- Email: [email protected]
Changes in Renal Peritubular Capillaries in Canine and Feline Chronic Kidney Disease
J. Comp. Path. 2018, Vol. 160, 79e83
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
Changes in Renal Peritubular Capillaries in Canine and Feline Chronic Kidney Disease R. Nakamura*, A. Yabuki*, O. Ichii†, H. Mizukawa‡, N. Yokoyamax and O. Yamato* *Laboratory of Veterinary Clinical Pathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, † Laboratory of Anatomy, Department of Basic Veterinary Sciences, ‡ Division of Environmental Veterinary Sciences and x Laboratory of Veterinary Internal Medicine, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan
Summary Renal capillary rarefaction is a crucial event that leads to tubulointerstitial damage during the progression of chronic kidney disease (CKD). In the present study, changes in CD34-positive renal capillaries were investigated in dogs and cats with CKD. A significant decrease in CD34-positive capillaries was observed in canine diseased kidneys, even at the early stage of disease. In cats, CD34-positive capillaries were well preserved in the diseased kidneys, with no link to the severity of CKD. Renal capillary rarefaction might be a trigger event that leads to the progression of CKD in dogs, rather than in cats. Ó 2018 Elsevier Ltd. All rights reserved. Keywords: capillary rarefaction; cat; chronic kidney disease; dog
Chronic kidney disease (CKD) can result from a variety of causes. Despite underlying causal differences, CKD progresses irreversibly to tubulointerstitial damage (TID), which is the final common mechanism that leads to end-stage kidney disease (Nangaku, 2004, 2006). TID is also involved in the progression of CKD in dogs and cats (Yabuki et al., 2010; Chakrabart et al., 2013; Lawson et al., 2015). However, while the progression of CKD is predominantly influenced by TID in cats, it appears to be mediated by both glomerulosclerosis and TID in dogs (Yabuki et al., 2010). Recent studies demonstrated that several factors such as transforming growth factor (TGF)-b, fibroblast growth factor 23 and transglutaminase 2 are involved in the progression of feline CKD (Geddes et al., 2013; SanchezLara et al., 2015; Lawson et al., 2016). Loss of peritubular capillaries (PTCs) in renal tissues, known as renal capillary rarefaction, is a crucial Correspondence to: A. Yabuki (e-mail: [email protected]). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2018.03.004
pathological event in the progression of TID in man (Mayer, 2011). The mechanism of capillary rarefaction is complicated. Pericyte damage and endothelial cell apoptosis play a pivotal role in the loss of renal microvasculature (Kida and Duffield, 2011; Ballermann and Obeidat, 2014; Kida et al., 2014). Many factors such as vascular endothelial growth factor and angiopoietin are also involved in this phenomenon (Woolf et al., 2009; Lin et al., 2011; Kida et al., 2014). A close relationship between renal capillary rarefaction and mononuclear infiltration was also demonstrated in a mouse model of TID (Masum et al., 2017). Renal capillary rarefaction induces hypoxia of the tubulointerstitial tissues, which are supplied by blood from these capillaries. This exacerbates the TID and eventually leads to the progression of CKD (Fine and Norman, 2008; Palm and Nordquist, 2011). In man, rarefaction of PTCs, which is closely correlated with the severity of renal fibrosis (Choi et al., 2000), is considered a predictive event and a new therapeutic target for CKD Ó 2018 Elsevier Ltd. All rights reserved.
