The Veterinary Journal 215 (2016) 21–29

Contents lists available at ScienceDirect

The Veterinary Journal

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l

Review

Advances in the evaluation of canine renal disease

Rachel Cianciolo a,*, Jessica Hokamp b, Mary Nabity b
a Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA
b Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA

A R T I C L E I N F O A B S T R A C T

Article history: Many recent advances in the evaluation of dogs with kidney disease have improved our diagnostic al-
Accepted 23 April 2016 gorithms and have impacted our therapeutic strategies. Non-invasive techniques, such as urinary and
serologic biomarker evaluation, can help a clinician diagnose and treat a patient that cannot undergo a
Keywords: renal biopsy for clinical or financial reasons. Some biomarkers might help localize the affected struc-
Acute kidney injury ture (glomerulus vs. tubule) and indicate the type or severity of injury present. Although more research
Biomarker
is needed, studies indicate that some biomarkers (e.g. urine protein to creatinine ratio and urinary im-
Glomerulus
munoglobulins) can be useful in predicting adverse outcomes. Importantly, the sensitivity and specificity
Renal
Urinary of biomarkers for renal injury should be established and clinicians need to understand the limitations
Dog of these assays.
If a renal biopsy is performed, then it should be evaluated by a specialty diagnostic service with ex-
pertise in nephropathology. A panel of special stains, immunofluorescence for the detection of
immunoglobulins and complement factors, and transmission electron microscopy can be routinely em-
ployed in cases of glomerular disease. These advanced diagnostics can be used to detect immune deposits
in order to definitively diagnose immune complex mediated glomerular disease. Integrating the results
of biomarker assays and comprehensive renal biopsy evaluation, the clinician can make informed ther-
apeutic decisions, such as whether or not to immunosuppress a patient.
© 2016 Elsevier Ltd. All rights reserved.

Introduction diagnostic relevance of the various urinary biomarkers in AKI will

Diseases of the kidney are common in small animals, and guide-
lines for the clinical staging and grading of renal disease have been
created and adopted by the International Renal Interest Society
(IRIS).1 Criteria for both acute kidney injury (AKI) and chronic kidney
disease (CKD) have helped identify cases in which the injury is re-
versible or can be mitigated by therapeutic intervention; however,
the veterinarian must first determine the nature of injury. Recent
advances in urinary and serum biomarker analysis and in the eval-
uation of renal tissue have markedly improved our diagnostic
algorithms. They have enhanced our ability to categorize diseases
based on the structure affected (glomerulus vs. tubule) and patho-
genesis (e.g. immune complex-mediated disease). These diagnostic
techniques are especially useful in the current era of hemodialy-
sis, plasmapheresis and immunosuppression for immune-mediated
diseases.
Because great strides have been made in the evaluation of
proteinuric kidney disease, the discussion here is focused on urinary
biomarkers in protein losing nephropathy. Where appropriate, the
* Corresponding author. Tel.: +1 614 292 9717.
E-mail address: cianciolo.14@osu.edu (R. Cianciolo).
1 See: http://www.iris-kidney.com/guidelines/index.html (accessed 2 May 2016).
be mentioned. This will be followed by discussion of serum
biomarkers of kidney disease. Finally, the techniques used for com-
prehensive evaluation of renal tissue (especially glomeruli) will be
presented. Much of the knowledge regarding urinary biomarkers
and comprehensive evaluation of renal biopsy specimens has been
gained by the authors’ experience in directing the International Vet-
erinary Renal Pathology Service (IVRPS), which is a collaborative
effort between the Ohio State University and Texas A&M Universi-
ty that routinely evaluates urine by gel electrophoresis and renal
tissue with transmission electron microscopy (TEM), immunofluo-
rescence (IF) and histopathology using a panel of special stains. For
simplicity’s sake, the term ‘renal biopsy’ will imply comprehen-
sive analysis using these modalities.
Proteinuric kidney disease
Renal proteinuria is commonly observed in dogs with kidney
disease. While proteinuria can result from either glomerular or
tubular disease, a urine protein to creatinine ratio (UPC) >2.0 is
strongly indicative of glomerular disease. Moreover, the role of pro-
teinuria in the development of CKD is likely under-appreciated
because affected dogs are identified late in their disease course. Renal
biopsy (discussed below) is the gold standard for evaluating canine
glomerular disease; however, many dogs are not candidates for the

http://dx.doi.org/10.1016/j.tvjl.2016.04.012
1090-0233/© 2016 Elsevier Ltd. All rights reserved.
22 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29

procedure due to health or financial reasons. Using less invasive and

inexpensive diagnostic methods not only provides evidence for the
presence or absence of glomerular disease, but the results might
also influence the decision to perform a renal biopsy. Several ex-
tensive reviews discuss the pathophysiologic mechanisms and
appropriate interpretation of proteinuria in small animals (Lees et al,
2005; Grauer, 2007, 2011; Harley and Langston, 2012). Despite con-
sensus statements and reviews about the importance of evaluating,
monitoring, and treating renal proteinuria, proteinuria is often still
overlooked in small animals as an early marker of kidney disease.
In some cases, this may lead to clinicians not detecting renal disease
until development of azotemia, missing the opportunity for timely
therapeutic intervention.
Proteinuria is defined by the detection of an excessive amount
of protein in the urine by means of semiquantitative tests on uri-
nalysis (dipstick) or quantitative measurements of UPC or urinary
albumin concentration (Lees et al, 2005). The origin of protein-
uria, its persistence and its magnitude must be established (Lees
et al, 2005). A step by step guide to localize the source of protein-
uria is discussed in detail in the 2005 ACVIM consensus statement
(Lees et al, 2005). Persistent renal proteinuria (UPC ≥0.5 in at least
three samples two or more weeks apart without a contributing
Fig. 1. Image from Bis-Tris gel electrophoresis demonstrating urine protein banding
prerenal or postrenal cause) could be glomerular, tubular, or both.
patterns obtained from dogs with primary tubular damage (lanes 2 and 3: low mag-
In the discussion below, ‘proteinuria’ refers to ‘renal proteinuria’, nitude of proteinuria with predominantly low molecular weight bands), primary
assuming pre- and post-renal causes of proteinuria have been ruled glomerular damage (lanes 6, 7, 8 and 9: relatively large magnitude of proteinuria
out. with predominantly intermediate and high molecular weight bands and few low
molecular weight bands), and mixed glomerular and tubular damage (lanes 4 and
5: mixture of prominent bands ranging from low to high molecular weight).

