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Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts
Research in Veterinary Science 88 (2010) 294–299
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts A. Yabuki a,*, S. Mitani a, M. Fujiki b, K. Misumi b, Y. Endo c, N. Miyoshi d, O. Yamato a a
Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Kagoshima University, Japan Laboratory of Veterinary Surgery, Department of Veterinary Medicine, Kagoshima University, Japan c Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Kagoshima University, Japan d Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Kagoshima University, Japan b
a r t i c l e
i n f o
Article history: Accepted 14 September 2009
Keywords: Cats Chronic kidney disease Dogs Myofibroblasts
a b s t r a c t We investigated the kidneys of dogs and cats to clarify whether renal myofibroblasts induction is associated with the severity of chronic kidney disease (CKD). Immunohistochemical expression of myofibroblast markers, a-smooth muscle actin (SMA) and vimentin, were evaluated quantitatively. The degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration, and interstitial fibrosis were also evaluated quantitatively. The plasma creatinine (pCre) concentrations correlated with glomerulosclerosis, cell infiltration, and fibrosis in dogs, and only with fibrosis in cats. The a-SMA expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, and with pCre and fibrosis in cats. Tubular vimentin expression correlated with fibrosis in cats, but not in dogs. Interstitial vimentin expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, but only with pCre in cats. In conclusion, it was suggested that the severity of CKD in dogs and cats was mediated by different pathways associated with myofibroblasts expression. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Chronic kidney disease (CKD) is one of the critical concerns in small animal as well as human medicine. The etiology of CKD is heterogeneous, and numerous causes of CKD have been identified. For example, immunologic disorders, amyloidosis, neoplasia, inflammation, infection, and urinary outflow obstruction have been recognized as possible causes for induction of CKD in dogs and cats (Grauer, 2009). These disorders induce the decrease of the number of functional nephrons via pathophysiological changes of glomeruli, tubules, vessels, or interstitium. Previous studies have focused on the pathological changes in glomeruli of dogs and cats as well as humans (Minkus et al., 1994; Scaglione et al., 2008; Slauson and Lewis, 1979), and the pathological changes of glomeruli are classified according to their histopathological or cytopathological features. Tubulointerstitial damage (TID), and not glomerulopathy, is known to play a critical role in the final progression of CKD to an end-stage renal disease; the prevalence of TID is independent of the primary insults (Nangaku, 2002, 2004). TID is induced by massive proteinuria or chronic hypoxia, and worsening of TID induces a decrease in glomerular filtration rate (GFR) via impair* Corresponding author. Tel./fax: +81 99 285 3561. E-mail address: [email protected] (A. Yabuki). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.09.003
ment of the tubuloglomerular feedback system and obliteration of the post-glomerular capillaries, eventually leading to renal failure (Nangaku, 2002, 2004). Pathological changes involved in TID include mononuclear cell infiltration, tubular injury, and interstitial fibrosis (Kelly and Neilson, 1996). Detailed studies using experimental animal models have elucidated that the complex mechanism underlying TID involves the induction and proliferation of myofibroblasts (Bascands and Schanstra, 2005; Zoja et al., 2006). These myofibroblasts are responsible for the generation of extracellular matrix (ECM), the overproduction of which can lead to renal sclerosis. These cells possess the characteristics of both fibroblasts and smooth muscle cells, and are closely involved in not only wound contraction and healing but also in tissue fibrosis of various organs, including the kidney (Gabbiani, 1992). Morphologically, myofibroblasts are extremely variable and heterogeneous in contrast to the normal quiescent fibroblast, and typical myofibroblasts contain abundant and inflated rough endoplasmatic reticulum (Kaissling and Le Hir, 2008). Immunohistochemistry is the most powerful tool for the identification of myofibroblasts. a-Smooth muscle actin (SMA) has been used as a reliable marker for identifying myofibroblasts (Kaissling and Le Hir, 2008), and the intermediate filament protein vimentin has also been used as a useful marker of these cells. These two proteins are not expressed in the normal fibroblasts in the kidney (Kaissling and Le Hir, 2008).