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(Steegh et al., 2011; Chade et al., 2016). However, rarefaction of PTCs in kidney diseases has still not been investigated in small animal medicine. Therefore, the aim of the present study was to evaluate the change in renal capillaries in canine and feline CKD. Kidneys from 15 dogs and 11 cats with CKD were collected during post-mortem examination performed at Kagoshima University, Japan. The study was approved by the Committee for Animal Experimentation of Kagoshima University, Japan (VM15020). Wax blocks of kidney tissue obtained from healthy beagles (n ¼ 5) and healthy cats (n ¼ 4) were used as normal controls. Control blocks from healthy cats were obtained from Hokkaido University, Japan. Diagnosis of CKD was performed using medical records based on the continuous elevation of plasma creatinine (pCre) concentrations. Plasma concentrations of blood urea nitrogen (BUN) were also measured in all cases. In cases with normal pCre, CKD was determined by evaluating histopathological changes in the kidney tissue. Briefly, cases with apparent interstitial fibrosis were regarded as an early stage of CKD. The animals in the present study had been given necessary treatment for the underlying or concurrent disorders. Cases with acute kidney injury, including acute-on-chronic CKD, were excluded from the present study. All tissue samples were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (3 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff, Jones’ methenamine silver and Masson’s trichrome (MT) stains for conventional histopathology. Serial sections were processed for immunohistochemistry (IHC) using CD34 as a marker of vascular endothelial cells. Antigen retrieval was performed by microwave heating with 10 mM citrate buffer (pH 6.0). The sections were then treated with 3% H2O2 and blocked with 0.25% casein (SigmaeAldrich Corp., St. Louis, Missouri, USA). The primary reagent, goat anti-CD34 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA), was diluted at 1 in 50 for dogs and 1 in 100 for cats. The sections were incubated with primary antibody overnight at 4 C. Biotinylated rabbit anti-goat IgG (Vector Laboratories, Burlingame, California, USA) as the secondary antibody was diluted at 1 in 200 and incubated for 30 min. Following a further 30 min incubation with peroxidase-conjugated streptavidin (Ready to use, KPL, Gaithersburg, Maryland, USA), labelling was detected using 3, 30 diaminobenzidine (DAB) as chromogen (DABbuffer tablet; Merck, Darmstadt, Germany). For the negative control sections, normal goat IgG
(Santa Cruz Biotechnology) was used instead of the primary antibody. Randomized quantitative analysis with a point counting method was performed (Mitani et al., 2014). Briefly, to count CD34-positive PTCs, digital images were captured at 200 magnification from the renal cortex (six images/section). Then, grid lines were drawn on the images using Adobe Photoshop Elements 9 (Adobe Systems, San Jose, California, USA) at 50 pixel intervals. Each image was composed of 768 circles (4,608 circles/section). Circles with CD34-positive PTCs covering over one-quarter of the area were counted as positive. Glomeruli and large vessels were excluded from the counts. The percentage of positive points was estimated as the index for CD34-positive PTCs. A point counting method similar to that used for MT-stained sections was used to evaluate interstitial fibrosis. Digital images were captured at 200 magnification and grid lines were at 80 pixel intervals. Each image was composed of 300 circles. The percentage of fibrotic points was estimated as the index for interstitial fibrosis. Statistical analyses were performed using PASW software program for Windows (IBM SPSS Statistics, Armonk, New York, USA). The significance of group differences was assessed using the ManneWhitney U test. Spearman’s rank correlation coefficients were used to evaluate correlation between parameters. Statistical significance was defined as P <0.05. In dogs, CD34-positive signals were detected in the glomerular capillaries and PTCs (Fig. 1). There were few positive PTCs in many cases of CKD, even at the early stage of disease (Fig. 2). The quantitative score in kidneys from dogs with CKD was significantly lower than that in the normal controls (Fig. 3). There
Fig. 1. Immunohistochemical detection of CD34 in a normal canine kidney. Bar, 80 mm.
81
Peritubular Capillary Changes in Chronic Kidney Disease Table 1 Correlations between the CD34-positive PTCs and the severity of CKD
Dogs Cats
BUN
pCre
Interstitial fibrosis
0.039 (0.889) 0.164 (0.631)
0.032 (0.909) 0.257 (0.446)
0.011 (0.970) 0.323 (0.332)
PTCs, peritubular capillaries; CKD, chronic kidney disease; BUN, blood urea nitrogen; pCre, plasma creatinine. Data represent the correlation coefficients. Value in parenthesis represents P-value. Spearman’s rank correlation coefficients were used to evaluate the correlation between the parameters.
Fig. 2. Immunohistochemical detection of CD34 in a diseased kidney from a dog with CKD. There are few CD34-positive peritubular capillaries. Bar, 80 mm.