Pathophysiology of renal proteinuria

In the healthy kidney there are several mechanisms that prevent
protein loss into the urine. The glomerular filtration barrier, com-
posed of the fenestrated endothelium and glycocalyx, trilaminar
glomerular basement membrane (GBM), and podocytes with slit dia-
phragms, is the main mechanism for preventing proteinuria (D’Amico
and Bazzi, 2003). The ultrastructural morphology of the glomeru-
lar filtration barrier will be described below. This barrier allows
proteins <40 kDa (low molecular weight [LMW] proteins) to filter
freely; however, intermediate molecular weight (IMW) proteins are
largely restricted, and high molecular weight (HMW) proteins
(>100 kDa) are almost completely restricted from glomerular fil-
tration (D’Amico and Bazzi, 2003). A second mechanism preventing
proteinuria is the ability of healthy proximal tubular epithelial cells
to reabsorb proteins normally present in the urinary filtrate (D’Amico
and Bazzi, 2003).
When renal damage occurs, the mechanisms that prevent
proteinuria are compromised. Glomerular damage increases the
permeability of the filtration barrier, allowing increased filtration
of IMW and HMW proteins (D’Amico and Bazzi, 2003). Tubular
damage can result in decreased protein reabsorption, leakage of
proteins from tubular epithelial cells, and increased production of
proteins involved in injury and repair (D’Amico and Bazzi, 2003).
Glomerular damage often results in massive proteinuria
whereas tubular damage is thought to result in mild proteinuria.
However, even today, the magnitude of proteinuria expected in
purely tubulointerstitial disease is under debate. Conflicting
experimental data, predominantly in rodents, have been used to
support arguments both for and against the development
of mild to moderate proteinuria when proximal tubular epithelial
cells are injured or lost (Comper et al., 2008; Navar, 2009;
Tanner, 2009). Treatment to reduce proteinuria has been shown
to mitigate progression of renal disease in dogs (Brown et al., 2003;
Lees et al, 2005). The reader is referred to additional sources
with detailed discussions of treatment protocols for reducing
proteinuria in dogs (Lees et al, 2005; Harley and Langston,
2012).
Patterns of proteinuria
When urine from dogs with renal injury is analyzed with sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) or
similar methods, the pattern of protein banding can help deter-
mine if glomerular damage, tubular damage, or both are contributing
to the proteinuria (D’Amico and Bazzi, 2003; Zini et al., 2004).
Primary tubular damage with limited or no glomerular damage will
generate predominantly LMW proteins, while primary glomerular
damage with minimal or no tubular damage generates a pattern of
IMW and HMW protein bands (Schultze and Jensen, 1998; D’Amico
and Bazzi, 2003; Zini et al., 2004; Giori et al., 2011). Mixed pat-
terns with protein bands in low, intermediate, and high molecular
weight ranges, however, are the predominant patterns seen in
proteinuric dogs, as concurrent damage to both glomerular and
tubular components commonly occurs (Schultze and Jensen, 1998;
Zini et al., 2004; Giori et al., 2011). Fig. 1 demonstrates different urine
protein banding patterns from proteinuric dogs evaluated by the
IVRPS.
Urine protein: Creatinine ratio
Once proteinuria is deemed to be persistent and renal in origin,
evaluation and monitoring of UPC are the important steps in de-
termining the presence and severity of glomerular damage. A
persistent UPC between 0.2 and 0.5 is classified as borderline pro-
teinuria while UPC ≥0.5 is considered proteinuric.2 UPC values ≥2
are generally considered to be indicative of glomerular protein-
uria, while values <2 are thought to occur more often with tubular
proteinuria (Center et al., 1985; Lees et al., 2005). Certainly, in the
authors’ experience, a UPC ≥2 is typically associated with at least
some injury to the glomerular filtration barrier, with significant glo-
merular lesions identified in 97.6% of 501 renal specimens from dogs
2 See: http://www.iris-kidney.com/guidelines/staging.html (accessed 2 May 2016).
 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29
biopsied for the clinical indication of proteinuria (Schneider et al.,
2013). However, it is also from our experience that a proportion of
proteinuric dogs with UPC <2 have primary glomerular damage dis-
covered on renal biopsy (Hokamp et al., 2016). Therefore, the
magnitude of the UPC cannot always localize renal proteinuria to
primary glomerular or tubulointerstitial damage.
UPC has prognostic value in azotemic CKD dogs, wherein a UPC
≥1.0 at initial evaluation was associated with increased risk of uremic
crisis, and the relative risk of uremia or death was ~1.5 times greater
for every 1-unit increase in UPC (Jacob et al., 2005). In contrast, a
study of predominantly proteinuric CKD dogs did not find UPC to
be associated with time to death due to renal disease; however, ap-
proximately 50% of the dogs were not azotemic (Hokamp et al.,
2016). Additionally, it is possible that treatment differences between
the two studies contributed in contrasting findings. The study by
Jacob et al. (2005) standardized treatment protocols for manage-
ment of CKD during the study and excluded dogs if they had
previously received treatment for CKD. In contrast the study by
Hokamp et al. (2016) did not exclude dogs based on current or past
treatments.
Individual biomarkers
While measurement of the UPC is an important first step in the
evaluation of renal proteinuria, its limitations include low speci-
ficity for determining the source of proteinuria and the severity of
injury. Therefore, several specific urine proteins are being investi-
gated as markers of glomerular or tubular damage, and these are
typically normalized to urine creatinine concentration.
Protein biomarkers of glomerular damage
Immunoglobulins
Immunoglobulins G (IgG), M (IgM), and A (IgA) are HMW pro-
teins in the serum that are unable to pass through the glomerular
filtration barrier in the healthy kidney. When the glomerular fil-
tration barrier is compromised, these immunoglobulins can pass
into the urinary filtrate.
Urinary IgG/creatinine (uIgG/c) is generally very low in healthy
dogs (<3 mg/g) and is significantly lower than uIgG/c observed in
dogs with either AKI or CKD (Maddens et al., 2010a, 2011; Defauw
et al., 2012; Nabity et al., 2012; Hrovat et al., 2013). Urinary IgM/
creatinine (uIgM/c) is also significantly lower in healthy dogs
compared to dogs with naturally occurring proteinuric CKD (un-
published data – Hokamp and Nabity).
Dogs with AKI due to a variety of natural causes, including Babesia
rossi, leishmaniasis, leptospirosis, snake envenomation, and pyo-
metra, have increased urinary IgG +/− IgA (Zaragoza et al., 2003a,
2003b; Maddens et al., 2010a, 2011; Defauw et al., 2012; Hrovat
et al., 2013). Urinary IgG is also significantly increased in dogs with
CKD due to X-linked hereditary nephropathy (XLHN), and in these
dogs, uIgG/c often increased before an increase in UPC (Nabity et al.,
2012). uIgG/c and uIgM/c are significantly correlated with both glo-
merular and tubulointerstitial lesions based on renal histopathology
in dogs with CKD due to various causes (Nabity et al., 2012; Hokamp
et al., 2016).
We recently reported that markedly increased urinary IgM and
IgG are significantly associated with immune complex mediated glo-
merulonephritis (ICGN) compared with other glomerular and tubular
disease categories in dogs with proteinuric CKD (Hokamp et al.,
2016). A combination of serum and urine measurements might also
be informative, as increased fractional excretions of both IgM and
IgG were significantly associated with decreased survival time in
dogs with proteinuric CKD that died or were euthanized due to renal
disease (Hokamp et al., 2016).
23
Protein and enzyme biomarkers of tubular damage
Protein markers of tubular damage and dysfunction can be in-
creased in the urine due to decreased tubular reabsorption (e.g.
retinol binding protein [RBP], neutrophil gelatinase-associated
lipocalin [NGAL]), increased production of proteins involved in injury
and repair (e.g. NGAL, Kidney Injury Molecule 1 [KIM-1]), and release
from damaged tubular epithelial cells (e.g. enzymes). A few pro-
teins that are present in the urine of healthy dogs, such as Tamm–
Horsfall protein, might decrease in the urine due to a decreased
production by damaged tubules (Raila et al., 2014).
Retinol binding protein (RBP)
Retinol binding protein is a LMW protein in plasma that circu-
lates bound to transthyretin. When unbound, RBP passes through
the glomerular filtration barrier into the renal filtrate and is reab-
sorbed by healthy proximal tubular epithelial cells (Raila et al.,
2003b). RBP is undetectable or present in very low amounts in the
urine of healthy dogs (Maddens et al., 2010b; Raila et al., 2010;
Nabity et al., 2011, 2012). If renal tubules are damaged, or if there
is competition for reabsorption due to large amounts of protein in
the urinary filtrate, RBP is not reabsorbed efficiently and can be de-
tected in the urine.
Urinary RBP increases in naturally occurring AKI in dogs (e.g. due
to pyometra, babesiosis, and snake envenomation), suggesting
tubular dysfunction (Forterre et al., 2004; Maddens et al., 2010b,
2011; Hrovat et al., 2013). Dogs with CKD have increased urinary
RBP and fractional excretion of RBP compared to healthy dogs (Raila
et al., 2003a, 2010; Nabity et al., 2012). In one study, urinary RBP
was particularly promising as a marker of CKD progression; it cor-
related strongly with histologic lesions and appeared to be less
influenced by magnitude of proteinuria than other urine biomarkers
evaluated (Nabity et al., 2012). Urinary RBP was also significantly
correlated with glomerular and tubulointerstitial damage, and both
uRBP/c and its fractional excretion were significantly associated with
time to death due to renal disease in dogs with naturally occur-
ring CKD due to a variety of causes (Nabity et al., 2012; Hokamp
et al., 2016). However, another study suggested that proteinuria
might be more important than decreased renal function in deter-
mining urinary RBP because urinary RBP was higher in proteinuric
dogs than azotemic, non-proteinuric dogs, and was not associated
with decreased glomerular filtration rate (Raila et al., 2010).
NGAL
NGAL is a LMW protein present in neutrophils and many organs
including the kidney. In health, NGAL filters freely through the glo-
merular filtration barrier and is reabsorbed by the proximal tubular
epithelial cells (Mori et al., 2005). However, glomerular damage with
resulting urinary protein overload can hinder tubular reabsorp-
tion of NGAL. Additionally, damaged tubular epithelial cells can
increase production and release of NGAL (Kuwabara et al., 2009).
Urinary NGAL increases due to, and thus is a marker of, both renal
and extra-renal (lower urinary tract) diseases. In experimentally-
induced AKI in dogs using nephrotoxic drugs (e.g. gentamicin),
urinary NGAL was shown to be an early marker of nephrotoxicity
and to correlate with severity of tubular damage (Kai et al., 2013;
Burt et al., 2014; Luo et al., 2014; Sasaki et al., 2014; Zhou et al.,
2014). Urinary NGAL is also increased in dogs with naturally oc-
curring AKI and/or sepsis (Kuwabara et al., 2009; Lee et al., 2012;
Segev et al., 2013; Hsu et al., 2014; Steinbach et al., 2014; Cortellini
et al., 2015). Dogs with various causes of CKD have significantly more
urinary NGAL compared with healthy dogs and dogs with lower
urinary tract diseases and infections (Nabity et al., 2012; Segev et al.,
2013; Hsu et al., 2014; Steinbach et al., 2014). Additionally, urinary
24 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29