A. Yabuki et al. / Research in Veterinary Science 88 (2010) 294–299
The expression of renal myofibroblasts and their apparent role in the TID has been demonstrated in various types of human nephropathies (Danilewicz et al., 1999; Goumenos et al., 2001; Hewitson and Becker, 1995). In small animals, although a few reports suggest the pathological importance of renal myofibroblasts in the progression of CKD (Ide et al., 2001; Sawashima et al., 2000), it is not clear whether the underlying mechanism is different in dogs and cats. To clarify this, we performed histopathological and immunohistochemical analysis of the kidneys of dogs and cats with various stages of CKD and compared the pathological mechanisms in each species, particularly, the relationship between glomerular damage, TID, and myofibroblast expression. 2. Materials and methods 2.1. Samples The kidney samples of 14 dogs and 10 cats were obtained between 2002 and 2006. Kidney samples from all cats and 11 dogs were collected during postmortem examination performed in Kagoshima University, Japan. The remaining 3 samples were obtained from clinically healthy Beagles that were euthanized after being used in other surgical experiments. The experiments in this study were performed in accordance with the Guidelines for Animal Experimentation of Kagoshima University, Japan. The age of the dogs was approximately in the range of 2–20 years and that of the cats was in the range of 6 months–17 years. The median values of the plasma creatinine concentrations (pCre) were 1.15 (interquartile range: 0.6, 3.4) mg/dl in dogs and 1.5 (interquartile range: 1.1, 4.6) mg/dl in cats. According to the IRIS (International Renal Interest Society) staging system on the basis of pCre (Grauer, 2009), 4 dogs and 4 cats were of stage 1 (<1.4 mg/dl in dog, <1.6 mg/dl in cat), 1 dog and 2 cats were of stage 2 (1.4–2.0 mg/ dl in dog, 1.6–2.8 mg/dl in cats), 5 dogs and 1 cat were of stage 3 (2.1–5.0 mg/dl in dog, 2.9–5.0 mg/dl in cat), and 1 dog and 2 cats were of stage 4 (>5.0 mg/dl in dog and cat). No renal injuries were detected in the remaining 3 dogs (healthy beagles) and 1cat (6month-old cat with feline infectious peritonitis). No cases of acute renal failure were included in the present study. 2.2. Tissue preparation The collected kidney samples were fixed in 10% neutral buffered formalin. After the samples were thoroughly washed in 0.1 M phosphate buffer (pH 7.4), they were embedded in paraffin according to the standard procedure. The samples were cut into 3 lmthick sections, and subsequently treated with hematoxylin-eosin, periodic-acid Schiff, periodic-acid methenamine-silver, and Masson’s trichrome stains, and analyzed by immunohistochemistry. Immunohistochemical procedure involved the following steps: (1) deparaffinization and rehydration, (2) antigen retrieval by heating the sample in a 10 mM citrate buffer (pH 6.0) in a microwave under the following conditions: pre-warming, 5 min; heating, 10 min; and cooling, 20 min, (3) treatment with 3% H2O2 in distilled water for 30 min, (4) washing in 10 mM phosphate-buffered saline (PBS; pH 7.4), (5) blocking with 0.25% casein in PBS for 60 min, (6) overnight incubation at 4 °C with rabbit anti-a-SMA polyclonal antibody (NeoMarkers, Fremont, CA, USA) diluted at 1:100 or mouse anti-vimentin monoclonal antibody (clone V9, NeoMarkers) diluted at 1:200, (7) washing in PBS, (8) incubation for 30 min with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) or horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted at 1:200, (9) washing in PBS, (10) incubation for 15 min with peroxidase-conjugated streptavidin (DakoCytomation, Glostrup, Denmark), (11) washing in PBS,
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(12) detection of immunoreactivity using a 3,30 -diaminobenzidine (DAB) system (DAB Buffer tablet; MERCK, Darmstadt, Germany), and (13) termination of the reaction with distilled water. The sections were counterstained using Mayer’s hematoxylin. For negative control sections, non-immunized rabbit IgG (DakoCytomation) and PBS were used instead of primary antibody. 2.3. Quantitative analysis Randomized quantitative analysis was performed according to the previously established methods. Sections stained with periodic-acid Schiff or Masson’s trichrome were analyzed as follows. (1) The diameter of the renal corpuscles: renal corpuscles with the vascular pole or the urinary pole were selected, and the widest distance of the glomerular capsule was measured (Yabuki et al., 1999). (2) The extent of glomerulosclerosis was evaluated using a semiquantitative scoring system described previously (Raij et al., 1984). Briefly, we examined approximately 100 glomeruli per animal, and graded the severity of the sclerotic lesions from 0 to +4, where 0 = normal, +1 = up to 25% sclerosis, +2 = 26–50% sclerosis, +3 = 51–75% sclerosis, and +4 = 76–100% sclerosis. The glomerulosclerosis score for each animal was then evaluated in the following manner: if among the total 100 glomeruli, 10 were of grade +1, 20 were of grade +2, 15 were of grade +3, and 5 were of grade +4, the final score was calculated as {(1 10/100) + (2 20/100) + (3 15/100) + (4 5/100)} 100 = 115. (3) The degree of interstitial fibrosis and cell infiltration, and the number of a-SMA-positive cells and vimentin-positive cells were also evaluated using a similar semiquantitative scoring system. Briefly, we examined approximately 20 non-overlapping cortical fields (magnification 200) per animal, and graded the severity of each parameter from 0 to +4: 0 = normal, +1 = mild, +2 = moderate, +3 = severe, and +4 = very severe. The score of each parameter was then determined for each animal. For example, in the case of interstitial fibrosis, if out of the 20 fields examined, 5 exhibited grade + 1 changes; 3, grade + 2; 2, grade + 3; and 1, grade + 4, the final score of interstitial fibrosis was calculated as: {(1 5/20) + (2 3/20) + (3 2/20) + (4 1/20)} 100 = 105. The correlation coefficients and their P-values between all these parameters were evaluated using the JMP 5.1 software program for Windows (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Histopathological findings The histopathological findings of glomeruli and the degree of the glomerular damage were different between cases. Expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM) were clearly observed in the kidneys of most dogs and cats (Fig. 1). The proliferation of mesangial and endothelial cells, and formation of spikes or bubbling in the GBM were observed in some cases. The grades of TID varied in most of the kidney samples (Fig. 1). The histopathological characteristics common to both dogs and cats were interstitial mononuclear cell infiltration (predominantly, macrophages and lymphoplasma cells), tubular atrophy or dilation, and interstitial fibrosis. 3.2. Immunohistochemical findings The interstitial cells were predominantly positive for a-SMA (Fig. 2A and B). The glomeruli of all samples were negative for aSMA. The cells of the segments from the proximal tubules to the collecting ducts, and mononuclear cells infiltrating the interstitium were also negative for a-SMA. The vascular smooth muscle cells, which were known as a-SMA-positive cells in normal tissue,
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However, the segments that exhibited such signals were different between cases. Moreover, the infiltrating mononuclear cells also exhibited vimentin-positive signals. Glomerular epithelial cells (podocytes), which were known as vimentin-positive cells in normal kidney, showed positive signals in all cases. Other glomerular components were found to be negative for vimentin. The positive signals in the infiltrated cells were clearly stronger in dogs than in cats. No immuno-positive signals were detected in any animals when non-immunized IgG and PBS were used instead of primary antibody. 3.3. Quantitative analysis
Fig. 1. Light micrograph of a typical renal lesion from a dog with stage-4 CKD. The glomeruli show sclerotic changes. Tubulointerstitial damage is severe, as indicated by tubular atrophy or dilation, interstitial mononuclear cell infiltration, and interstitial fibrosis. Periodic-acid Schiff stain. Bar: 150 lm.
