Hypercortisolism affects glomerular and tubular function in dogs
P.M.Y. Smetsa,⇑, H.P. Lefebvreb, H.S. Kooistrac, E. Meyerd, S. Croubelsd, B.E.J. Maddensd, S. Vandenabeelea,
J.H. Saunderse, S. Damineta
aDepartment of Small Animal Medicine and Clinical Biology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
bDepartment of Clinical Sciences and UMR181 de Physiopathologie et Toxicologie Expérimentales INRA, École Nationale Vétérinaire de Toulouse (ENVT), 23 Chemin des Capelles,
BP 87614, 31076 Toulouse Cedex 3, France
cDepartment of Clinical Sciences of Companion Animals, Utrecht University, Yalelaan 108, 3584 CM Utrecht, The Netherlands
dDepartment of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
eDepartment of Medical Imaging, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
a r t i c l ei n f o
Accepted 25 May 2011
Glomerular filtration rate
a b s t r a c t
Renal function was assessed in 25 dogs with Cushing’s syndrome and in 12 healthy controls. Routine
renal parameters and glomerular filtration rate (GFR) were measured and urinary biomarkers such as uri-
nary albumin (uALB), urinary immunoglobulin G (uIgG), and urinary retinol-binding protein (uRBP) were
assessed by ELISA. Urinary N-acetyl-b-D-glucosaminidase activity (uNAG) was determined colorimetri-
cally. All urinary markers were indexed to urinary creatinine concentration (c). Plasma exo- (Clexo) and
endo-iohexol (Clendo) clearance were used to measure GFR. Based on a Mann–Whitney U test, urea and
Clexodid not differ, sCr was significantly lower, and UPC, uALB/c, uIgG/c, uRBP/c, uNAG/c and Clendowere
higher in the dogs with Cushing’s syndrome when compared with controls. The findings indicate that glo-
merular and tubular function are both altered in dogs with Cushing’s syndrome. Further longitudinal
studies will be required to elucidate the pathogenesis of the changes in GFR.
? 2011 Elsevier Ltd. All rights reserved.
Up to 75% of dogs with untreated Cushing’s syndrome have pro-
teinuria and up to 86% have systemic hypertension (Hurley and
Vaden, 1998; Ortega et al., 1996). Frequently these clinical signs
do not respond to therapy (Hurley and Vaden, 1998) and are cen-
tral to the development of chronic kidney disease (CKD) (Jacob
et al., 2003, 2005). Dogs with Cushing’s syndrome may thus be at
risk of developing renal complications, and in human patients
decreased glomerular filtration rate (GFR) occurs (Haentjens
et al., 2005). However, as the precise effect of Cushing’s syndrome
on renal function remains unclear, the present study was under-
taken to assess renal function in dogs with Cushing’s syndrome,
using routine renal parameters as well as GFR and glomerular
and tubular urinary biomarkers.
Following Ethical Committee approval (Ethical Committee of
the Faculty of Veterinary Medicine and Bioscience Engineering,
Ghent University, authorisation number: EC2008/066) and in-
formed owner consent, 25 dogs with Cushing’s syndrome and 12
bodyweight matched controls P7 years old were selected for
study. Cushing’s syndrome was diagnosed based on clinical signs,
physical examination, and consistent results on a combination of
screening methodologies: low-dose dexamethasone suppression-
test; urinary corticoid-to-creatinine ratio (UCCR); adrenocortico-
trophic hormone (ACTH)-stimulation test; and differentiation tests
(UCCR after oral administration of dexamethasone, plasma ACTH-
concentration, abdominal ultrasound, computed tomography). A
complete blood count, biochemistry profile and urinalysis (includ-
ing bacterial culture), were performed on all dogs.
Systolic blood pressure (SBP) was measured according to the
American College of Veterinary Internal Medicine guidelines
(Brown et al., 2007). Morning urine samples were taken by cysto-
centesis (10 mL) using a 22 G needle. After centrifugation, the ur-
ine supernatant was stored at ?80 ?C. Glomerular filtration rate
was determined by plasma clearance of exo- (Clexo) and endo-
iohexol (Clendo), using a high performance liquid chromatogra-
phy-method adapted from van Hoek et al. (2007).
Biomarkers of glomerular function, urinary albumin (uALB) and
immunoglobulin G (uIgG), were determined by ELISA (Dog albu-
min and Dog IgG ELISA, Immunology Consultants Laboratory).
