Dietary Hydroxyproline Induced Calcium Oxalate Lithiasis and Associated Renal Injury in the Porcine Model

Abstract

Background and purpose:
We previously reported hyperoxaluria and calcium oxalate calculi in adult pigs (sows) fed hydroxyproline (HP). The purpose of this study was to grossly and histopathologically characterize intrarenal effects in this model.

Methods:
In the swine facility at our campus, we maintained 21 gestating sows, of which 15 received daily treatment (5% HP mixed with dry feed) and 6 received no treatment (controls). Nine were sacrificed at 21 d (three control, six HP). All kidneys were extracted and examined grossly and for radiographic evidence of stones (GE CT scanner, 80kV, 400MA, 1 sec rotation, 0.625 mm slices). Papillary and cortical samples were processed for histologic analysis.

Results:
Kidneys from treated sows showed significant calculi distributed within the renal papilla on CT, appeared mottled in the renal cortex and papillary areas, and had less distinct corticomedullary borders. Tiny crystals and mucinous debris lined the papillary tips, calices, and pelvis in kidneys from four of six treated sows, and multiple stones were noted at the papillary tips. Hematoxylin and eosin stain revealed crystals in collecting tubules and papillary tips in treated kidneys and none in controls. Yasue staining confirmed crystals in proximal periglomerular tubules of treated but not control animals. Tubular dilation and inflammatory/fibrotic changes were identified in kidneys from treated animals; none of these changes were evident in control kidneys.

Conclusions:
We report renal damage as a result of dietary-induced hyperoxaluria in adult sows. Specifically, we found crystalluria in proximal periglomerular tubules and collecting ducts, with tubular damage at all segments.

Full text

Dietary Hydroxyproline Induced Calcium Oxalate Lithiasis
and Associated Renal Injury in the Porcine Model
Sri Sivalingam, MD,
1
Stephen Y. Nakada, MD,
1
Priyanka D. Sehgal, BSc(Hons),
1
Thomas D. Crenshaw, PhD,
2
and Kristina L. Penniston, PhD
1
Abstract
Background and Purpose: We previously reported hyperoxaluria and calcium oxalate calculi in adult pigs
(sows) fed hydroxyproline (HP). The purpose of this study was to grossly and histopathologically characterize
intrarenal effects in this model.
Methods: In the swine facility at our campus, we maintained 21 gestating sows, of which 15 received daily
treatment (5% HP mixed with dry feed) and 6 received no treatment (controls). Nine were sacrificed at 21 d
(three control, six HP). All kidneys were extracted and examined grossly and for radiographic evidence of stones
(GE CT scanner, 80kV, 400MA, 1 sec rotation, 0.625mm slices). Papillary and cortical samples were processed for
histologic analysis.
Results: Kidneys from treated sows showed significant calculi distributed within the renal papilla on CT,
appeared mottled in the renal cortex and papillary areas, and had less distinct corticomedullary borders. Tiny
crystals and mucinous debris lined the papillary tips, calices, and pelvis in kidneys from four of six treated sows,
and multiple stones were noted at the papillary tips. Hematoxylin and eosin stain revealed crystals in collecting
tubules and papillary tips in treated kidneys and none in controls. Yasue staining confirmed crystals in proximal
periglomerular tubules of treated but not control animals. Tubular dilation and inflammatory/fibrotic changes
were identified in kidneys from treated animals; none of these changes were evident in control kidneys.
Conclusions: We report renal damage as a result of dietary-induced hyperoxaluria in adult sows. Specifically, we
found crystalluria in proximal periglomerular tubules and collecting ducts, with tubular damage at all segments.
Introduction
Nephrolithiasis carries a lifetime risk of 15% ac-
cording to recent population-based studies from the
United States.
1,2
Up to 80% of renal calculi in adults are pre-
dominantly calcium based,
3
but mechanisms for the patho-
genesis of idiopathic calcium oxalate lithiasis remain unclear.
Animal models facilitate the understanding of pathophysi-
ology. Although rodent models have been prototypical for
studying urolithiaisis, the relevant renal anatomy and phys-
iology differ substantially from humans.
4
Accordingly, more
comparable animal models of urolithiasis are desirable. The
porcine model is useful in this regard because porcine kidneys
are anatomically and physiologically akin to the human kid-
ney, with multipyramidal systems and an undivided cortex,
with each medullary pyramid forming a separate papilla with
some compound papilla.
5,6
Oxalate excreted in the urine is derived in approximately
equal proportions from diet and endogenous synthesis.
7
Hydroxyproline (HP) is a precursor for endogenous oxalate
synthesis, and the metabolism of HP primarily occurs in the
mitochondria of hepatocytes and renal proximal tubule cells.
8
Collagen degradation is the major source of HP and has a daily
turnover in humans of 2 to 3 g/day.
9
We have previously
demonstrated sustained, predictable, and reproducible hyper-
oxaluria through dietary manipulation in our ongoing devel-
opment of a porcine model.
10,11
In this study, we sought to
characterize crystal formation with HP feeding and to describe
associated renal changes in gestating sows. As a secondary
objective, we sought to verify that nongestating sows responded
similarly to HP feeding with respect to stone formation.
We hypothesized that (1) 21 days is sufficient for studying
HP-induced stone formation and associated urinary and renal
changes in this model, and (2) kidneys from sows forming
stones would have histologic evidence of renal damage.
Methods
The study was approved by the University of Wisconsin
(UW)-Madison Research Animal Resource Committee.
1
Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
2
Department of Animal Sciences, University of Wisconsin–Madison, Madison, Wisconsin.
JOURNAL OF ENDOUROLOGY
Volume 27, Number 12, December 2013
ªMary Ann Liebert, Inc.
Pp. 1493–1498
DOI: 10.1089/end.2013.0185
1493