showed positive signals in all cases. Thus, no apparent species-specific differences were observed in a-SMA immunohistochemistry. The interstitial cells were also observed to be predominantly positive for vimentin (Fig. 2B and C). In addition, tubular epithelial cells of various segments exhibited vimentin-positive signals.
We analyzed the correlation between pCre and each histopathological parameter (Fig. 3). In the case of dogs, pCre was found to be significantly correlated with the scores of interstitial fibrosis, mononuclear cell infiltration, and glomerulosclerosis. In the case of cats, the pCre was significantly correlated with only the interstitial fibrosis scores. The remaining parameters in dogs and cats did not correlate significantly with pCre. The a-SMA expression level was found to be significantly correlated with pCre, and with mononuclear cell infiltration, glomerulosclerosis, and interstitial fibrosis in the case of dogs (Table 1). In the case of cats, the a-SMA expression level was found to be signifi-
Fig. 2. Immunohistochemistry for the detection of a-SMA and vimentin. A and C: dog kidneys. B and D: cat kidneys. A and B: a-SMA. Interstitial cells were positive for a-SMA in both cases. The glomeruli, tubular epithelium, and infiltrated cells were negative for a-SMA. C and D: vimentin. The interstitial cells, tubular epithelium, and infiltrated mononuclear cells are positive for vimentin. The signals in infiltrated mononuclear cells of dogs are more remarkable than those of cats. Glomerular epithelial cells are used as internal positive control cells for vimentin. Counter stain: Mayer’s hematoxylin. Bar: 100 lm.
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Fig. 3. Correlations between pCre and each of the histopathological parameters. A–D: sections from dogs. E–F: sections from cats. A and E: pCre vs. glomerulosclerosis. B and F: pCre vs. interstitial mononuclear cell infiltration. C and G: pCre vs. interstitial fibrosis. D and H: pCre vs. renal corpuscular diameter.
cantly correlated with pCre and interstitial fibrosis. In the case of both dogs and cats, the remaining parameters did not correlate significantly with a-SMA expression. The vimentin expression levels in tubules and interstitium were evaluated separately (Table 1). The tubular vimentin expression level was found to be significantly correlated with the interstitial fibrosis scores in the case of cats, but not with other parameters in the case of both dogs and cats. The interstitial vimentin expression level in dogs and cats was significantly correlated with pCre. The interstitial expression of vimentin in the case of dogs was found to be significantly correlated with interstitial fibrosis, glomerulosclerosis, and mononuclear infiltration; however, the expression of this marker in cats did not correlate significantly with any of the histopathological parameters evaluated. 4. Discussion The findings of the present comparative study suggest that the pathogenesis of CKD is different in dogs and cats. In veterinary medicine, pCre is considered to be the most reliable biochemical marker in blood for evaluating renal function, and canine and feline CKD is now evaluated on the basis of pCre (Grauer, 2009). Although pCre reflects the change of GFR, the present study demonstrated that pCre correlated significantly with the degree of interstitial fibrosis in the case of both dogs and cats. Interstitial fibrosis is a typical feature of TID. With respect to humans, the rate of deterioration of renal function is now widely accepted to be strongly correlated with the degree of TID (Nangaku 2002). TID in-
duces a decrease in GFR via several mechanisms, and eventually leads to an ischemic renal injury and a decrease in the number of functional nephrons. Since the significance of pCre as a prognostic factor of feline CKD has been demonstrated recently (King et al., 2007; Jepson et al., 2009), the findings of this study strongly suggest that interstitial fibrosis is closely related to the severity of feline CKD. Similar relation might be presented in canine CKD. Furthermore a correlation was also observed between the degree of glomerulosclerosis and pCre in the case of dogs; therefore, a reduction in GFR as a direct impact of structural loss of glomerular capillaries was thought to be a possible mechanism involved in the progression of CKD in the case of dogs. These findings statistically supported the previous histopathological surveys of kidney disease of dogs and cats (Macdougall et al., 1986; DiBartola et al., 1987). In these previous surveys, glomerular disease and TID were shown as common histopathological features in dogs and cats, respectively. Myofibroblasts are ECM-producing cells and share the characteristics of both fibroblasts and smooth muscle cells. These unique cells play an important role in the pathogenesis of tissue fibrosis and scarring (Gabbiani, 1992). Under an electron microscope, myofibroblasts are visualized as large satellite cells with heterogeneous morphological characteristics (Kaissling and Le Hir, 2008); however, conventional light microscopy cannot be used to distinguish myofibroblasts from quiescent fibroblasts. These limitations deem it necessary to use marker proteins for the identification of myofibroblasts. a-SMA has been used as the most reliable immunohistochemical marker of myofibroblasts (Kaissling and Le Hir, 2008; Muchaneta-Kubara and el Nahas, 1997). Hence, we used this mar-