Table 1
Selected non-invasive markers of kidney disease.

Marker Information provideda Measured in serum/plasma

or urineb

Serum creatinine Estimate of GFR Serum/plasma

Cystatin C Estimate of GFR Serum/plasma
Symmetric dimethylarginine Estimate of GFR Serum/plasma
Gel electrophoresis (e.g. SDS-PAGE) Glomerular and/or tubular damage/dysfunction Urine
Urine protein:creatinine Glomerular and/or tubular damage/dysfunction Urine
Albumin Glomerular and/or tubular damage/dysfunction Urine
C-reactive protein Glomerular damage/dysfunction Urine
Immunoglobulins A, G, and M Glomerular damage/dysfunction Urine
N-acetyl-β-D-glucosaminidase Tubular damage/dysfunction (but also increases with Urine
glomerular damage/dysfunction)
Clusterin Tubular damage/dysfunction Urine
Cystatin C Tubular damage/dysfunction Urine
Gamma glutamyl-transpeptidase Tubular damage/dysfunction Urine
Kidney injury molecule-1 Tubular damage/dysfunction Urine
Neutrophil gelatinase-associated lipocalin Tubular damage/dysfunction Urine
Retinol binding protein Tubular damage/dysfunction Urine
Tamm–Horsfall protein Tubular damage/dysfunction Urine

SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; GFR, glomerular filtration rate.
a
For all protein measurements in urine, interpretation assumes absence of post-renal disease (i.e. inactive sediment).
b
For UPC and all individual urinary biomarkers, urine creatinine concentration must also be measured for normalization purposes.