Animals were housed atthe UW Swine Research and Teaching
Center, an Association for Assessment and Accreditation of
Laboratory Animal Care-accredited swine facility with full-
time staff support.
Experimental design
The overall experiment had two parts: study #1 was de-
signed to study the renal effects of the dietary intervention in
gestating sows and was carried through to 42 days; study #2
was a secondary experiment to verify our model in non-
gestating sows.
Study #1 – dietary intervention, gestating sows. The treat-
ment diet (TD) consisted of 5% HP (100 g in 2 kg) evenly
mixed with the standard UW gestation diet comprised of
corn, soybean meal, mineral and vitamin supplements,
while the control diet (CD) consisted of 5% (100 g in 2 kg)
starch mixed with the standard UW diet. The gestating
sows were initially acclimated to a 2 kg basal diet for 2
weeks before day 1 of the experiment. At day 1, 21 sows
were randomly allotted to the TD (n=15) and CD (n=6)
groups. The TD group was further divided into 21 days
(TD-21)and42days(TD-42)ofdietaryintervention,with
six and nine sows, respectively. Animals in each group
were sacrificed at the end of the intervention using standard
U.S. Department of Agriculture methods (USDA Food
Safety and Inspection Service Directive 6900.2, revision 2; 8/
15/2011), and their kidneys were extracted for imaging and
evaluation.
Study #2 – verification of model in nongestating sows.
We assessed stone formation in four nongestating sows of
similar age and weight (two CD and two TD sows) as above.
Treatment sows were maintained on the TD for 21 days,
and all animals were sacrificed at 21 days, and kidneys were
extracted as described above.
Tissue processing
The extracted kidneys were immediately flushed with hep-
arinized saline, maintained in ice, and taken to the CT suite.
The ex-vivo kidneys were scanned on a GE Discovery CT750
HD CT scanner using a protocol optimized for stone detection
(80kV, 400MA, 1 sec rotation, 0.625 mm slices). Images from the
unlabeled kidneys were then examined for radiographic evi-
dence of stones using standard bone windows.
After scanning, the kidneys were bivalved and examined.
Gross appearance was recorded and digital photographs
were taken; loose stone material was documented, collected in
individual jars, and sent for analysis. Whole kidneys were
fixed in 10% formalin for 48 hours, and subsequently, four
transverse sections of each of the following were taken:
Upper, midpolar, and lower pole papilla and renal cortex.
Paraffin embedded blocks of each representative region were
prepared, sectioned at 5 microns, and processed for the fol-
lowing stains: Hematoxylin and eosin (H&E), Yasue metal
substitution, and periodic acid Schiff. The stained sections
were then evaluated microscopically under incandescent and
polarized light with the assistance of an experienced research
pathologist; digital photographs were obtained.
Results
Study #1 – Dietary intervention, gestating sows
The TD kidneys had extensive hyperattenuating material
distributed throughout the medullary tips and papilla and
within the calices (Fig. 1A); there was no evidence of cortical
calcifications. No calcifications were evident in the CD kid-
neys. The calculated stone size ranged from submillimeter
crystals up to 2 mm. The Hounsfield unit density in the larger
stones was in the range of 400 to 500.
Kidneys from CD animals were normal in overall gross
appearance (Fig. 2A). In the TD kidneys, the renal cortex and
papillary areas appeared mottled (Fig 2B); corticomedullary
FIG. 1. CT scans showing
calcifications in the renal col-
lecting system of a pair of
treatment diet-21 kidneys.
(A) Representative thin slice
coronal view. (B, C) Maximal
Intensity Projection images
showing all scanned layers
superimposed, highlighting
all calcifications in a single
image.
1494 SIVALINGAM ET AL.