Fig. 3. Score for CD34-positive PTCs in dogs; shown as a box plot. ManneWhitney U test.
were no significant correlations between the score of the PTCs and the levels of the BUN, pCre and interstitial fibrosis (Table 1). These results implied that capillary rarefaction of renal tissue was occurring prior to the progression of CKD and it was suggested that such rarefaction could be a trigger for the development of TID, which induces progression of canine CKD. However, in the present study the influence of age-related changes in the renal capillaries remains unclear. In cats, CD34-positive PTCs were not reduced in most cases of CKD (Supplementary Figs. 1 and 2). The quantitative score in the kidneys of cats with CKD was not significantly different from that in normal controls (Supplementary Fig. 3). There were no correlations between the score of PTCs and the levels of BUN, pCre and interstitial fibrosis
(Table 1). These results implied that the renal microvascular system of cats was well preserved in CKD, suggesting that renal capillary rarefaction was not a crucial factor for the progression of feline CKD. Since TID is a typical renal pathological event in feline CKD (Brown et al., 2016), we had originally hypothesized that renal capillary rarefaction, which leads to TID, might be more prominent in feline CKD than in canine CKD. However, the findings from the present study disproved our hypothesis. The reasons for the maintenance of the microvascular system of the kidneys of cats during the progression of CKD remain unclear. It is possible that this could be attributed to histopathological features of feline CKD. In cats, primary glomerular disease is a rare cause of CKD (Chakrabarti et al., 2013; Brown et al., 2016). Our previous study demonstrated that the severity of glomerular sclerosis correlated with the elevation of pCre in canine CKD, but not in feline CKD (Yabuki et al., 2010). Since PTCs are derived from the efferent arterioles of the glomeruli, sclerotic changes in the glomeruli would reduce the blood supply to the PTCs, leading to regression of the postglomerular capillary network (Ballermann and Obeidat, 2014). Therefore, resistance to glomerular damage in feline CKD might lead directly to the maintenance of blood flow to the PTCs, preventing hypoxic damage of renal microvasculature. Retention or activation of renoprotective factors might also be involved in the attenuation of microvascular loss in feline CKD. For example, using IHC we investigated the intrarenal expression of angiotensinconverting enzyme (ACE)-2 in canine and feline CKD. During CKD, ACE-2 reactivity was well preserved in feline compared with canine tissues (Mitani et al., 2014). ACE-2 is a newly recognized homologue of ACE, which plays an important physiological role in regulating renal haemodynamics, glomerular filtration and tubular reabsorption via the generation of Ang-(1e7) from angiotensin II (Donoghue et al., 2000; Tipnis et al., 2000; Sim~ oes et al., 2012; Zimmerman and Burns, 2012). Apart from ACE-2, the expression of other renoprotective
R. Nakamura et al.
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substances, such as cardiolipin and sirtuin 1, in damaged kidneys has also been reported (Eirin et al., 2014; Yang et al., 2016). The association of these renoprotective factors with the attenuation of microvascular loss in feline kidneys should be investigated in future studies.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (grant numbers 16K08056, 15K1613205) from the Japan Society for the Promotion of Science.
Conflict of Interest Statement The authors declare no conflict of interest with respect to publication of this manuscript.
Supplementary Data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcpa.2018.03.004.
References Ballermann BJ, Obeidat M (2014) Tipping the balance from angiogenesis to fibrosis in CKD. Kidney International Supplements, 4, 45e52. Brown CA, Elliott J, Schmiedt CW, Brown SA (2016) Chronic kidney disease in aged cats: clinical features, morphology, and proposed pathogeneses. Veterinary Pathology, 53, 309e326. Chade AR, Tullos NA, Harvey TW, Mahdi F, Bidwell GL 3rd (2016) Renal therapeutic angiogenesis using a bioengineered polymer-stabilized vascular endothelial growth factor construct. Journal of the American Society of Nephrology, 27, 1741e1752. Chakrabarti S, Syme HM, Brown CA, Elliott J (2013) Histomorphometry of feline chronic kidney disease and correlation with markers of renal dysfunction. Veterinary Pathology, 50, 147e155. Choi YJ, Chakraborty S, Nguyen V, Nguyen C, Kim BK (2000) Peritubular capillary loss is associated with chronic tubulointerstitial injury in human kidney: altered expression of vascular endothelial growth factor. Human Pathology, 31, 1491e1497. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M et al. (2000) A novel angiotensin-converting enzymerelated carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1e9. Circulation Research, 87, E1eE9. Eirin A, Ebrahimi B, Zhang X, Zhu XY, Woollard JR et al. (2014) Mitochondrial protection restores renal function in swine atherosclerotic renovascular disease. Cardiovascular Research, 103, 461e472. Fine LG, Norman JT (2008) Chronic hypoxia as a mechanism of progression of chronic kidney diseases: from hy-
pothesis to novel therapeutics. Kidney International, 74, 867e872. Geddes RF, Finch NC, Elliott J, Syme HM (2013) Fibroblast growth factor 23 in feline chronic kidney disease. Journal of Veterinary Internal Medicine, 27, 234e241. Kida Y, Duffield JS (2011) Pivotal role of pericytes in kidney fibrosis. Clinical and Experimental Pharmacology and Physiology, 38, 467e743. Kida Y, Tchao BN, Yamaguchi I (2014) Peritubular capillary rarefaction: a new therapeutic target in chronic kidney disease. Pediatric Nephrology, 29, 333e342. Lawson J, Elliott J, Wheeler-Jones C, Syme H, Jepson R (2015) Renal fibrosis in feline chronic kidney disease: known mediators and mechanisms of injury. The Veterinary Journal, 203, 18e26. Lawson JS, Syme HM, Wheeler-Jones CP, Elliott J (2016) Urinary active transforming growth factor b in feline chronic kidney disease. Veterinary Journal, 214, 1e6. Lin SL, Chang FC, Schrimpf C, Chen YT, Wu CF et al. (2011) Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis. American Journal of Pathology, 178, 911e923. Masum MA, Ichii O, Elewa YHA, Nakamura T, Kon Y (2017) Local CD34-positive capillaries decrease in mouse models of kidney disease associating with the severity of glomerular and tubulointerstitial lesions. BMC Nephrology, 18, 280. Mayer G (2011) Capillary rarefaction, hypoxia, VEGF and angiogenesis in chronic renal disease. Nephrology Dialysis Transplantation, 26, 1132e1137. Mitani S, Yabuki A, Sawa M, Chang HS, Yamato O (2014) Intrarenal distributions and changes of angiotensin-converting enzyme and angiotensinconverting enzyme 2 in feline and canine chronic kidney disease. Journal of Veterinary Medical Science, 76, 45e50. Nangaku M (2004) Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Internal Medicine, 43, 9e17. Nangaku M (2006) Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. Journal of the American Society of Nephrology, 17, 17e25. Palm F, Nordquist L (2011) Renal tubulointerstitial hypoxia: cause and consequence of kidney dysfunction. Clinical and Experimental Pharmacology and Physiology, 38, 474e480. S anchez-Lara AC, Elliott J, Syme HM, Brown CA, Haylor JL (2015) Feline chronic kidney disease is associated with upregulation of transglutaminase 2: a collagen cross-linking enzyme. Veterinary Pathology, 52, 513e523. Sim~ oes E Silva AC, Flynn JT (2012) The reninangiotensin-aldosterone system in 2011: role in hypertension and chronic kidney disease. Pediatric Nephrology, 27, 1835e1845.
Peritubular Capillary Changes in Chronic Kidney Disease
Steegh FM, Gelens MA, Nieman FH, van Hooff JP, Cleutjens JP et al. (2011) Early loss of peritubular capillaries after kidney transplantation. Journal of the American Society of Nephrology, 22, 1024e1029. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G et al. (2000) A human homolog of angiotensinconverting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. Journal of Biological Chemistry, 275, 33238e33243. Woolf AS, Gnudi L, Long DA (2009) Roles of angiopoietins in kidney development and disease. Journal of the American Society of Nephrology, 20, 239e244. Yabuki A, Mitani S, Fujiki M, Misumi K, Endo Y et al. (2010) Comparative study of chronic kidney disease in dogs and cats: induction of myofibroblasts. Research in Veterinary Science, 88, 294 249.
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Yang SY, Lin SL, Chen YM, Wu VC, Yang WS et al. (2016) A low-salt diet increases the expression of renal sirtuin 1 through activation of the ghrelin receptor in rats. Scientific Reports, 6, 32787. Zimmerman D, Burns KD (2012) Angiotensin-(1-7) in kidney disease: a review of the controversies. Clinical Science, 123, 333e346.