NGAL/creatinine correlates strongly with glomerular and
tubulointerstitial damage on histology (Nabity et al., 2012; Hsu et al.,
2014; Steinbach et al., 2014).
While renal diseases affect urinary NGAL, it should be noted that
pyuria, particularly with concurrent infection, can markedly in-
crease urinary NGAL concentrations (Lee et al., 2012; Daure et al.,
2013; Segev et al., 2013; Proverbio et al., 2015). Therefore caution
should be taken when interpreting increased urinary NGAL in dogs
with active urine sediments or histories of lower urinary tract
diseases.
N-acetyl-β-D-glucosaminidase (NAG)
NAG is a lysosomal enzyme in the renal tubular epithelial cells
that is released when renal damage occurs. Therefore, NAG has his-
torically been considered a marker of tubular damage. Urinary NAG
activity should be low in healthy dogs, although it is generally greater
in intact male than female dogs, likely because of spermatic enzymes,
since NAG activity decreases after castration (Higashiyama
et al., 1983; Reusch et al., 1991; Sato et al., 2002; Brunker et al.,
2009).
Urinary NAG increases in both AKI and CKD in dogs. Urinary NAG/
creatinine (uNAG/c) increases as early as 24 h after administration
of nephrotoxic doses of gentamycin, and studies support that uNAG/c
and urinary gamma-glutamyl transpeptidase/creatinine are more
sensitive for detection of drug induced AKI than markers of glo-
merular filtration rate, such as serum creatinine concentration and/
or 24-hour endogenous creatinine clearance (Greco et al., 1985;
Grauer et al., 1995; Rivers et al., 1996; Sasaki et al., 2014; Zhou et al.,
2014). While studies are few in number, naturally occurring AKI also
increases urinary NAG, as shown in dogs with pyometra and snake
envenomation (Sato et al., 2002; Maddens et al., 2010a, 2011;
Palviainen et al., 2013).
Urinary NAG also increases in most dogs with CKD compared to
healthy controls, and several studies have found that NAG trends
with proteinuria (Sato et al., 2002; Smets et al., 2010; Nabity et al.,
2012; Hokamp et al., 2016). Mild increases in uNAG/c were seen
before increased UPC in dogs with CKD due to XLHN (Nabity et al.,
2012). In dogs with XLHN, uNAG/c correlated moderately to strongly
with both glomerular and tubulointerstitial damage based on his-
tology (Nabity et al., 2012); however, in dogs with a variety of causes
of naturally occurring, proteinuric CKD, uNAG/c was moderately cor-
related with glomerular damage but not with tubulointerstitial
damage (Hokamp et al., 2016). Furthermore, uNAG/c was moder-
ately to strongly correlated with UPC (Nabity et al., 2012; Hokamp
et al., 2016). Thus, uNAG/c might be a better marker of glomerular
than tubular damage in proteinuric CKD, possibly due to in-
creased tubular epithelial lysosomal activity or by loss of NAG from
the serum across a leaky glomerular filtration barrier.
In summary, urinary biomarkers appear promising for non-
invasively providing information about the glomeruli and tubules,
including the severity of damage and potentially even the type of
glomerular disease present. Furthermore, a large number of urinary
biomarkers in addition to those discussed above are currently being
evaluated in a research setting, a partial listing of which is in-
cluded in Table 1. However, more studies are needed before clinical
recommendations can be made, and most urinary biomarker assays
are not yet widely available to practitioners. Lastly, it is impera-
tive that only urine samples with renal proteinuria and not samples
with active sediments (e.g. hematuria, pyuria, or bacteriuria) be used
for accurate interpretation of urinary biomarkers.
Serum biomarkers of kidney disease
As discussed, proteinuria is the earliest marker of glomerular
disease, but urine is often not evaluated in many patients for a variety
of reasons. Additionally, primary tubulointerstitial disease might not
be present with clinically evident proteinuria. Therefore, serum
biomarkers remain to be important tools for the diagnosis of kidney
disease, serving as endogenous markers of glomerular filtration rate
(GFR). In order for decreased GFR to be clinically detectable, sub-
stantial losses of nephrons and decreases in overall kidney function
are necessary, with estimates of up to 50% or more decrease in func-
tion occurring before azotemia develops. However, improved
interpretation of serum creatinine (sCr) can lead to an earlier di-
agnosis of kidney disease, and new biomarkers of GFR show promise
as early indicators of decreased kidney function. It is important to
remember, however, that markers of GFR should always be inter-
preted in light of the urine specific gravity and clinical history,
because hydration/blood volume status and urinary tract obstruc-
tion can influence these values.
Historically, sCr has been interpreted in the context of a refer-
ence interval. Particularly in dogs, where a variety of breeds and
sizes are represented, use of a standard, laboratory-based refer-
ence interval is an insensitive method for determining whether sCr
is increased in a particular patient, given that muscle mass is the
 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29
major determinant of baseline sCr concentration. More appropri-
ately, a baseline healthy adult measurement would be obtained,
followed by monitoring at regular intervals (i.e. during routine yearly
health checks). This allows for recognition of small, progressive in-
creases that can indicate relatively large decrements in kidney
function. Certainly, CKD can be detected much earlier by such ‘trend-
ing’ of sCr, as evidenced in one study in dogs with CKD due to XLHN,
where an average decrease in GFR of approximately 25% was iden-
tified compared with 50% when using a reference interval (Nabity
et al., 2015). Earlier identification of AKI is also possible using trend-
ing, and this is reflected by the IRIS AKI grading guidelines, wherein
an increase in sCr of 0.3 mg/dL within 48 h is used to diagnose Grade
1 or 2 AKI.3 Of course, such subtle increases in sCr require excel-
lent laboratory and instrument precision and this can present a
problem, particularly with benchtop instruments commonly used
in veterinary clinics (Braun et al., 2008; Ulleberg et al., 2011). Muscle
wasting in a patient over time can also interfere with the ability to
identify small, but clinically significant increases in sCr. For in-
stance, one study found cats >15 years old to have lower sCr but
also lower GFR than cats <15 years old (Hall et al., 2014b).
In an effort to overcome some of the limitations of sCr in the eval-
uation of kidney function, cystatin C and symmetric dimethylarginine
(SDMA) have been evaluated as new markers of GFR. Cystatin C is
a small protein, while SDMA is a methylated amino acid of similar
size to creatinine. In the few published studies on cystatin C in dogs,
it correlated as well or better than sCr with measured GFR using
clearance techniques in dogs with renal disease, and it was a more
sensitive indicator of decreased GFR than sCr (Almy et al., 2002;
Wehner et al., 2008; Miyagawa et al., 2009). Readers are referred
to a recent comprehensive review of cystatin C for additional in-
formation (Ghys et al., 2014). Thus far, two studies have shown SDMA
to be consistently more sensitive than sCr for detecting decreased
GFR in both dogs and cats when sCr was interpreted based on a ref-
erence interval (Hall et al., 2014a; Nabity et al., 2015). It was also
more sensitive than sCr based on trending in young, growing dogs
(Nabity et al., 2015), possibly because of SDMA’s lack of influence
by muscle mass (Hall et al., 2014a, 2014b).
SDMA is now widely available to practitioners, offered by IDEXX
Laboratories; however, cystatin C is still limited to the research
setting. While studies with both of these markers appear promis-
ing, they are thus far limited in the scope of injuries examined.
Furthermore, non-renal influences are still largely unknown, al-
though the major limitation of sCr (muscle mass) does not appear
to influence either cystatin C or SDMA. As we gain more experi-
ence with these new markers, and in particular, SDMA, we will learn
more about their limitations as well as their advantages as com-
pared with sCr.
Comprehensive evaluation of renal biopsy specimens from
proteinuric patients
Most renal biopsy specimens submitted to the IVRPS are for the
evaluation of proteinuric kidney disease. Based on the evaluation
of more than 1000 canine renal specimens by the IVRPS, there
appears to be two broad categories of glomerular disease: ICGN and
diseases that are not mediated by immune complexes, hereafter re-
ferred to as non-immune complex mediated glomerulonephropathy
(or non-ICGN). Examples of non-ICGN include: amyloidosis, dis-
eases of podocyte injury such as focal segmental glomerulosclerosis
and diseases of the GBM. Recently, the World Small Animal Veter-
inary Association sponsored a collaboration of pathologists and
veterinary nephrologists, designed to identify and define renal lesions
3 See http://www.iris-kidney.com/guidelines/grading.html (accessed 2 May 2016).
25
in proteinuric dogs. After scoring a battery of such lesions in 89 dogs,
hierarchical cluster analysis was employed to discern patterns of
disease phenotypes identified with light microscopy (LM), TEM and
IF. There were two branches of the dendrogram, and the presence
of immune complex deposits was the main difference between clus-
ters, supporting the concept of dividing glomerular diseases into
those that are immune complex-mediated and those that are not.
Interestingly, when the hierarchical analysis was performed on the
same cases using only the data from the LM evaluation, cases were
not clustered based on whether or not immune complexes were
identified. In fact, 24.7% of cases were misclassified when only LM
data were used. The misclassifications (or ‘misdiagnosis’) resulted
in ICGN cases clustering with non-ICGN cases and vice versa. This
study clearly showed that different pathogeneses of lesions can result
in similar histologic phenotypes (Cianciolo et al., 2015). Since ther-
apeutic decisions rest on whether or not the glomerular disease is
mediated by immune complexes, determining their presence or
absence should be a priority in the evaluation of renal biopsy
specimens.
Unfortunately, histologic evaluation of glomeruli using routine
hematoxylin and eosin staining is often insufficient for the correct
diagnosis of either category. Additional staining techniques such as
Periodic acid Schiff, Masson’s trichrome, Congo red and Jones me-
thenamine silver can be used to highlight various features of
glomerular disease (Table 2). Importantly, all histochemical tech-
niques (with the exception of the Congo red method) should be
performed on well-fixed tissues sectioned at 3 μm thickness. Thin
sections enable identification of hypercellularity and remodeling of
the GBM. The section used for the Congo red method should be
8–10 μm thick, in order to detect small amounts of amyloid. Even
with the appropriate panel of stains, the histopathologic diagno-
sis can be difficult. In fact, 25% of the 501 renal biopsy specimens
from proteinuric dogs submitted to the IVRPS required TEM for a
final diagnosis (Schneider et al., 2013). Examples of renal biopsies
that required IF and TEM for the final diagnosis are shown in Figs. 2
and 3.
Immunofluorescence is performed on unfixed tissue and allows
identification of immune complex deposits in glomeruli. Samples
for IF need to be placed in Michel’s transport media and should be
washed, embedded in optimal cutting temperature (OCT) and frozen
within 5 days. If the transport media is unavailable, samples can
be placed in saline but they need to be embedded in OCT within 1
day. Direct IF for the detection of immunoglobulins and comple-
ment components is performed on frozen sections. At the IVRPS,
fluorescent antibodies directed against canine IgG, IgM, IgA and C3
are routinely used. Fluorescent antibodies against human C1q, kappa
light chain and lambda light chain cross react with canine tissue
and can provide robust labeling of immunoglobulins. Evaluation of
IF staining requires sufficient training (Walker, 2009) because the
pattern of staining is often more important than the intensity. For
example granular staining indicates immune complexes, whereas
splotchy staining is usually non-specific (Fig. 2, panel f).
TEM is performed on well-fixed tissue. Ideally, samples are sub-
mitted in glutaraldehyde; however, if glutaraldehyde is unavailable,
small cores/cubes placed in formalin can be post-fixed in glutar-
aldehyde once they arrive in the renal biopsy laboratory. Samples
should be small (1–2 mm3) in order to ensure adequate fixation by
either fixative. Oftentimes one to three glomeruli are evaluated ul-
trastructurally (Fig. 2, panel e). This study is used to detect electron
dense deposits, which can be located between the GBM and
podocytes (subepithelial), within the GBM (intramembranous),
between the GBM and endothelial cells (subendothelial), within the
mesangial matrix (mesangial) or along the GBM that encircles the
mesangium (paramesangial). In many cases there are deposits in
more than one location. An interesting correlation between ultra-
structure and histology is that deposits located in subendothelial
26 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29

Table 2
Special stains used for the evaluation of renal tissue.