borders were less distinct compared with controls. Tiny
crystals (yellow-white in appearance) and mucinous debris
lined the papillary tips and calices in 7 of 12 kidneys from
treated sows (Figs. 2B, 2C). Transverse sections of the papilla
showed crystals aligned in the direction of the medullary
collecting ducts (Fig. 3A) and numerous stones at the papil-
lary tips (Fig. 3B). Yellow calcified deposits were seen to
project from the tips of the papilla, plugging the openings of
the distal collecting ducts (Fig. 3C). There was no visible ev-
idence of Randall plaques in any of the papilla.
Study #2 – Verification of model in nongestating sows
While the two TD sows were initially fed 10% HP diets, their
intake declined after 5 days. Therefore, the HP concentration
was reduced to 5%, and their tolerance and regular feed con-
sumption was reestablished. After 21 days, all animals were
sacrificed and their kidneys were extracted. CT scanning of the
kidneys demonstrated calculi near the papillary tips of all four
kidneys from the TD sows, which were up to 1.6mm in max-
imal diameter; no cortical calcifications were identified. Kid-
neys from the CD sows had no identifiable stones.
Stone analysis
The retrieved stone material ranged in size from <1mm
crystals to 2.1 mm with a pale white-yellow appearance. Five
separate specimens from five of the treated sows were sent for
analysis by micro CT ( James Williams, PhD, Indiana Uni-
versity). Each appeared as a cluster or aggregate of calcium
oxalate particles and crystals. Micro-CT examination of the
largest of these (1.9 mm in length) revealed polyhedral crys-
tals, consistent with calcium oxalate dihydrate, and a smaller
area of round lumps, which had an x-ray attenuation consis-
tent with calcium oxalate and which were presumed to be
calcium oxalate monohydrate (Fig. 4).
Histology
H&E staining revealed crystals within the collecting tu-
bules and at papillary tips of two-thirds (67%) of TD kidneys
but in none of the controls (Figs. 5A, 5B). Yasue staining
confirmed that these crystals contained calcium (Figs. 5C, 5D).
Tubular epithelial damage indicated by epithelial attenuation
and ulceration was observed with H&E in some areas with
heavy intratubular crystals (Figs. 6B, 6C). Notably, the spo-
radic presence of crystals in cortical tubules and interstitium
was observed (Fig. 6B). In some of the TD kidneys, many
crystal aggregates were not clearly associated with tubules
and appeared embedded in the interstitium of the papilla (and
cortex), although loss of discernible tubular structures with
apparent interstitial localization of crystals is possible. The
interstitially localized crystals were often surrounded by
multinucleate giant cells (Fig. 6C), and occasionally the same
was observed with intratubular crystals.
FIG. 2. Bivalved kidneys at 21 days. (A) Control diet kidney with normal appearing papilla and cortex; (B) treatment diet
(TD) kidney with mottled appearance and debris within the calyx (arrow); (C) crystals and stones (arrows) in collecting
system of TD kidney.
FIG. 3. Gross dissection of treatment diet kidneys. (A) Transverse section of renal papilla showing crystals along the
medullary collecting ducts (arrow); (B) numerous stones at papillary tip; (C) stone extruding from ducts of Bellini (arrow).
PORCINE MODEL OF CALCIUM OXALATE LITHIASIS 1495