February 7th, 2018 ½ Received, Accepted, March 24th, 2018
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
SPONTANEOUSLY ARISING DISEASE
Changes in Renal Peritubular Capillaries in Canine and Feline Chronic Kidney Disease R. Nakamura*, A. Yabuki*, O. Ichii†, H. Mizukawa‡, N. Yokoyamax and O. Yamato* *Laboratory of Veterinary Clinical Pathology, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, † Laboratory of Anatomy, Department of Basic Veterinary Sciences, ‡ Division of Environmental Veterinary Sciences and x Laboratory of Veterinary Internal Medicine, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan
Summary Renal capillary rarefaction is a crucial event that leads to tubulointerstitial damage during the progression of chronic kidney disease (CKD). In the present study, changes in CD34-positive renal capillaries were investigated in dogs and cats with CKD. A significant decrease in CD34-positive capillaries was observed in canine diseased kidneys, even at the early stage of disease. In cats, CD34-positive capillaries were well preserved in the diseased kidneys, with no link to the severity of CKD. Renal capillary rarefaction might be a trigger event that leads to the progression of CKD in dogs, rather than in cats. Ó 2018 Elsevier Ltd. All rights reserved. Keywords: capillary rarefaction; cat; chronic kidney disease; dog
Chronic kidney disease (CKD) can result from a variety of causes. Despite underlying causal differences, CKD progresses irreversibly to tubulointerstitial damage (TID), which is the final common mechanism that leads to end-stage kidney disease (Nangaku, 2004, 2006). TID is also involved in the progression of CKD in dogs and cats (Yabuki et al., 2010; Chakrabart et al., 2013; Lawson et al., 2015). However, while the progression of CKD is predominantly influenced by TID in cats, it appears to be mediated by both glomerulosclerosis and TID in dogs (Yabuki et al., 2010). Recent studies demonstrated that several factors such as transforming growth factor (TGF)-b, fibroblast growth factor 23 and transglutaminase 2 are involved in the progression of feline CKD (Geddes et al., 2013; SanchezLara et al., 2015; Lawson et al., 2016). Loss of peritubular capillaries (PTCs) in renal tissues, known as renal capillary rarefaction, is a crucial Correspondence to: A. Yabuki (e-mail: [email protected]). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2018.03.004
pathological event in the progression of TID in man (Mayer, 2011). The mechanism of capillary rarefaction is complicated. Pericyte damage and endothelial cell apoptosis play a pivotal role in the loss of renal microvasculature (Kida and Duffield, 2011; Ballermann and Obeidat, 2014; Kida et al., 2014). Many factors such as vascular endothelial growth factor and angiopoietin are also involved in this phenomenon (Woolf et al., 2009; Lin et al., 2011; Kida et al., 2014). A close relationship between renal capillary rarefaction and mononuclear infiltration was also demonstrated in a mouse model of TID (Masum et al., 2017). Renal capillary rarefaction induces hypoxia of the tubulointerstitial tissues, which are supplied by blood from these capillaries. This exacerbates the TID and eventually leads to the progression of CKD (Fine and Norman, 2008; Palm and Nordquist, 2011). In man, rarefaction of PTCs, which is closely correlated with the severity of renal fibrosis (Choi et al., 2000), is considered a predictive event and a new therapeutic target for CKD Ó 2018 Elsevier Ltd. All rights reserved.