Stain/method Specific uses in renal tissue Caveats

Hematoxylin and eosin Allows identification of inflammatory cells Cell cytoplasm blends with GBM and mesangial matrix
(intraglomerular and interstitial)
Periodic acid Schiff Facilitates localization of hypercellularity (endocapillary Inflammatory cells and red blood cells often stain poorly
vs. mesangial or both); stains basement membranes,
allowing detection of some types of GBM remodeling;
hyalinosis (insudated plasma is bright pink); highlights
the tubular apical brush border for detecting acute
tubular epithelial injury
Masson’s trichrome Allows identification of scarring (both in the interstitium Difficult to identify inflammatory cells; can be difficult to
and glomerulus); large immune complex deposits will differentiate interstitial edema from interstitial fibrosis
stain red; hyalinosis stains red-orange; amyloid stains
mottled peach-blue; fibrin stains red-orange and is
fibrillary
Jones methenamine silver Stains basement membranes black, enabling the Difficult to identify inflammatory cells
identification of GBM remodeling (double contours,
spikes, holes); amyloid does not take up silver, therefore
mesangial amyloid will be non-staining
Congo red Stains amyloid peach which demonstrates apple green Should be performed on sections 8–10 μm thick in order
birefringence when viewed with polarized light to identify small amyloid deposits

GBM, glomerular basement membrane.

zones often activate the complement pathway near the capillary
lumen, thereby attracting inflammatory cells to the glomerular tuft
to result in endocapillary hypercellularity. Similarly, mesangial de-
posits can induce mesangial cells to become activated and replicate,
leading to mesangial hypercellularity. Therefore, when mesangial
and subendothelial deposits are present, the histologic pattern tends
to be ‘membranoproliferative’. In contrast, when deposits are sub-
epithelial (as opposed to subendothelial), the histologic pattern
usually lacks hypercellularity and is called ‘membranous’ (Nangaku
and Couser, 2005; Nangaku et al., 2005). Similar relationships
between deposit location and histologic pattern occur in human renal
biopsies (Jennette et al., 2007). There are exceptions to the rule,
however, wherein deposits can be identified in all of the above lo-
cations and have either a membranous or membranoproliferative
histologic pattern. In the end, it is still unknown whether the his-
tologic pattern or the ultrastructural pattern is more important with
respect to the clinical prognosis.
In addition to demonstrating the presence of electron dense de-
posits, TEM evaluation is useful to examine the GBM structure and
the podocytes. Certainly for cases without electron dense depos-
its, the TEM examination focuses on evaluating the integrity of the
components of the glomerular filtration barrier: namely endothe-
lial cells, GBM and podocytes with their slit diaphragms. Endothelial
cells are the most interior portion of the filtration barrier. Al-
though they are fenestrated (containing transcellular pores
throughout their attenuated cytoplasm), they are covered by a
glycocalyx, which can be likened to a complex meshwork of neg-
atively charged membrane-bound proteoglycans and glycoproteins.
Plasma molecules must first negotiate the glycocalyx before they
can pass through the transcellular fenestrae. Unfortunately, the
glycocalyx cannot be easily evaluated even with TEM and requires