Table 1 Correlations between immunohistochemical parameters and histopathological parameters. Dogs
pCre Glomerulosclerosis Cell infiltration Interstitial fibrosis Diameter of RC
Cats
a-SMA
t-Vimentin
i-Vimentin
a-SMA
t-Vimentin
i-Vimentin
0.65* 0.59* 0.72** 0.58** NS
NS NS NS NS NS
0.83** 0.74** 0.79** 0.87** NS
0.88** NS NS 0.72* NS
NS NS NS 0.70* NS
0.84** NS NS NS NS
Values represent the correlation coefficient. t-Vimentin: tubular vimentin, i-vimentin: interstitial vimentin. P < 0.05. ** P < 0.01. RC: renal corpuscles. NS: not significant. *
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ker for investigating the expression of myofibroblasts in the kidneys of dogs and cats. The interstitial cells of the damaged kidneys were positive for a-SMA. These positive cells were believed to be myofibroblasts, since previous reports had suggested that a-SMA is a good marker of renal myofibroblasts in dogs and cats (Ide et al., 2001; Sawashima et al., 2000). It is believed that a-SMA-positive myofibroblasts play a critical role in the progression of renal fibrosis in humans (Gabbiani, 1992). A previous report in cats has suggested that the levels of interstitial a-SMA expression and pCre are significantly correlated, and that a-SMA would be a useful histological marker for severity of feline CKD (Sawashima et al., 2000). In the present study, we demonstrated that a-SMA can be used as a histological marker for severity of CKD in not only cats but also dogs. The a-SMA-positive cells were detected in the interstitium; thus, the expected association between a-SMA expression and interstitial fibrosis was clearly established by quantitative analysis. Tubular expression of vimentin was demonstrated previously in dogs with glomerulonephritis; vimentin-positive signals were considered to be indicative of epithelial–mesenchymal transition (EMT) in the renal tubules (Aresu et al., 2007). In addition, expression of vimentin in the damaged and regenerating tubular cells of was demonstrated in rats with chronic progressive nephropathy (Nakatsuji et al., 1998). In the present study, we found that the level of tubular vimentin expression correlated significantly with the degree of interstitial fibrosis in the case of cats, but no such correlation was observed in the case of dogs. Tubular EMT is a process in which renal tubular cells lose their epithelial phenotype and acquire new characteristic features of mesenchymal cells. It is an important event in the progression of renal fibrosis and is being recently recognized as a novel therapeutic target for CKD (Liu, 2004; Yang et al., 2005). Our findings suggested that tubular EMT plays an important role in the progression of renal fibrosis in cats, and therefore, the suppression of EMT might be a possible therapeutic approach for feline CKD. The usefulness of vimentin as a marker for myofibroblasts is widely accepted (Kaissling and Le Hir, 2008; Muchaneta-Kubara and el Nahas, 1997). In our study, the level of interstitial vimentin expression was found to be significantly correlated to pCre levels as expected; further, the level of a-SMA expression was also correlated to pCre levels. In the case of dogs, the degrees of glomerulosclerosis, interstitial fibrosis, and mononuclear cell infiltration correlated with the level of interstitial vimentin expression, and we speculated that these findings may be attributed to the source of renal myofibroblasts. In the present immunohistochemical study, vimentin-positive signals were detected not only in the interstitial cells but also in the tubular epithelial and the infiltrated mononuclear cells, and therefore vimentin was a marker of myofibroblasts as well as a marker of progenitor cells that transform into myofibroblasts. The origin of myofibroblasts in the kidney has been studied extensively and tubular EMT, transformation of resident fibroblasts, and infiltration of bone marrow derived precursor cells have been considered as the sources of renal myofibroblasts (Li et al., 2007; Picard et al., 2008). In addition, a recent study that used a mouse model revealed that pericytes were also an important source of renal myofibroblasts (Lin et al., 2008). Although the immunohistochemical findings of this study could not clearly classify the type of vimentin-positive cells, the resident fibroblasts, bone marrow derived precursors, and pericytes were considered as other possible precursors of renal myofibroblasts in canine CKD. Even though no correlation was observed between the level of interstitial vimentin expression and the histopathological parameters in the case of cats, tubular vimentin expression was correlated significantly with interstitial fibrosis. Therefore, tubular EMT was considered as a possible source of renal myofibroblasts in the feline CKD.
