granules

Positive droplets Positive Mild Negative

HF494

Anthraquinone

Rat

Positive Positive Negative granules


HM191

Anthraquinone

Mouse

Negative Negative Negative Positive

Marked Negative

HM125

Anthraquinone

Mouse

Negative Negative Negative Positive

Marked Negative

Figure 3.

Rat Kidney,

Negative control,

myoglobin,

A75723, 40X

Figure 4.

Rat Kidney,

myoglobin,

A75725, 40X

Figure 5.

Mouse Kidney,

myoglobin,

A75726, 40X

Figure 6.

Rat Kidney,

myoglobin,

A75727, 40X

Figure 7.

Mouse Kidney,

myoglobin,

A75729, 40X

Figure 8.

Rat kidney,

Schmorl’s,

A75730, 40X

Figure 9.

Mouse, Schmorl’s,

A75732, 40X

Figure 10.

Rat, Schmorl’s,

A75734, 40X

Figure 11.

Rat, PAS,

A75735, 40X

Figure 12.

Mouse, PAS,

A75737, 40X

Figure 13.

Rat, PAS,

A75738, 40X

Figure 14.

Mouse, PAS,

A75739, 40X

Figure 17.

Rat, Prussian

Blue, A75742,

40X

Figure 18.

Mouse, Prussian

Blue, A75743,

40X

Figure 15, 16

Figure 15.

Rat, Prussian Blue,

A75740, 40X

Figure 16.

Rat, Prussian Blue,

A75741, 40X

Figure 1. Rat Heart. Image A75722, 40X objective. Negative myoglobin control

Figure 2. Rat Heart. Image A75721, 40X objective. Positive myoglobin control

Figure 3. Emodin rat HM181. Image A75723, 40X objective. Negative control

myoglobin. The brown pigment in Emodin rats was mainly small intracytoplasmic

granules in proximal tubules. Rats also had some pigment which was seen

interstitially and luminally, where it was less granular and more amorphous and

“dusty”.

Figure 4. Emodin rat HF473. Image A75725, 40X objective. Myoglobin positive.

The immunohistochemical reaction for myoglobin resulted in positive (reddish-

purple) staining of distinct small intracellular granules of brown pigment only within

the proximal renal tubule epithelial cells of all the rat kidney sections treated with

Emodin.

Figure 5. Emodin mouse MM185. Image A75726, 40X objective. Myoglobin

negative. In Emodin in mice the brown pigment was less intracytoplasmic and more

luminal and interstitial in coarser clumps.

Figure 6. Anthraquinone rat HM239. Image A75727, 40X objective. Myoglobin

positive. The immunohistochemical reaction for myoglobin resulted in positive

(reddish-purple) staining of distinct small intracellular granules of brown pigment

only within the proximal renal tubule epithelial cells of all the rat kidney sections

treated with anthraquinone.

Figure 7. Anthraquinone mouse HM191. Image A75729, 40X objective. Myoglobin

negative. In anthraquinone mice the pigment in was intracytoplasmic and formed

small golden brown granules. It was more uniformly distributed throughout the

cortex than the pigment in Emodin rats and mice and anthraquinone rats.

Figure 8. Emodin rat HM181. Image A75730, 40X objective. Schmorl’s stain. The

Schmorl’s reaction (teal blue) for lipofuscin, when positive, was associated with

larger droplets and luminal smudgy pigment and not the small distinct granules.

Figure 9. Emodin mouse MM185. Image A75732, 40X objective. Schmorl’s stain.

The Schmorl’s reaction (teal blue) for lipofuscin, when positive, was associated with

larger droplets, interstitial, and luminal smudgy pigment and not the small distinct

granules.

Figure 10. Anthraquinone rat HF494. Image A75734, 40X objective. Schmorl’s

stain. The Schmorl’s reaction (teal blue) for lipofuscin, when positive, was

associated with larger droplets and luminal smudgy pigment and not the small

distinct granules.

Figure 11. Emodin rat HM181. Image A75735, 40X objective. PAS stain. The PAS

reaction (red) was positive for larger droplets and negative for small granules in

Emodin rats.

Figure 12. Emodin mouse MM185. Image A75737, 40X objective. PAS stain.

Negative staining of the brown particles.

Figure 13. Anthraquinone rat HM239. Image A75738, 40X objective. PAS stain.

The PAS reaction (red) was positive for larger droplets and negative for small

granules in Anthraquinone rats.

Figure 14. Anthraquinone mouse HM191. Image A75739, 40X objective. PAS

stain. Negative staining of the brown particles.

Figure 15. Emodin rat HM181. Image A75740, 40X objective. Prussian Blue stain.

Negative staining of brownish granules.

Figure 16. Anthraquinone rat HM239. Image A75741, 40X objective. Prussian

Blue stain. Mostly positive.

Figure 17. Anthraquinone rat HF494. Image A75742, 40X objective. Prussian Blue

stain. Negative for small brown granules.

Figure 18. Anthraquinone mouse HM125. Image A75743, 40X objective. Prussian

Blue stain. The Prussian Blue reaction (blue) in Anthraquinone mice was strongly

positive for the iron stain and uniformly distributed intracytoplasmically throughout

the renal cortical epithelium.

Figure 1.

Rat Heart,

Negative control,

myoglobin,

A75722, 40X

Figure 2.

Rat Heart,

Positive control,

myoglobin,

A75721, 40X

This may indicate that there is a coexisting muscle lesion

present in rats. Senna has been associated with muscle

lesions in other species.

Conclusion