Fig. 2. Photomicrographs of a glomerulus from a 1.7-year-old castrated male Cav-

alier King Charles spaniel dog with a 1-month history of proteinuria (UPC 11.3) and
historical azotemia (SCr 3.1 mg/dL) which had resolved at the time of biopsy (SCr
0.8 mg/dL). The dog was not hypertensive. (a) Hematoxylin and eosin, (b) Periodic
acid Schiff, (c) Masson’s trichrome, (d) Jones methenamine silver stain all demon-
strate minimal light microscopic changes. Glomeruli are not hypercellular and the
glomerular basement membranes are of normal thickness. There is mild podocyte
hypertrophy. All scale bars = 20 μm. (e) Transmission electron microscopy reveals
small electron dense deposits (arrows) along the subepithelial aspects of the glo-
merular basement membranes. Scale bar = 2 μm. (f) Immunofluorescence using a
FITC-labeled antibody against IgG demonstrates positive granular labeling along cap-
illary walls. Scale bar = 20 μm. Taken together, the biopsy is diagnostic for early
immune complex mediated membranous glomerulonephropathy.
Fig. 3. Photomicrographs of a glomerulus from a 12.8-year-old spayed female Lab-
rador retriever dog with a 1-year history of proteinuria (UPC >4) without azotemia
(SCr 1.04 mg/dL) or hypertension. (a) Hematoxylin and eosin, (b) Periodic acid Schiff,
(c) Masson’s trichrome, (d) Jones methenamine silver stain all demonstrate seg-
mental sclerosis (arrow) of the tuft. There is also a focal synechia. Three of 22 glomeruli
were segmentally sclerotic. All scale bars = 20 μm. (e) Transmission electron mi-
croscopy reveals global foot process effacement of podocytes and microvillus
transformation of the podocyte cytoplasm. Scale bar = 5 μm. (f) Higher magnifica-
tion demonstrates the lack of electron dense deposits along the capillary wall. Scale
bar = 2 μm. IF studies did not demonstrate positive staining for any of the
immunoreactants tested (data not shown). Taken together, this biopsy is consis-
tent with focal segmental glomerulosclerosis.
 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29
special processing steps to demonstrate its presence (Salmon et al.,
2012). Experimentally and in certain diseases, when the glomeru-
lar endothelial glycocalyx is cleaved, there is marked proteinuria.
Although there is active research in the experimental models of
glycocalyx and endothelial injury (Salmon and Satchell, 2012), little
is known regarding spontaneous disease of these structures in small
animals.
The GBM is a trilaminar structure, composed of the lamina rara
interna, lamina densa and lamina rara externa. The endothelial cells
and podocytes are responsible for synthesizing the GBM and main-
taining its structure. In general, the TEM evaluation is used to identify
and characterize GBM remodeling. The most common type of re-
modeling occurs in response to the presence of immune complexes;
however, it can also be seen secondary to other factors. In humans,
these ‘other factors’ include hypertension, endothelial cell damage,
diabetes mellitus or genetic mutations in the proteins that com-
prise the GBM (Bonsib, 2007). In dogs, only mutations in GBM
proteins have been verified as a cause of GBM abnormalities (XLHN)
(Tsai et al., 2007). Hypertension and dyslipidemia are also sus-
pected to alter the GBM structure in dogs but a comprehensive study
of the renal biopsy findings from non-proteinuric dogs with these
conditions has not yet been performed.
Podocytes are intricate cells with large cell bodies and exten-
sive primary and secondary processes that encircle the abluminal
surface of the glomerular capillaries. These processes have small del-
icate ‘foot processes’ that are oriented perpendicular to the GBM,
contacting its outer surface. Foot processes from different podocytes
are interdigitated and connected to one another by a molecular sieve,
called the ‘slit diaphragm’. This molecular network allows some mol-
ecules that have traversed the GBM to pass through into the urinary
space.
Most proteinuric animals will have some degree of podocyte foot
process effacement. Importantly, foot process effacement is a re-
versible process (Kriz et al., 2013). However, if the podocyte
undergoes irreversible cell damage, it will detach and be lost into
the urinary filtrate. The denuded GBM will either adhere to Bow-
man’s capsule (synechia) or its surface will be covered by the foot
processes of a neighboring podocyte. In order to cover more GBM
surface area, remaining podocytes have to hypertrophy, a process
that will eventually become unsustainable. Hypertrophied cells are
at a higher risk for injury, leading to a vicious cycle of podocyte injury,
detachment and loss. Lobules of the glomerular tuft that are no
longer covered by a sufficient number of podocytes become scle-
rotic or ‘scarred’. This process might start segmentally in a few
glomeruli, but can progress to become a global diffuse process. Ex-
perimentally, the threshold of podocyte loss that will ultimately lead
to segmental sclerosis in rodents has been determined to be ap-
proximately 40% (Wharram et al., 2005).
The main diagnostic dilemma that arises when evaluating renal
biopsy specimens is elucidating whether or not glomerulosclero-
sis is secondary to ICGN-induced podocyte injury. Immune deposits,
especially subepithelial ones, can activate complement resulting in
sub-lethal to lethal podocyte injury (Nangaku et al., 2005). There-
fore, chronic ICGN can result in a sclerosing glomerular lesion on
histopathology, underscoring the need for comprehensive evalua-
tion of renal biopsy specimens with TEM and IF.
Evaluation of the panel of special stains is also crucial for de-
tecting acute tubular epithelial injury and estimating interstitial
fibrosis and tubular atrophy. In the former scenario, loss of the apical
brush border of proximal tubular epithelial cells is easily ob-
served in PAS and trichrome-stained sections. With chronic tubular
injury, the interstitium widens due to collagen deposition noted on
the trichrome stain. Additionally tubules degenerate and atrophy,
characterized by a thick PAS tubular basement membrane and at-
tenuated epithelium. In humans, the extent of interstitial fibrosis
and tubular atrophy correlates well with the likelihood that the
27
patient will progress to renal insufficiency and CKD
(Mackensen-Haen et al., 1981; Farris et al., 2011). This is especial-
ly important in patients (dogs or humans) who might have fewer
nephrons than normal (so-called ‘nephron paucity’) due to renal mal-
development. Numerous examples of abnormal renal development
in dogs have been reported in the literature and represent approx-
imately 15% of the renal biopsy submissions to the IVRPS
(unpublished data).
Taken together, disturbance of any of the components of the glo-
merular filtration barrier – loss of the endothelial glycocalyx,
abnormalities/remodeling of the GBM, electron dense deposits along
the GBM, podocyte foot process fusion or podocyte loss – can result
in proteinuria. The goal of a comprehensive renal biopsy is to assess
which lesions are present, if they are irreversible and whether or
not they warrant immunosuppression.
Conclusion
Recent advances in the detection of canine kidney disease can
lead to earlier diagnosis and therapeutic intervention. Biomarker
evaluations are minimally invasive and can be performed repeat-
edly to monitor the course of disease and the response to treatment.
In addition to assessing the severity of disease, some of the assays
can even be used to help discern the type of damage (glomerular
vs. tubulointerstitial) that has occurred. The comprehensive renal
biopsy evaluation is still considered the gold standard for diagno-
sis of proteinuric kidney disease. Importantly, it can distinguish
between ICGN and non-ICGN, which will help the clinician develop
a treatment plan that is effective and does not expose non-ICGN pa-
tients to unneeded immunosuppression.
Conflict of interest statement
None of the authors of this paper has a financial or personal re-
lationship with other people or organizations that could
inappropriately influence or bias the content of the paper.
Acknowledgements
The authors would like to thank Alan Flechtner, Anne Saulsbery
and Florinda Jaynes from the Comparative Mouse Phenotyping
Shared Resource (Cancer Center Support Grant – P30 CA016058)
for histopathology, immunofluorescence and electron microscopy
specimen preparation. Additionally, the authors gratefully thank Mary
Sanders (Department of Small Animal Clinical Sciences, Texas A&M