Some crystals appeared to expand between the renal tubular
epithelium and the underlying interstitial connective tissue.
The interstitium surrounding some regions of crystal accu-
mulation in the cortex was infiltrated by small to moderate
numbers of mononuclear leukocytes; inflammation surround-
ing crystals in the papilla was more commonly limited to
multinucleate giant cells, although sometimes lymphoid cells
and polymorphonuclear leukocytes were present. The cortices
of several TD kidneys exhibited narrow radiating regions of
mild widening of the interstitium with extracellular matrix
interpreted as fibrotic changes (Fig. 6D). These radiating zones
were both associated and unassociated with crystals in or near
the lesions and often had mild to moderate mononuclear in-
flammatory infiltrates.
In addition to crystals, several of the animals had aggre-
gates of lamellar eosinophilic and basophilic material in the
papilla interpreted as mineral. Striking polymorphonuclear
and mononuclear inflammation of the papilla was noted in
one animal with crystal accumulation at the papillary tip with
evidence suggestive of ascending pyelonephritis. CD kidneys
exhibited minimal changes generally limited to mild occa-
sional lymphocytic interstitial infiltrates, minimal interstitial
widening with fibrous tissue, rare individual inflamed or re-
generative renal tubules, and occasional nonpolarizing ag-
gregate accumulation (interpreted as mineral) in the papilla
(note that only six CD kidneys were examined).
Discussion
We have developed a porcine model in which sustained
hyperoxaluria and renal crystal formation is achieved with
dietary intervention.
10,11
In the present study, we reproduced
calcium oxalate crystal formation in our model within 21 days
of HP feeding and identified renal derangements associated
with hyperoxaluria. Specifically, we demonstrated renal stone
formation in adult swine, crystals in proximal tubules adja-
cent to glomeruli and in collecting ducts, and signs of in-
flammatory tubular damage at all segments. This damage
was characterized by giant multinucleated cells, degeneration
of tubular epithelium, expansion of the interstitium with
matrix deposition, and rarely the extrusion of crystal into the
interstitium of TD but not CD kidneys—such changes indicate
an acute inflammatory response, presumably to the stress of
hyperoxaluria and crystalluria, which appeared to culminate
in an early fibrotic process. Although the most apparent
FIG. 4. Micro-CT demonstration of an extracted stone,
confirming a combination of calcium oxalate dihydrate and
monohydrate crystals.
FIG. 5. Papillary tips at 21
days, 4 ·magnification. He-
matoxylin and eosin stain:
(A) No crystals in control
diet (CD) kidney and (B)
crystals in treatment diet
(TD) kidney (arrows); Yasue
stain: (C) no crystals in CD
kidney and (D) crystals in
TD kidney (arrows).
1496 SIVALINGAM ET AL.