Markers of renal tubular function, urinary retinol-binding protein
(uRBP) and urinary N-acetyl-b-D-glucosaminidase activity (uNAG),
were measured by ELISA (Human Retinol-Binding Protein ELISA,
Immunology Consultants Laboratory), or using a colorimetric assay
(b-N-Acetyl-glucosaminidase Assay kit, Sigma–Aldrich), respec-
tively, and were ‘indexed’ to urinary creatinine (c), as previously
validated (Maddens et al., 2010; Smets et al., 2010).
Bodyweight, age, SBP and renal parameters were compared be-
tween affected and control dogs using a Mann–Whitney U test
1090-0233/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +32 9 264 7689.
E-mail address: email@example.com (P.M.Y. Smets).
The Veterinary Journal 192 (2012) 532–534
Contents lists available at ScienceDirect
The Veterinary Journal
journal homepage: www.elsevier.com/locate/tvjl
(Systat, version 12.00.08) with a significance level set at 5%. Be-
cause two dogs in the Cushing’s syndrome group had concurrent
CKD, the analysis was repeated excluding the data from these ani-
mals. Descriptive statistics and significant differences between the
diseased and control groups are presented in Table 1. The plasma
exo- and endo-iohexol concentration–time curves from both
groups are illustrated in Fig. 1. When the dogs with concurrent
Cushing’s syndrome and CKD were excluded from the analysis,
the overall results remained similar.
This is the first study to determine the GFR and the levels of glo-
merular and tubular biomarkers in dogs with Cushing’s syndrome.
Although the highest urine protein-to-creatinine ratio (UPC) was
16.8 and was found in a dog with both Cushing’s syndrome and
CKD, three animals with Cushing’s syndrome also had UPCs >10,
with normal urine sediment. Renal proteinuria can be caused by
increased glomerular permeability, impaired tubular reabsorption
of filtered protein, or both. Marked increases in UPC, uALB/c and
uIgG/c in the diseased group indicates proteinuria of glomerular
origin, which may result from glomerular hypertension or altered
glomerular permselectivity. High uIgG/c suggests the latter, since
the glomerular barrier is normally impermeable to this high
molecular-weight protein. Although the SBP was not increased in
the dogs with Cushing’s syndrome compared to the controls, this
does not preclude the possibility that glomerular hypertension
contributed to the proteinuria (Ortega et al., 1996).
Measures were taken to reduce any ‘white-coat effect’, but we
cannot completely exclude the influence of handling stress in dogs
in both groups. Furthermore, increased uRBP/c and uNAG/c in the
Cushing’s dogs indicates proteinuria of tubular origin. It is difficult
to elucidate whether this proteinuria is related solely to the Cush-
ing’s syndrome or to underlying glomerular disease. Follow-up
studies in these dogs are ongoing to investigate this finding further.
Plasma Clexowas not significantly different between the two
groups of dogs, whereas plasma Clendowas higher in the animals
with Cushing’s syndrome. Differences in the plasma clearance of
the two stereo-isomers indicate that their dispositions may vary
in different clinical settings for reasons that currently remain un-
clear (van Hoek et al., 2007). Exo-iohexol is the major stereo-iso-
mer and is typically considered a marker of GFR in dogs (Laroute
et al., 1999). Short term administration of glucocorticoids increases
the GFR in dogs (Kubota et al., 2001). In contrast, a long-term con-
sequence of Cushing’s syndrome in human patients can be de-
creased GFR (Haentjens et al., 2005). Therefore, the variability of
GFR according to stage of disease in the dogs with Cushing’s syn-
drome could possibly explain why differences in Clexowere not
In conclusion, this study found evidence of both renal glomeru-
lar and tubular dysfunction in dogs with untreated Cushing’s syn-
drome. Longitudinal studies will be required to determine the
effects of treatment on this renal dysfunction and to investigate
further the longer-term variations in GFR and kidney function.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could inappro-
priately influence or bias the content of the paper.
This work was funded by a grant from the Fonds voor
Wetenschappelijk Onderzoek (FWO) Vlaanderen. The authors
thank S. Galac, J.J.C.W.M. Buijtels and B.P. Meij for their help with
Details of the descriptive statistics (median [range]) for the dogs with Cushing’s
syndrome and controls.
Variable Dogs with Cushing’s syndromeControls
Number of dogs
4 F, 9 FN, 10 M, 2 MN
2 F, 4 FN, 3 M, 3 MN
F, female (intact); FN, female (neutered); M, male (intact); MN, male (neutered).
