“Renal Tubular Pigmentation Associated with Senna-Related Metabolites.” Willson GA (presenter) Malarkey DE, Allison N, Harris N, Miller RA. The 54th Annual Society of Toxicology Meeting. San Diego, CA. March 25, 2015. For full-resolution viewing, please open or save as a PDF.

1. 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, 4Results
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
Positive droplets
Negative Negative
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
Positive droplets
Negative Negative
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