80
R. Nakamura et al.
(Steegh et al., 2011; Chade et al., 2016). However, rarefaction of PTCs in kidney diseases has still not been investigated in small animal medicine. Therefore, the aim of the present study was to evaluate the change in renal capillaries in canine and feline CKD. Kidneys from 15 dogs and 11 cats with CKD were collected during post-mortem examination performed at Kagoshima University, Japan. The study was approved by the Committee for Animal Experimentation of Kagoshima University, Japan (VM15020). Wax blocks of kidney tissue obtained from healthy beagles (n ¼ 5) and healthy cats (n ¼ 4) were used as normal controls. Control blocks from healthy cats were obtained from Hokkaido University, Japan. Diagnosis of CKD was performed using medical records based on the continuous elevation of plasma creatinine (pCre) concentrations. Plasma concentrations of blood urea nitrogen (BUN) were also measured in all cases. In cases with normal pCre, CKD was determined by evaluating histopathological changes in the kidney tissue. Briefly, cases with apparent interstitial fibrosis were regarded as an early stage of CKD. The animals in the present study had been given necessary treatment for the underlying or concurrent disorders. Cases with acute kidney injury, including acute-on-chronic CKD, were excluded from the present study. All tissue samples were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (3 mm) were stained with haematoxylin and eosin (HE), periodic acideSchiff, Jones’ methenamine silver and Masson’s trichrome (MT) stains for conventional histopathology. Serial sections were processed for immunohistochemistry (IHC) using CD34 as a marker of vascular endothelial cells. Antigen retrieval was performed by microwave heating with 10 mM citrate buffer (pH 6.0). The sections were then treated with 3% H2O2 and blocked with 0.25% casein (SigmaeAldrich Corp., St. Louis, Missouri, USA). The primary reagent, goat anti-CD34 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA), was diluted at 1 in 50 for dogs and 1 in 100 for cats. The sections were incubated with primary antibody overnight at 4 C. Biotinylated rabbit anti-goat IgG (Vector Laboratories, Burlingame, California, USA) as the secondary antibody was diluted at 1 in 200 and incubated for 30 min. Following a further 30 min incubation with peroxidase-conjugated streptavidin (Ready to use, KPL, Gaithersburg, Maryland, USA), labelling was detected using 3, 30 diaminobenzidine (DAB) as chromogen (DABbuffer tablet; Merck, Darmstadt, Germany). For the negative control sections, normal goat IgG
(Santa Cruz Biotechnology) was used instead of the primary antibody. Randomized quantitative analysis with a point counting method was performed (Mitani et al., 2014). Briefly, to count CD34-positive PTCs, digital images were captured at 200 magnification from the renal cortex (six images/section). Then, grid lines were drawn on the images using Adobe Photoshop Elements 9 (Adobe Systems, San Jose, California, USA) at 50 pixel intervals. Each image was composed of 768 circles (4,608 circles/section). Circles with CD34-positive PTCs covering over one-quarter of the area were counted as positive. Glomeruli and large vessels were excluded from the counts. The percentage of positive points was estimated as the index for CD34-positive PTCs. A point counting method similar to that used for MT-stained sections was used to evaluate interstitial fibrosis. Digital images were captured at 200 magnification and grid lines were at 80 pixel intervals. Each image was composed of 300 circles. The percentage of fibrotic points was estimated as the index for interstitial fibrosis. Statistical analyses were performed using PASW software program for Windows (IBM SPSS Statistics, Armonk, New York, USA). The significance of group differences was assessed using the ManneWhitney U test. Spearman’s rank correlation coefficients were used to evaluate correlation between parameters. Statistical significance was defined as P <0.05. In dogs, CD34-positive signals were detected in the glomerular capillaries and PTCs (Fig. 1). There were few positive PTCs in many cases of CKD, even at the early stage of disease (Fig. 2). The quantitative score in kidneys from dogs with CKD was significantly lower than that in the normal controls (Fig. 3). There
Fig. 1. Immunohistochemical detection of CD34 in a normal canine kidney. Bar, 80 mm.
81
Peritubular Capillary Changes in Chronic Kidney Disease Table 1 Correlations between the CD34-positive PTCs and the severity of CKD
Dogs Cats
BUN
pCre
Interstitial fibrosis
0.039 (0.889) 0.164 (0.631)
0.032 (0.909) 0.257 (0.446)
0.011 (0.970) 0.323 (0.332)
PTCs, peritubular capillaries; CKD, chronic kidney disease; BUN, blood urea nitrogen; pCre, plasma creatinine. Data represent the correlation coefficients. Value in parenthesis represents P-value. Spearman’s rank correlation coefficients were used to evaluate the correlation between the parameters.
Fig. 2. Immunohistochemical detection of CD34 in a diseased kidney from a dog with CKD. There are few CD34-positive peritubular capillaries. Bar, 80 mm.