crystal formation was seen within expected regions of the
kidney (ie, renal papilla and distal tubules), we also noted
proximal tubular damage at the level of the renal cortex.
Observations similar to ours have been reported in rodent
studies,
12,13
whereby a short-term induction of hyperoxaluria
and crystalluria led to tubular epithelial destruction and
widened interstitial spaces
14
and migration of calcium oxalate
crystals from the tubular lumen into the interstitium.
15
The
striking similarities of our results with those using established
rodent models of urolithiasis strengthen the validity of our
model.
Although not a major amino acid in the human diet, HP is
consumed in small amounts in meats and gelatin containing
foods.
16
Ours is not the first study to use HP as a nutrition
intervention. Mandel and colleagues
17
demonstrated short-
term hyperoxaluria, peaking at day 6, in young swine fed 10%
HP.
17
Whereas Mandel and associates
16
used young growing
pigs, which have different metabolic capacity than adult pigs,
we used adult sows because they may more closely resemble
adult humans. In developing our model, we devised a reliable
method for sustaining sows’ consumption of HP-enriched
feed, for obtaining 24-hour urine collections, and for mea-
suring urinary oxalate excretion. In Kaplon and coworkers,
11
we reported hyperoxaluria in adult sows fed HP for 5 days in
both their standard feed and in feed that was acidified to
mimic the acid load of the typical Western diet. Thereafter,
Patel and associates
10
demonstrated that both short (5 days)
and long-term (21 days) hyperoxaluria (similar in scale to
human values) was reliably induced with HP and also with
gelatin (the latter of which was used by Knight and col-
leagues
16
in a similar human study). A single kidney from a
single treated animal revealed a small calcium oxalate stone.
10
Using this paradigm, we now report stone formation in
both gestating and nongestating sows. We observed calculus
deposits (‘‘plugs’’) at the papillary tips and occasional
submucosal crystal deposits at the papilla, similar to findings
from human intestinal bypass as reported by Evan and col-
leagues.
18
Our results also showed fibrotic changes in the
cortical tubules adjacent to the glomeruli of treated sows,
suggesting that renal damage occurs in the initial portion of
proximal renal tubules. Despite the inflammatory effects we
observed, only one of the TD animals had histologic signs of
infection, suggesting that these changes are sequelae of hy-
peroxaluria and not of infection.
Results from this model may have clinical relevance. Because
our model increases endogenous oxalate, it may be relevant for
studying primary hyperoxaluria. Calcium oxalate crystal for-
mation in these patients usually occurs initially, however, in the
renal parenchyma as nephrocalcinosis and frequently pro-
gresses to reduced renal function, leading to extrarenal oxalate
deposits. In our study, we observed crystals primarily within
the renal tubules and minimal to no evidence of intracellular
crystals as would be expected with nephrocalcinosis. Accord-
ingly, although our model is not characterized by an intestinal
challenge, our results are similar to those observed in patients
with short bowel,
18
whose intestinal calcium absorption is
usually very low and whose urinary oxalate excretion is sub-
sequently extremely high. Our model has potential relevance
for studying enteric calcium oxalate stone disease.
A limitation of our study is the possibility that direct po-
tential toxicity induced by renal reabsorption and cortical
metabolism of HP (as suggested by Knight and coworkers
16
)
might contribute to some of the renal effects observed. This
could mean that the changes may not necessarily be attrib-
utable to hyperoxaluria and/or to intratubular calcium crys-
tal deposition. Nonetheless, we detected similar tissue
architectural changes to those seen in human calcium oxalate
stone formers,
18
thereby supporting the theory that these
changes are truly from crystalluria. Further studies are nee-
ded to assess the direct impact HP might have on renal tissue.
FIG. 6. Cortical section, 21
days, H&E 20 ·.(A) Normal
glomerulus in control kidney;
(B) periglomerular inflam-
mation and fibrotic changes
(white arrows), with crystal
in proximal tubule (black
arrow); (C) tubules sur-
rounded by multinucleated
giant cells (arrows); (D) 10 ·
magnification, radiating re-
gions in renal cortex with
widening of interstitium
with extracellar matrix
(arrows) interpreted as
fibrotic changes.
PORCINE MODEL OF CALCIUM OXALATE LITHIASIS 1497