Involvement of myofibroblasts in the pathogenesis of TID was well investigated using laboratory rodents and reviewed previously (Liu, 2004; Bascands and Schanstra, 2005). Myofibroblast play an important role for progression of TID as the ECM-producing cells, and induction and proliferation of myofibroblast are stimulated by growth factors/cytokines such as transforming growth factor-b. Although the present study demonstrated the expression of myofibroblast in the kidneys of dogs and cats, detail pathological roles of renal myofibroblast remains unclear in canine and feline nephropathy. Further studies targeting ECM-production and growth factors/cytokines-mediation in canine and feline nephropathy are required. In conclusion, the present comparative study demonstrated that pathological mechanisms underlying the severity of CKD are different in dogs and cats. The severity of CKD in dogs appeared to be mediated by both glomerulosclerosis and TID, whereas in cats it was mediated by TID predominantly, particularly by interstitial fibrosis. a-SMA-positive myofibroblasts were detected in the kidneys of both dogs and cats. Further, vimentin immunohistochemistry suggested that the precursors of renal myofibroblasts were different for dogs and cats.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Comparative study of chronic kidney disease in dogs and cats: Induction of myofibroblasts A. Yabuki a,*, S. Mitani a, M. Fujiki b, K. Misumi b, Y. Endo c, N. Miyoshi d, O. Yamato a a
Laboratory of Veterinary Clinical Pathology, Department of Veterinary Medicine, Kagoshima University, Japan Laboratory of Veterinary Surgery, Department of Veterinary Medicine, Kagoshima University, Japan c Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Kagoshima University, Japan d Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Kagoshima University, Japan b
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Article history: Accepted 14 September 2009
Keywords: Cats Chronic kidney disease Dogs Myofibroblasts
a b s t r a c t We investigated the kidneys of dogs and cats to clarify whether renal myofibroblasts induction is associated with the severity of chronic kidney disease (CKD). Immunohistochemical expression of myofibroblast markers, a-smooth muscle actin (SMA) and vimentin, were evaluated quantitatively. The degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration, and interstitial fibrosis were also evaluated quantitatively. The plasma creatinine (pCre) concentrations correlated with glomerulosclerosis, cell infiltration, and fibrosis in dogs, and only with fibrosis in cats. The a-SMA expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, and with pCre and fibrosis in cats. Tubular vimentin expression correlated with fibrosis in cats, but not in dogs. Interstitial vimentin expression correlated with pCre, glomerulosclerosis, cell infiltration, and fibrosis in dogs, but only with pCre in cats. In conclusion, it was suggested that the severity of CKD in dogs and cats was mediated by different pathways associated with myofibroblasts expression. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Chronic kidney disease (CKD) is one of the critical concerns in small animal as well as human medicine. The etiology of CKD is heterogeneous, and numerous causes of CKD have been identified. For example, immunologic disorders, amyloidosis, neoplasia, inflammation, infection, and urinary outflow obstruction have been recognized as possible causes for induction of CKD in dogs and cats (Grauer, 2009). These disorders induce the decrease of the number of functional nephrons via pathophysiological changes of glomeruli, tubules, vessels, or interstitium. Previous studies have focused on the pathological changes in glomeruli of dogs and cats as well as humans (Minkus et al., 1994; Scaglione et al., 2008; Slauson and Lewis, 1979), and the pathological changes of glomeruli are classified according to their histopathological or cytopathological features. Tubulointerstitial damage (TID), and not glomerulopathy, is known to play a critical role in the final progression of CKD to an end-stage renal disease; the prevalence of TID is independent of the primary insults (Nangaku, 2002, 2004). TID is induced by massive proteinuria or chronic hypoxia, and worsening of TID induces a decrease in glomerular filtration rate (GFR) via impair* Corresponding author. Tel./fax: +81 99 285 3561. E-mail address: [email protected] (A. Yabuki). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.09.003
ment of the tubuloglomerular feedback system and obliteration of the post-glomerular capillaries, eventually leading to renal failure (Nangaku, 2002, 2004). Pathological changes involved in TID include mononuclear cell infiltration, tubular injury, and interstitial fibrosis (Kelly and Neilson, 1996). Detailed studies using experimental animal models have elucidated that the complex mechanism underlying TID involves the induction and proliferation of myofibroblasts (Bascands and Schanstra, 2005; Zoja et al., 2006). These myofibroblasts are responsible for the generation of extracellular matrix (ECM), the overproduction of which can lead to renal sclerosis. These cells possess the characteristics of both fibroblasts and smooth muscle cells, and are closely involved in not only wound contraction and healing but also in tissue fibrosis of various organs, including the kidney (Gabbiani, 1992). Morphologically, myofibroblasts are extremely variable and heterogeneous in contrast to the normal quiescent fibroblast, and typical myofibroblasts contain abundant and inflated rough endoplasmatic reticulum (Kaissling and Le Hir, 2008). Immunohistochemistry is the most powerful tool for the identification of myofibroblasts. a-Smooth muscle actin (SMA) has been used as a reliable marker for identifying myofibroblasts (Kaissling and Le Hir, 2008), and the intermediate filament protein vimentin has also been used as a useful marker of these cells. These two proteins are not expressed in the normal fibroblasts in the kidney (Kaissling and Le Hir, 2008).