University, College Station, TX) for her expert technical assistance.
References
Almy, F.S., Christopher, M.M., King, D.P., Brown, S.A., 2002. Evaluation of cystatin C
as an endogenous marker of glomerular filtration rate in dogs. Journal of
Veterinary Internal Medicine 16, 45–51.
Bonsib, S.M., 2007. Renal anatomy and histology. In: Heptinstall’s Pathology of the
Kidney, Sixth Ed. Lippincott and Wilkins, Philadelphia, pp. 1–70.
Braun, J.P., Cabe, E., Geffre, A., Lefebvre, H.P., Trumel, C., 2008. Comparison of plasma
creatinine values measured by different veterinary practices. The Veterinary
Record 162, 215–216.
Brown, S.A., Finco, D.R., Brown, C.A., Crowell, W.A., Alva, R., Ericsson, G.E., Cooper,
T., 2003. Evaluation of the effects of inhibition of angiotensin converting enzyme
with enalapril in dogs with induced chronic renal insufficiency. American Journal
of Veterinary Research 64, 321–327.
Brunker, J.D., Ponzio, N.M., Payton, M.E., 2009. Indices of urine N-acetyl-beta-D-
glucosaminidase and gamma-glutamyl transpeptidase activities in clinically
normal adult dogs. American Journal of Veterinary Research 70, 297–301.
Burt, D., Crowell, S.J., Ackley, D.C., Magee, T.V., Aubrecht, J., 2014. Application of
emerging biomarkers of acute kidney injury in development of kidney-sparing
polypeptide-based antibiotics. Drug and Chemical Toxicology 37, 204–212.
Center, S.A., Wilkinson, E., Smith, C.A., Erb, H., Lewis, R.M., 1985. 24-Hour urine
protein/creatinine ratio in dogs with protein-losing nephropathies. Journal of
the American Veterinary Medical Association 187, 820–824.
28 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29
Cianciolo, R.E., Mohr, F.C., Aresu, L., Brown, C.A., James, C., Jansen, J.H., Spangler, W.L.,
van der Lugt, J.J., Kass, P.H., Brovida, C., et al., 2015. World small animal veterinary
association renal pathology initiative: Classification of glomerular diseases in
dogs. Veterinary Pathology 53, 113–135.
Comper, W.D., Hilliard, L.M., Nikolic-Paterson, D.J., Russo, L.M., 2008. Disease-
dependent mechanisms of albuminuria. American Journal of Physiology – Renal
Physiology 295, F1589–F1600.
Cortellini, S., Pelligand, L., Syme, H., Chang, Y.M., Adamantos, S., 2015. Neutrophil
gelatinase-associated lipocalin in dogs with sepsis undergoing emergency
laparotomy: A prospective case-control study. Journal of Veterinary Internal
Medicine 29, 1595–1602.
Daure, E., Belanger, M.C., Beauchamp, G., Lapointe, C., 2013. Elevation of neutrophil
gelatinase-associated lipocalin (NGAL) in non-azotemic dogs with urinary tract
infection. Research in Veterinary Science 95, 1181–1185.
D’Amico, G., Bazzi, C., 2003. Pathophysiology of proteinuria. Kidney International 63,
809–825.
Defauw, P., Schoeman, J.P., Smets, P., Goddard, A., Meyer, E., Liebenberg, C.,
Daminet, S., 2012. Assessment of renal dysfunction using urinary markers in
canine babesiosis caused by Babesia rossi. Veterinary Parasitology 190,
326–332.
Farris, A.B., Adams, C.D., Brousaides, N., Della Pelle, P.A., Collins, A.B., Moradi, E., Smith,
R.N., Grimm, P.C., Colvin, R.B., 2011. Morphometric and visual evaluation of
fibrosis in renal biopsies. Journal of the American Society of Nephrology 22,
176–186.
Forterre, S., Raila, J., Schweigert, F.J., 2004. Protein profiling of urine from dogs with
renal disease using ProteinChip analysis. Journal of Veterinary Diagnostic
Investigation 16, 271–277.
Ghys, L., Paepe, D., Smets, P., Lefebvre, H., Delanghe, J., Daminet, S., 2014. Cystatin
C: A new renal marker and its potential use in small animal medicine. Journal
of Veterinary Internal Medicine 28, 1152–1164.
Giori, L., Tricomi, F.M., Zatelli, A., Roura, X., Paltrinieri, S., 2011. High-resolution gel
electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine
samples for qualitative analysis of proteinuria in dogs. Journal of Veterinary
Diagnostic Investigation 23, 682–690.
Grauer, G.F., 2007. Measurement, interpretation, and implications of proteinuria and
albuminuria. Veterinary Clinics of North America: Small Animal Practice 37,
283–295.
Grauer, G.F., 2011. Proteinuria: Measurement and interpretation. Topics in Companion
Animal Medicine 26, 121–127.
Grauer, G.F., Greco, D.S., Behrend, E.N., Mani, I., Fettman, M.J., Allen, T.A., 1995.
Estimation of quantitative enzymuria in dogs with gentamicin-induced
nephrotoxicosis using urine enzyme/creatinine ratios from spot urine samples.
Journal of Veterinary Internal Medicine 9, 324–327.
Greco, D.S., Turnwald, G.H., Adams, R., Gossett, K.A., Kearney, M., Casey, H., 1985.
Urinary gamma-glutamyl transpeptidase activity in dogs with gentamicin-
induced nephrotoxicity. American Journal of Veterinary Research 46,
2332–2335.
Hall, J.A., Yerramilli, M., Obare, E., Yerramilli, M., Jewell, D.E., 2014a. Comparison of
serum concentrations of symmetric dimethylarginine and creatinine as kidney
function biomarkers in cats with chronic kidney disease. Journal of Veterinary
Internal Medicine 28, 1676–1683.
Hall, J.A., Yerramilli, M., Obare, E., Yerramilli, M., Yu, S., Jewell, D.E., 2014b. Comparison
of serum concentrations of symmetric dimethylarginine and creatinine as kidney
function biomarkers in healthy geriatric cats fed reduced protein foods enriched
with fish oil, L-carnitine, and medium-chain triglycerides. The Veterinary Journal
202, 588–596.
Harley, L., Langston, C., 2012. Proteinuria in dogs and cats. Canadian Veterinary Journal
53, 631–638.
Higashiyama, N., Nishiyama, S., Itoh, T., Nakamura, M., 1983. Effect of castration on
urinary N-acetyl-beta-D-glucosaminidase levels in male beagles. Renal Physiology
6, 226–231.
Hokamp, J.A., Cianciolo, R.E., Boggess, M.M., Lees, G.E., Benali, S.L., Kovarsky, M., Nabity,
M.B., 2016. Correlation of urine and serum biomarkers with renal damage and
survival in dogs with naturally occurring chronic kidney disease. Journal of
Veterinary Internal Medicine 30, 591–601.
Hrovat, A., Schoeman, J.P., de Laat, B., Meyer, E., Smets, P., Goddard, A., Nagel, S.,
Daminet, S., 2013. Evaluation of snake envenomation-induced renal dysfunction
in dogs using early urinary biomarkers of nephrotoxicity. The Veterinary Journal
198, 239–244.
Hsu, W.L., Lin, Y.S., Hu, Y.Y., Wong, M.L., Lin, F.Y., Lee, Y.J., 2014. Neutrophil gelatinase-
associated lipocalin in dogs with naturally occurring renal diseases. Journal of
Veterinary Internal Medicine 28, 437–442.
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.
Jennette, J.C., Schwartz, M.M., Olson, J.L., Silva, F.G., 2007. Primer on the pathologic
diagnosis of renal disease. In: Heptinstall’s Pathology of the Kidney, Sixth Ed.
Lippincott Williams and Wilkins, Philadelphia, PA, pp. 97–123.
Kai, K., Yamaguchi, T., Yoshimatsu, Y., Kinoshita, J., Teranishi, M., Takasaki, W., 2013.
Neutrophil gelatinase-associated lipocalin, a sensitive urinary biomarker of acute
kidney injury in dogs receiving gentamicin. The Journal of Toxicological Sciences
38, 269–277.
Kriz, W., Shirato, I., Nagata, M., LeHir, M., Lemley, K.V., 2013. The podocyte’s response
to stress: The enigma of foot process effacement. American Journal of Physiology
304, F333–F347.
Kuwabara, T., Mori, K., Mukoyama, M., Kasahara, M., Yokoi, H., Saito, Y., Yoshioka,
T., Ogawa, Y., Imamaki, H., Kusakabe, T., et al., 2009. Urinary neutrophil gelatinase-
associated lipocalin levels reflect damage to glomeruli, proximal tubules, and
distal nephrons. Kidney International 75, 285–294.
Lee, Y.J., Hu, Y.Y., Lin, Y.S., Chang, C.T., Lin, F.Y., Wong, M.L., Kuo-Hsuan, H., Hsu, W.L.,
2012. Urine neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker
for acute canine kidney injury. BMC Veterinary Research 8, 248.
Lees, G.E., Brown, S.A., Elliott, J., Grauer, G.E., Vaden, S.L., American College of
Veterinary Internal Medicine, 2005. Assessment and management of proteinuria
in dogs and cats: 2004 ACVIM Forum Consensus Statement (small animal). Journal
of Veterinary Internal Medicine 19, 377–385.
Luo, X.Q., Yang, X., Hu, R., Huang, W.T., Lan, B., Tu, R.X., Liu, J.Y., 2014. Effects of methyl
cantharidimide tablets on urinary protein and enzymes in Beagle dogs. Zhongguo
Zhong Yao Za Zhi 39, 4426–4429.
Mackensen-Haen, S., Bader, R., Grund, K.E., Bohle, A., 1981. Correlations between renal
cortical interstitial fibrosis, atrophy of the proximal tubules and impairment of
the glomerular filtration rate. Clinical Nephrology 15, 167–171.
Maddens, B., Daminet, S., Smets, P., Meyer, E., 2010a. Escherichia coli pyometra induces
transient glomerular and tubular dysfunction in dogs. Journal of Veterinary
Internal Medicine 24, 1263–1270.