In considering the use of animal models for studying uro-
lithiasis, our study is important for a number of reasons: (1)
Irrespective of how calcium oxalate lithiasis is induced, the renal
effects of hyperoxaluria, stone formation and growth can be
studied in this model; (2) similar urinary and renal changes,
along with calculus formation, are evident in both gestating and
nongestating sows, confirming that both groups respond simi-
larly; (3) we can reliably induce stone formation with 21 days of
dietary manipulation, obviating the need for longer, more ex-
pensive interventions; (4) our results (a) concur with currently
upheld theories of stone formation derived from human studies,
specifically regarding papillary extrusion of crystals through
ducts of Bellini and submucosal crystal deposition,
18
and (b)
corroborate the histologic tissue changes seen in established
rodent models.
14,15,19
The overall findings of this study hold
promise for further development and use of our porcine model
to study the pathophysiology of calcium oxalate urolithiasis.
Conclusion
We demonstrated crystal formation and described the as-
sociated stress-induced histopathology in a porcine model of
urolithiasis. This is the initial report of dietary-induced renal
calculi in adult sows, with crystalluria in cortical proximal
tubules and throughout the collecting ducts and with tubular
damage at all segments. Further studies will allow us to elu-
cidate the etiology of renal damage and identify potential
preventive mechanisms.
Acknowledgments
The authors thank the University of Wisconsin Swine Re-
search and Teaching Center staff for their exemplarycare of the
research animals; Ruth Sullivan, VMD, PhD, for her assistance
with histology review and photos; Frank N. Ranallo, PhD, for
his assistance with CT protocols and image reconstruction; and
Lisa Sampson for her technical assistance with CT imaging and
tissue handling.
Disclosure Statement
No competing financial interests exist.
References
1. Sierakowski R, Finlayson B, Landes RR, et al. The frequency
of urolithiasis in hospital discharge diagnoses in the United
States. Invest Urol 1978;15:438–441.
2. Stamatelou KK, Francis ME, Jones CA, et al. Time trends in
reported prevalence of kidney stones in the United States:
1976–1994. Kidney Int 2003;63:1817–1823.
3. Moe OW. Kidney stones: Pathophysiology and medical
management. Lancet 2006;367:333–344.
4. Khan SR. Animal models of kidney stone formation: An
analysis. World J Urol 1997;15:236–243.
5. Thomsen HS, Larsen S, Talner LB. Papillary morphology in
adult human kidneys and in baby and adult pig kidneys.
Eur Urol 1983;9:170–180.
6. Sachs DH. The pig as a potential xenograft donor. Vet Im-
munol Immunopathol 1994;43:185–191.
7. Holmes RP, Goodman HO, Assimos DG. Contribution of
dietary oxalate to urinary oxalate excretion. Kidney Int
2001;59:270–276.
8. Knight J, Holmes RP. Mitochondrial hydroxyproline me-
tabolism: Implications for primary hyperoxaluria. Am J
Nephrol 2005;25:171–175.
9. Phang J, Hu C, Valle D. Disorders of proline and hydro-
xyproline metabolism. In: Beaudet AL, Sly WS, Vallee D,
Childs B, Kinzler KW, Vogelstein B, eds. The Metabolic and
Molecular Bases of Inherited Disease. New York: McGraw-
Hill; 2001, pp 1821–1938.
10. Patel SR, Penniston KL, Iwicki L, et al. Dietary induction of
long-term hyperoxaluria in the porcine model. J Endourol
2012;26:433–438.
11. Kaplon DM, Penniston KL, Darriet C, et al. Hydroxyproline-
induced hyperoxaluria using acidified and traditional diets
in the porcine model. J Endourol 2010;24:355–359.
12. O’Connor RC, Worcester EM, Evan AP, et al. Nephrolithiasis
and nephrocalcinosis in rats with small bowel resection. Urol
Res 2005;33:105–115.
13. Worcester EM, Chuang M, Laven B, et al. A new animal
model of hyperoxaluria and nephrolithiasis in rats with
small bowel resection. Urol Res 2005;33:380–382.
14. Sarica K, Yagci F, Bakir K, et al. Renal tubular injury induced
by hyperoxaluria: Evaluation of apoptotic changes. Urol Res
2001;29:34–37.
15. Khan SR, Shevock PN, Hackett RL. Acute hyperoxaluria,
renal injury and calcium oxalate urolithiasis. J Urol 1992;147:
226–230.
16. Knight J, Jiang J, Assimos DG, Holmes RP. Hydroxyproline
ingestion and urinary oxalate and glycolate excretion. Kid-
ney Int 2006;70:1929–1934.
17. Mandel NS, Henderson JD Jr, Hung LY, et al. A porcine
model of calcium oxalate kidney stone disease. J Urol 2004;
171:1301–1303.
18. Evan AP, Lingeman JE, Worcester EM, et al. Renal histo-
pathology and crystal deposits in patients with small bowel
resection and calcium oxalate stone disease. Kidney Int 2010;
78:310–317.
19. Khan SR. Crystal-induced inflammation of the kidneys:
Results from human studies, animal models, and tissue-
culture studies. Clin Exp Nephrol 2004;8:75–88.
Address correspondence to:
Kristina L. Penniston, PhD
Department of Urology
University of Wisconsin School of
Medicine and Public Health
3258 MFCB
1685 Highland Avenue
Madison, WI, 53705
E-mail: Penn@urology.wisc.edu
Abbreviations Used
CD ¼control diet
CT ¼computed tomography
H&E ¼hematoxylin and eosin
HP ¼hydroxyproline
TD ¼treatment diet
USDA ¼United States Department of Agriculture
1498 SIVALINGAM ET AL.