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The expression of renal myofibroblasts and their apparent role in the TID has been demonstrated in various types of human nephropathies (Danilewicz et al., 1999; Goumenos et al., 2001; Hewitson and Becker, 1995). In small animals, although a few reports suggest the pathological importance of renal myofibroblasts in the progression of CKD (Ide et al., 2001; Sawashima et al., 2000), it is not clear whether the underlying mechanism is different in dogs and cats. To clarify this, we performed histopathological and immunohistochemical analysis of the kidneys of dogs and cats with various stages of CKD and compared the pathological mechanisms in each species, particularly, the relationship between glomerular damage, TID, and myofibroblast expression. 2. Materials and methods 2.1. Samples The kidney samples of 14 dogs and 10 cats were obtained between 2002 and 2006. Kidney samples from all cats and 11 dogs were collected during postmortem examination performed in Kagoshima University, Japan. The remaining 3 samples were obtained from clinically healthy Beagles that were euthanized after being used in other surgical experiments. The experiments in this study were performed in accordance with the Guidelines for Animal Experimentation of Kagoshima University, Japan. The age of the dogs was approximately in the range of 2–20 years and that of the cats was in the range of 6 months–17 years. The median values of the plasma creatinine concentrations (pCre) were 1.15 (interquartile range: 0.6, 3.4) mg/dl in dogs and 1.5 (interquartile range: 1.1, 4.6) mg/dl in cats. According to the IRIS (International Renal Interest Society) staging system on the basis of pCre (Grauer, 2009), 4 dogs and 4 cats were of stage 1 (<1.4 mg/dl in dog, <1.6 mg/dl in cat), 1 dog and 2 cats were of stage 2 (1.4–2.0 mg/ dl in dog, 1.6–2.8 mg/dl in cats), 5 dogs and 1 cat were of stage 3 (2.1–5.0 mg/dl in dog, 2.9–5.0 mg/dl in cat), and 1 dog and 2 cats were of stage 4 (>5.0 mg/dl in dog and cat). No renal injuries were detected in the remaining 3 dogs (healthy beagles) and 1cat (6month-old cat with feline infectious peritonitis). No cases of acute renal failure were included in the present study. 2.2. Tissue preparation The collected kidney samples were fixed in 10% neutral buffered formalin. After the samples were thoroughly washed in 0.1 M phosphate buffer (pH 7.4), they were embedded in paraffin according to the standard procedure. The samples were cut into 3 lmthick sections, and subsequently treated with hematoxylin-eosin, periodic-acid Schiff, periodic-acid methenamine-silver, and Masson’s trichrome stains, and analyzed by immunohistochemistry. Immunohistochemical procedure involved the following steps: (1) deparaffinization and rehydration, (2) antigen retrieval by heating the sample in a 10 mM citrate buffer (pH 6.0) in a microwave under the following conditions: pre-warming, 5 min; heating, 10 min; and cooling, 20 min, (3) treatment with 3% H2O2 in distilled water for 30 min, (4) washing in 10 mM phosphate-buffered saline (PBS; pH 7.4), (5) blocking with 0.25% casein in PBS for 60 min, (6) overnight incubation at 4 °C with rabbit anti-a-SMA polyclonal antibody (NeoMarkers, Fremont, CA, USA) diluted at 1:100 or mouse anti-vimentin monoclonal antibody (clone V9, NeoMarkers) diluted at 1:200, (7) washing in PBS, (8) incubation for 30 min with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) or horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) diluted at 1:200, (9) washing in PBS, (10) incubation for 15 min with peroxidase-conjugated streptavidin (DakoCytomation, Glostrup, Denmark), (11) washing in PBS,
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(12) detection of immunoreactivity using a 3,30 -diaminobenzidine (DAB) system (DAB Buffer tablet; MERCK, Darmstadt, Germany), and (13) termination of the reaction with distilled water. The sections were counterstained using Mayer’s hematoxylin. For negative control sections, non-immunized rabbit IgG (DakoCytomation) and PBS were used instead of primary antibody. 2.3. Quantitative analysis Randomized quantitative analysis was performed according to the previously established methods. Sections stained with periodic-acid Schiff or Masson’s trichrome were analyzed as follows. (1) The diameter of the renal corpuscles: renal corpuscles with the vascular pole or the urinary pole were selected, and the widest distance of the glomerular capsule was measured (Yabuki et al., 1999). (2) The extent of glomerulosclerosis was evaluated using a semiquantitative scoring system described previously (Raij et al., 1984). Briefly, we examined approximately 100 glomeruli per animal, and graded the severity of the sclerotic lesions from 0 to +4, where 0 = normal, +1 = up to 25% sclerosis, +2 = 26–50% sclerosis, +3 = 51–75% sclerosis, and +4 = 76–100% sclerosis. The glomerulosclerosis score for each animal was then evaluated in the following manner: if among the total 100 glomeruli, 10 were of grade +1, 20 were of grade +2, 15 were of grade +3, and 5 were of grade +4, the final score was calculated as {(1 10/100) + (2 20/100) + (3 15/100) + (4 5/100)} 100 = 115. (3) The degree of interstitial fibrosis and cell infiltration, and the number of a-SMA-positive cells and vimentin-positive cells were also evaluated using a similar semiquantitative scoring system. Briefly, we examined approximately 20 non-overlapping cortical fields (magnification 200) per animal, and graded the severity of each parameter from 0 to +4: 0 = normal, +1 = mild, +2 = moderate, +3 = severe, and +4 = very severe. The score of each parameter was then determined for each animal. For example, in the case of interstitial fibrosis, if out of the 20 fields examined, 5 exhibited grade + 1 changes; 3, grade + 2; 2, grade + 3; and 1, grade + 4, the final score of interstitial fibrosis was calculated as: {(1 5/20) + (2 3/20) + (3 2/20) + (4 1/20)} 100 = 105. The correlation coefficients and their P-values between all these parameters were evaluated using the JMP 5.1 software program for Windows (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Histopathological findings The histopathological findings of glomeruli and the degree of the glomerular damage were different between cases. Expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM) were clearly observed in the kidneys of most dogs and cats (Fig. 1). The proliferation of mesangial and endothelial cells, and formation of spikes or bubbling in the GBM were observed in some cases. The grades of TID varied in most of the kidney samples (Fig. 1). The histopathological characteristics common to both dogs and cats were interstitial mononuclear cell infiltration (predominantly, macrophages and lymphoplasma cells), tubular atrophy or dilation, and interstitial fibrosis. 3.2. Immunohistochemical findings The interstitial cells were predominantly positive for a-SMA (Fig. 2A and B). The glomeruli of all samples were negative for aSMA. The cells of the segments from the proximal tubules to the collecting ducts, and mononuclear cells infiltrating the interstitium were also negative for a-SMA. The vascular smooth muscle cells, which were known as a-SMA-positive cells in normal tissue,
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However, the segments that exhibited such signals were different between cases. Moreover, the infiltrating mononuclear cells also exhibited vimentin-positive signals. Glomerular epithelial cells (podocytes), which were known as vimentin-positive cells in normal kidney, showed positive signals in all cases. Other glomerular components were found to be negative for vimentin. The positive signals in the infiltrated cells were clearly stronger in dogs than in cats. No immuno-positive signals were detected in any animals when non-immunized IgG and PBS were used instead of primary antibody. 3.3. Quantitative analysis
Fig. 1. Light micrograph of a typical renal lesion from a dog with stage-4 CKD. The glomeruli show sclerotic changes. Tubulointerstitial damage is severe, as indicated by tubular atrophy or dilation, interstitial mononuclear cell infiltration, and interstitial fibrosis. Periodic-acid Schiff stain. Bar: 150 lm.