Maddens, B., Heiene, R., Smets, P., Svensson, M., Aresu, L., van der Lugt, J., Daminet,
S., Meyer, E., 2011. Evaluation of kidney injury in dogs with pyometra based on
proteinuria, renal histomorphology, and urinary biomarkers. Journal of Veterinary
Internal Medicine 25, 1075–1083.
Maddens, B.E., Daminet, S., Demeyere, K., Demon, D., Smets, P., Meyer, E., 2010b.
Validation of immunoassays for the candidate renal markers C-reactive protein,
immunoglobulin G, thromboxane B2 and retinol binding protein in canine urine.
Veterinary Immunology and Immunopathology 134, 259–264.
Miyagawa, Y., Takemura, N., Hirose, H., 2009. Evaluation of the measurement of serum
cystatin C by an enzyme-linked immunosorbent assay for humans as a marker
of the glomerular filtration rate in dogs. Journal of Veterinary Medical Science
71, 1169–1176.
Mori, K., Lee, H.T., Rapoport, D., Drexler, I.R., Foster, K., Yang, J., Schmidt-Ott, K.M.,
Chen, X., Li, J.Y., Weiss, S., et al., 2005. Endocytic delivery of lipocalin-
siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.
Journal of Clinical Investigation 115, 610–621.
Nabity, M.B., Lees, G.E., Dangott, L.J., Cianciolo, R., Suchodolski, J.S., Steiner, J.M., 2011.
Proteomic analysis of urine from male dogs during early stages of
tubulointerstitial injury in a canine model of progressive glomerular disease.
Veterinary Clinical Pathology 40, 222–236.
Nabity, M.B., Lees, G.E., Cianciolo, R., Boggess, M.M., Steiner, J.M., Suchodolski, J.S.,
2012. Urinary biomarkers of renal disease in dogs with X-linked hereditary
nephropathy. Journal of Veterinary Internal Medicine 26, 282–293.
Nabity, M.B., Lees, G.E., Boggess, M.M., Yerramilli, M., Obare, E., Yerramilli, M.,
Rakitin, A., Aguiar, J., Relford, R., 2015. Symmetric dimethylarginine assay
validation, stability, and evaluation as a marker for the early detection of
chronic kidney disease in dogs. Journal of Veterinary Internal Medicine 29,
1036–1044.
Nangaku, M., Couser, W.G., 2005. Mechanisms of immune-deposit formation and
the mediation of immune renal injury. Clinical and Experimental Nephrology
9, 183–191.
Nangaku, M., Shankland, S.J., Couser, W.G., 2005. Cellular response to injury in
membranous nephropathy. Journal of the American Society of Nephrology 16,
1195–1204.
Navar, L.G., 2009. Glomerular permeability: A never-ending saga. American Journal
of Physiology 296, F1266–F1268.
Palviainen, M., Raekallio, M., Vainionpaa, M., Lahtinen, H., Vainio, O., 2013. Evaluation
of renal impairment in dogs after envenomation by the common European adder
(Vipera berus berus). The Veterinary Journal 198, 723–724.
Proverbio, D., Spada, E., Baggiani, L., Bagnagatti De Giorgi, G., Ferro, E., Martino, P.A.,
Perego, R., 2015. Short communication: Relationship between urinary neutrophil
gelatinase-associated lipocalin and noninfectious pyuria in dogs. Disease Markers
2015, 387825.
Raila, J., Forterre, S., Kohn, B., Brunnberg, L., Schweigert, F.J., 2003a. Effects of chronic
renal disease on the transport of vitamin A in plasma and urine of dogs. American
Journal of Veterinary Research 64, 874–879.
Raila, J., Neumann, U., Schweigert, F.J., 2003b. Immunochemical localization of
megalin, retinol-binding protein and Tamm-Horsfall glycoprotein in the kidneys
of dogs. Veterinary Research Communications 27, 125–135.
Raila, J., Brunnberg, L., Schweigert, F.J., Kohn, B., 2010. Influence of kidney function
on urinary excretion of albumin and retinol-binding protein in dogs with naturally
occurring renal disease. American Journal of Veterinary Research 71,
1387–1394.
Raila, J., Schweigert, F.J., Kohn, B., 2014. Relationship between urinary Tamm-Horsfall
protein excretion and renal function in dogs with naturally occurring renal
disease. Veterinary Clinical Pathology 43, 261–265.
Reusch, C., Vochezer, R., Weschta, E., 1991. Enzyme activities of urinary alanine
aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) in healthy
dogs. Zentralblatt fur Veterinarmedizin. Reihe A 38, 90–98.
Rivers, B.J., Walter, P.A., O’Brien, T.D., King, V.L., Polzin, D.J., 1996. Evaluation of urine
gamma-glutamyl transpeptidase-to-creatinine ratio as a diagnostic tool in an
experimental model of aminoglycoside-induced acute renal failure in the dog.
Journal of the American Animal Hospital Association 32, 323–336.
Salmon, A.H., Satchell, S.C., 2012. Endothelial glycocalyx dysfunction in disease:
Albuminuria and increased microvascular permeability. The Journal of Pathology
226, 562–574.
 R. Cianciolo et al. / The Veterinary Journal 215 (2016) 21–29
Salmon, A.H., Ferguson, J.K., Burford, J.L., Gevorgyan, H., Nakano, D., Harper, S.J., Bates,
D.O., Peti-Peterdi, J., 2012. Loss of the endothelial glycocalyx links albuminuria
and vascular dysfunction. Journal of the American Society of Nephrology 23,
1339–1350.
Sasaki, A., Sasaki, Y., Iwama, R., Shimamura, S., Yabe, K., Takasuna, K., Ichijo, T.,
Furuhama, K., Satoh, H., 2014. Comparison of renal biomarkers with glomerular
filtration rate in susceptibility to the detection of gentamicin-induced acute
kidney injury in dogs. Journal of Comparative Pathology 151, 264–270.
Sato, R., Soeta, S., Miyazaki, M., Syuto, B., Sato, J., Miyake, Y., Yasuda, J., Okada, K.,
Naito, Y., 2002. Clinical availability of urinary N-acetyl-beta-D-glucosaminidase
index in dogs with urinary diseases. Journal of Veterinary Medical Science 64,
361–365.
Schneider, S.M., Cianciolo, R.E., Nabity, M.B., Clubb, F.J., Jr., Brown, C.A., Lees, G.E.,
2013. Prevalence of immune-complex glomerulonephritides in dogs biopsied
for suspected glomerular disease: 501 cases (2007–2012). Journal of Veterinary
Internal Medicine 27 (Suppl. 1), 67–75.
Schultze, A.E., Jensen, R.K., 1998. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of canine urinary proteins for the analysis and differentiation
of tubular and glomerular diseases. Veterinary Clinical Pathology 18, 93–97.
Segev, G., Palm, C., LeRoy, B., Cowgill, L.D., Westropp, J.L., 2013. Evaluation of
neutrophil gelatinase-associated lipocalin as a marker of kidney injury in dogs.
Journal of Veterinary Internal Medicine 27, 1362–1367.
Smets, P.M., Meyer, E., Maddens, B.E., 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.
Steinbach, S., Weis, J., Schweighauser, A., Francey, T., Neiger, R., 2014. Plasma and
urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney
injury or chronic kidney disease. Journal of Veterinary Internal Medicine 28,
264–269.
Tanner, G.A., 2009. Glomerular sieving coefficient of serum albumin in the rat: A
two-photon microscopy study. American Journal of Physiology 296, F1258–F1265.
29
Tsai, K.L., Clark, L.A., Murphy, K.E., 2007. Understanding hereditary diseases using
the dog and human as companion model systems. Mammalian Genome 18,
444–451.
Ulleberg, T., Robben, J., Nordahl, K.M., Ulleberg, T., Heiene, R., 2011. Plasma creatinine
in dogs: Intra- and inter-laboratory variation in 10 European veterinary
laboratories. Acta Veterinaria Scandinavica 53, 25.
Walker, P.D., 2009. The renal biopsy. Archives of Pathology and Laboratory Medicine
133, 181–188.
Wehner, A., Hartmann, K., Hirschberger, J., 2008. Utility of serum cystatin C as a
clinical measure of renal function in dogs. Journal of the American Animal
Hospital Association 44, 131–138.
Wharram, B.L., Goyal, M., Wiggins, J.E., Sanden, S.K., Hussain, S., Filipiak, W.E.,
Saunders, T.L., Dysko, R.C., Kohno, K., Holzman, L.B., et al., 2005. Podocyte
depletion causes glomerulosclerosis: Diphtheria toxin-induced podocyte
depletion in rats expressing human diphtheria toxin receptor transgene. Journal
of the American Society of Nephrology 16, 2941–2952.
Zaragoza, C., Barrera, R., Centeno, F., Tapia, J.A., Duran, E., Gonzalez, M., Mane, M.C.,
2003a. SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis.
Veterinary Research 34, 137–151.
Zaragoza, C., Barrera, R., Centeno, F., Tapia, J.A., Mane, M.C., 2003b.
Characterization of renal damage in canine leptospirosis by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blotting of the urinary proteins. Journal of Comparative Pathology 129,
169–178.
Zhou, X., Ma, B., Lin, Z., Qu, Z., Huo, Y., Wang, J., Li, B., 2014. Evaluation of the
usefulness of novel biomarkers for drug-induced acute kidney injury in beagle
dogs. Toxicology and Applied Pharmacology 280, 30–35.
Zini, E., Bonfanti, U., Zatelli, A., 2004. Diagnostic relevance of qualitative proteinuria
evaluated by use of sodium dodecyl sulfate-agarose gel electrophoresis and
comparison with renal histologic findings in dogs. American Journal of Veterinary
Research 65, 964–971.