Fig. 3. Score for CD34-positive PTCs in dogs; shown as a box plot. ManneWhitney U test.
were no significant correlations between the score of the PTCs and the levels of the BUN, pCre and interstitial fibrosis (Table 1). These results implied that capillary rarefaction of renal tissue was occurring prior to the progression of CKD and it was suggested that such rarefaction could be a trigger for the development of TID, which induces progression of canine CKD. However, in the present study the influence of age-related changes in the renal capillaries remains unclear. In cats, CD34-positive PTCs were not reduced in most cases of CKD (Supplementary Figs. 1 and 2). The quantitative score in the kidneys of cats with CKD was not significantly different from that in normal controls (Supplementary Fig. 3). There were no correlations between the score of PTCs and the levels of BUN, pCre and interstitial fibrosis
(Table 1). These results implied that the renal microvascular system of cats was well preserved in CKD, suggesting that renal capillary rarefaction was not a crucial factor for the progression of feline CKD. Since TID is a typical renal pathological event in feline CKD (Brown et al., 2016), we had originally hypothesized that renal capillary rarefaction, which leads to TID, might be more prominent in feline CKD than in canine CKD. However, the findings from the present study disproved our hypothesis. The reasons for the maintenance of the microvascular system of the kidneys of cats during the progression of CKD remain unclear. It is possible that this could be attributed to histopathological features of feline CKD. In cats, primary glomerular disease is a rare cause of CKD (Chakrabarti et al., 2013; Brown et al., 2016). Our previous study demonstrated that the severity of glomerular sclerosis correlated with the elevation of pCre in canine CKD, but not in feline CKD (Yabuki et al., 2010). Since PTCs are derived from the efferent arterioles of the glomeruli, sclerotic changes in the glomeruli would reduce the blood supply to the PTCs, leading to regression of the postglomerular capillary network (Ballermann and Obeidat, 2014). Therefore, resistance to glomerular damage in feline CKD might lead directly to the maintenance of blood flow to the PTCs, preventing hypoxic damage of renal microvasculature. Retention or activation of renoprotective factors might also be involved in the attenuation of microvascular loss in feline CKD. For example, using IHC we investigated the intrarenal expression of angiotensinconverting enzyme (ACE)-2 in canine and feline CKD. During CKD, ACE-2 reactivity was well preserved in feline compared with canine tissues (Mitani et al., 2014). ACE-2 is a newly recognized homologue of ACE, which plays an important physiological role in regulating renal haemodynamics, glomerular filtration and tubular reabsorption via the generation of Ang-(1e7) from angiotensin II (Donoghue et al., 2000; Tipnis et al., 2000; Sim~ oes et al., 2012; Zimmerman and Burns, 2012). Apart from ACE-2, the expression of other renoprotective
R. Nakamura et al.
82
substances, such as cardiolipin and sirtuin 1, in damaged kidneys has also been reported (Eirin et al., 2014; Yang et al., 2016). The association of these renoprotective factors with the attenuation of microvascular loss in feline kidneys should be investigated in future studies.
Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research (grant numbers 16K08056, 15K1613205) from the Japan Society for the Promotion of Science.
Conflict of Interest Statement The authors declare no conflict of interest with respect to publication of this manuscript.
Supplementary Data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcpa.2018.03.004.
References Ballermann BJ, Obeidat M (2014) Tipping the balance from angiogenesis to fibrosis in CKD. Kidney International Supplements, 4, 45e52. Brown CA, Elliott J, Schmiedt CW, Brown SA (2016) Chronic kidney disease in aged cats: clinical features, morphology, and proposed pathogeneses. Veterinary Pathology, 53, 309e326. Chade AR, Tullos NA, Harvey TW, Mahdi F, Bidwell GL 3rd (2016) Renal therapeutic angiogenesis using a bioengineered polymer-stabilized vascular endothelial growth factor construct. Journal of the American Society of Nephrology, 27, 1741e1752. Chakrabarti S, Syme HM, Brown CA, Elliott J (2013) Histomorphometry of feline chronic kidney disease and correlation with markers of renal dysfunction. Veterinary Pathology, 50, 147e155. Choi YJ, Chakraborty S, Nguyen V, Nguyen C, Kim BK (2000) Peritubular capillary loss is associated with chronic tubulointerstitial injury in human kidney: altered expression of vascular endothelial growth factor. Human Pathology, 31, 1491e1497. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M et al. (2000) A novel angiotensin-converting enzymerelated carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1e9. Circulation Research, 87, E1eE9. Eirin A, Ebrahimi B, Zhang X, Zhu XY, Woollard JR et al. (2014) Mitochondrial protection restores renal function in swine atherosclerotic renovascular disease. Cardiovascular Research, 103, 461e472. Fine LG, Norman JT (2008) Chronic hypoxia as a mechanism of progression of chronic kidney diseases: from hy-
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February 7th, 2018 ½ Received, Accepted, March 24th, 2018