showed positive signals in all cases. Thus, no apparent species-specific differences were observed in a-SMA immunohistochemistry. The interstitial cells were also observed to be predominantly positive for vimentin (Fig. 2B and C). In addition, tubular epithelial cells of various segments exhibited vimentin-positive signals.
We analyzed the correlation between pCre and each histopathological parameter (Fig. 3). In the case of dogs, pCre was found to be significantly correlated with the scores of interstitial fibrosis, mononuclear cell infiltration, and glomerulosclerosis. In the case of cats, the pCre was significantly correlated with only the interstitial fibrosis scores. The remaining parameters in dogs and cats did not correlate significantly with pCre. The a-SMA expression level was found to be significantly correlated with pCre, and with mononuclear cell infiltration, glomerulosclerosis, and interstitial fibrosis in the case of dogs (Table 1). In the case of cats, the a-SMA expression level was found to be signifi-
Fig. 2. Immunohistochemistry for the detection of a-SMA and vimentin. A and C: dog kidneys. B and D: cat kidneys. A and B: a-SMA. Interstitial cells were positive for a-SMA in both cases. The glomeruli, tubular epithelium, and infiltrated cells were negative for a-SMA. C and D: vimentin. The interstitial cells, tubular epithelium, and infiltrated mononuclear cells are positive for vimentin. The signals in infiltrated mononuclear cells of dogs are more remarkable than those of cats. Glomerular epithelial cells are used as internal positive control cells for vimentin. Counter stain: Mayer’s hematoxylin. Bar: 100 lm.
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Fig. 3. Correlations between pCre and each of the histopathological parameters. A–D: sections from dogs. E–F: sections from cats. A and E: pCre vs. glomerulosclerosis. B and F: pCre vs. interstitial mononuclear cell infiltration. C and G: pCre vs. interstitial fibrosis. D and H: pCre vs. renal corpuscular diameter.
cantly correlated with pCre and interstitial fibrosis. In the case of both dogs and cats, the remaining parameters did not correlate significantly with a-SMA expression. The vimentin expression levels in tubules and interstitium were evaluated separately (Table 1). The tubular vimentin expression level was found to be significantly correlated with the interstitial fibrosis scores in the case of cats, but not with other parameters in the case of both dogs and cats. The interstitial vimentin expression level in dogs and cats was significantly correlated with pCre. The interstitial expression of vimentin in the case of dogs was found to be significantly correlated with interstitial fibrosis, glomerulosclerosis, and mononuclear infiltration; however, the expression of this marker in cats did not correlate significantly with any of the histopathological parameters evaluated. 4. Discussion The findings of the present comparative study suggest that the pathogenesis of CKD is different in dogs and cats. In veterinary medicine, pCre is considered to be the most reliable biochemical marker in blood for evaluating renal function, and canine and feline CKD is now evaluated on the basis of pCre (Grauer, 2009). Although pCre reflects the change of GFR, the present study demonstrated that pCre correlated significantly with the degree of interstitial fibrosis in the case of both dogs and cats. Interstitial fibrosis is a typical feature of TID. With respect to humans, the rate of deterioration of renal function is now widely accepted to be strongly correlated with the degree of TID (Nangaku 2002). TID in-
duces a decrease in GFR via several mechanisms, and eventually leads to an ischemic renal injury and a decrease in the number of functional nephrons. Since the significance of pCre as a prognostic factor of feline CKD has been demonstrated recently (King et al., 2007; Jepson et al., 2009), the findings of this study strongly suggest that interstitial fibrosis is closely related to the severity of feline CKD. Similar relation might be presented in canine CKD. Furthermore a correlation was also observed between the degree of glomerulosclerosis and pCre in the case of dogs; therefore, a reduction in GFR as a direct impact of structural loss of glomerular capillaries was thought to be a possible mechanism involved in the progression of CKD in the case of dogs. These findings statistically supported the previous histopathological surveys of kidney disease of dogs and cats (Macdougall et al., 1986; DiBartola et al., 1987). In these previous surveys, glomerular disease and TID were shown as common histopathological features in dogs and cats, respectively. Myofibroblasts are ECM-producing cells and share the characteristics of both fibroblasts and smooth muscle cells. These unique cells play an important role in the pathogenesis of tissue fibrosis and scarring (Gabbiani, 1992). Under an electron microscope, myofibroblasts are visualized as large satellite cells with heterogeneous morphological characteristics (Kaissling and Le Hir, 2008); however, conventional light microscopy cannot be used to distinguish myofibroblasts from quiescent fibroblasts. These limitations deem it necessary to use marker proteins for the identification of myofibroblasts. a-SMA has been used as the most reliable immunohistochemical marker of myofibroblasts (Kaissling and Le Hir, 2008; Muchaneta-Kubara and el Nahas, 1997). Hence, we used this mar-
Table 1 Correlations between immunohistochemical parameters and histopathological parameters. Dogs
pCre Glomerulosclerosis Cell infiltration Interstitial fibrosis Diameter of RC
Cats
a-SMA
t-Vimentin
i-Vimentin
a-SMA
t-Vimentin
i-Vimentin
0.65* 0.59* 0.72** 0.58** NS
NS NS NS NS NS
0.83** 0.74** 0.79** 0.87** NS