SBP, systolic blood pressure; sCr, serum creatinine; USG, urine specific gravity; UPC,
urine protein-to-creatinine ratio; uALB/c, urinary albumin-to-creatinine ratio;
uIgG/c, urinary immunoglobulin G-to-creatinine ratio; uRBP/c, urinary retinol-
binding protein-to-creatinine ratio; uNAG/c, urinary N-acetyl-b-D-glucosamini-
dase-to-creatinine ratio; Clexo, plasma clearance of exo-iohexol; Clendo, plasma
clearance of endo-iohexol.
*P < 0.05 between dogs with Cushing’s syndrome and controls.
**P < 0.001 between dogs with Cushing’s syndrome and controls.
Fig. 1. Plasma concentration–time curves of exo-iohexol (A) and endo-iohexol (B)
in dogs with Cushing’s syndrome (black squares) and in controls (white circles).
Plasma concentrations are expressed as mean ± SD.
P.M.Y. Smets et al./The Veterinary Journal 192 (2012) 532–534
case recruitment and S. De Baere, J. Lambrecht, M. Kramer and P. Download full-text
Van Broeck for excellent technical assistance.
Brown, S., Atkins, C., Bagley, R., Carr, A., Cowgill, L., Davidson, M., Egner, B., Elliott, J.,
Henik, R., Labato, M., Littman, M., Polzin, D., Ross, L., Snyder, P., Stepien, R., 2007.
Guidelines for the identification, evaluation, and management of systemic
hypertension in dogs and cats. Journal of Veterinary Internal Medicine 21, 542–
Haentjens, P., De Meirleir, L., Abs, R., Verhelst, J., Poppe, K., Velkeniers, B., 2005.
Glomerular filtration rate in patients with Cushing’s disease: A matched case-
control study. European Journal of Endocrinology 153, 819–829.
Hurley, K.J., Vaden, S.L., 1998. Evaluation of urine protein content in dogs with
pituitary-dependent hyperadrenocorticism. Journal of the American Veterinary
Medical Association 212, 369–373.
Jacob, F., Polzin, D.J., Osborne, C.A., Neaton, J.D., Lekcharoensuk, C., Allen, T.A., Kirk,
C.A., Swanson, L.L., 2003. Association between initial systolic blood pressure and
risk of developing a uremic crisis or of dying in dogs with chronic renal failure.
Journal of the American Veterinary Medical Association 222, 322–329.
Jacob, F., Polzin, D.J., Osborne, C.A., Neaton, J.D., Kirk, C.A., Allen, T.A., Swanson, L.L.,
2005. Evaluation of the association between initial proteinuria and morbidity
rate or death in dogs with naturally occurring chronic renal failure. Journal of
the American Veterinary Medical Association 226, 393–400.
Kubota, E., Hayashi, K., Matsuda, H., Honda, M., Tokuyama, H., Okubo, K., Naitoh, M.,
Arakawa, K., Saruta, T., 2001. Role of intrarenal angiotensin II in glucocorticoid-
induced renal vasodilation. Clinical and Experimental Nephrology 5, 186–192.
Laroute, V., Lefebvre, H.P., Costes, G., Toutain, P.L., 1999. Measurement of
glomerular filtration rate and effective renal plasma flow in the conscious
Beagle dog by single intravenous bolus of iohexol and p-aminohippuric acid.
Journal of Pharmacological and Toxicological Methods 41, 17–25.
Maddens, B.E., Daminet, S., Demeyere, K., Demon, D., Smets, P., Meyer, E., 2010.
Validation of immunoassays for the candidate renal markers C-reactive protein,
immunoglobulin G, thromboxane B(2) and retinol binding protein in canine
urine. Veterinary Immunology and Immunopathology 134, 259–264.
Ortega, T.M., Feldman, E.C., Nelson, R.W., Willits, N., Cowgill, L.D., 1996. Systemic
arterial blood pressure and urine protein/creatinine ratio in dogs with
Association 209, 1724–1729.
Smets, P.M.Y., Meyer, E., Maddens, B.E.J., Duchateau, L., Daminet, S., 2010. Urinary
markers in healthy young and aged dogs and dogs with chronic kidney disease.
Journal of Veterinary Internal Medicine 24, 65–72.
van Hoek, I., Vandermeulen, E., Duchateau, L., Lefebvre, H.P., Croubels, S., Peremans,
K., Polis, I., Daminet, S., 2007. Comparison and reproducibility of plasma
clearance of exogenous creatinine, exo-iohexol, endo-iohexol, and Cr-51-EDTA
in young adult and aged healthy cats. Journal of Veterinary Internal Medicine
the American VeterinaryMedical
P.M.Y. Smets et al./The Veterinary Journal 192 (2012) 532–534