0.88** NS NS 0.72* NS
NS NS NS 0.70* NS
0.84** NS NS NS NS
Values represent the correlation coefficient. t-Vimentin: tubular vimentin, i-vimentin: interstitial vimentin. P < 0.05. ** P < 0.01. RC: renal corpuscles. NS: not significant. *
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ker for investigating the expression of myofibroblasts in the kidneys of dogs and cats. The interstitial cells of the damaged kidneys were positive for a-SMA. These positive cells were believed to be myofibroblasts, since previous reports had suggested that a-SMA is a good marker of renal myofibroblasts in dogs and cats (Ide et al., 2001; Sawashima et al., 2000). It is believed that a-SMA-positive myofibroblasts play a critical role in the progression of renal fibrosis in humans (Gabbiani, 1992). A previous report in cats has suggested that the levels of interstitial a-SMA expression and pCre are significantly correlated, and that a-SMA would be a useful histological marker for severity of feline CKD (Sawashima et al., 2000). In the present study, we demonstrated that a-SMA can be used as a histological marker for severity of CKD in not only cats but also dogs. The a-SMA-positive cells were detected in the interstitium; thus, the expected association between a-SMA expression and interstitial fibrosis was clearly established by quantitative analysis. Tubular expression of vimentin was demonstrated previously in dogs with glomerulonephritis; vimentin-positive signals were considered to be indicative of epithelial–mesenchymal transition (EMT) in the renal tubules (Aresu et al., 2007). In addition, expression of vimentin in the damaged and regenerating tubular cells of was demonstrated in rats with chronic progressive nephropathy (Nakatsuji et al., 1998). In the present study, we found that the level of tubular vimentin expression correlated significantly with the degree of interstitial fibrosis in the case of cats, but no such correlation was observed in the case of dogs. Tubular EMT is a process in which renal tubular cells lose their epithelial phenotype and acquire new characteristic features of mesenchymal cells. It is an important event in the progression of renal fibrosis and is being recently recognized as a novel therapeutic target for CKD (Liu, 2004; Yang et al., 2005). Our findings suggested that tubular EMT plays an important role in the progression of renal fibrosis in cats, and therefore, the suppression of EMT might be a possible therapeutic approach for feline CKD. The usefulness of vimentin as a marker for myofibroblasts is widely accepted (Kaissling and Le Hir, 2008; Muchaneta-Kubara and el Nahas, 1997). In our study, the level of interstitial vimentin expression was found to be significantly correlated to pCre levels as expected; further, the level of a-SMA expression was also correlated to pCre levels. In the case of dogs, the degrees of glomerulosclerosis, interstitial fibrosis, and mononuclear cell infiltration correlated with the level of interstitial vimentin expression, and we speculated that these findings may be attributed to the source of renal myofibroblasts. In the present immunohistochemical study, vimentin-positive signals were detected not only in the interstitial cells but also in the tubular epithelial and the infiltrated mononuclear cells, and therefore vimentin was a marker of myofibroblasts as well as a marker of progenitor cells that transform into myofibroblasts. The origin of myofibroblasts in the kidney has been studied extensively and tubular EMT, transformation of resident fibroblasts, and infiltration of bone marrow derived precursor cells have been considered as the sources of renal myofibroblasts (Li et al., 2007; Picard et al., 2008). In addition, a recent study that used a mouse model revealed that pericytes were also an important source of renal myofibroblasts (Lin et al., 2008). Although the immunohistochemical findings of this study could not clearly classify the type of vimentin-positive cells, the resident fibroblasts, bone marrow derived precursors, and pericytes were considered as other possible precursors of renal myofibroblasts in canine CKD. Even though no correlation was observed between the level of interstitial vimentin expression and the histopathological parameters in the case of cats, tubular vimentin expression was correlated significantly with interstitial fibrosis. Therefore, tubular EMT was considered as a possible source of renal myofibroblasts in the feline CKD.
Involvement of myofibroblasts in the pathogenesis of TID was well investigated using laboratory rodents and reviewed previously (Liu, 2004; Bascands and Schanstra, 2005). Myofibroblast play an important role for progression of TID as the ECM-producing cells, and induction and proliferation of myofibroblast are stimulated by growth factors/cytokines such as transforming growth factor-b. Although the present study demonstrated the expression of myofibroblast in the kidneys of dogs and cats, detail pathological roles of renal myofibroblast remains unclear in canine and feline nephropathy. Further studies targeting ECM-production and growth factors/cytokines-mediation in canine and feline nephropathy are required. In conclusion, the present comparative study demonstrated that pathological mechanisms underlying the severity of CKD are different in dogs and cats. The severity of CKD in dogs appeared to be mediated by both glomerulosclerosis and TID, whereas in cats it was mediated by TID predominantly, particularly by interstitial fibrosis. a-SMA-positive myofibroblasts were detected in the kidneys of both dogs and cats. Further, vimentin immunohistochemistry suggested that the precursors of renal myofibroblasts were different for dogs and cats.
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