Background
Kidney disease causes a systemic deterioration in health. Chronic kidney disease, in particular, is a substantial public health burden because it is associated with end stage renal disease and cardiovascular disease [
1]. Various intrinsic and extrinsic factors, such as genomic factors, infections, and drug use, affect the progression of kidney diseases. The kidney is a highly vascularized organ, renal vasculature is also believed to play important roles in both renal function and tissue homeostasis. Altered vasculature can cause several renal injuries mediated by hypoxia-associated processes [
2‐
4]. Local hypoxia in the kidney also results in tubular atrophy, inflammation, and interstitial accumulation of extracellular matrix [
3,
4]. Loss of vascular endothelial growth factor (VEGF) and chronic hypoxia lead to microvascular dysfunction in kidneys [
3‐
6].
The mammalian glomerulus has a well-developed capillary tuft, and these capillaries are lined by thin fenestrated endothelial cells and podocytes that play critical coordinating roles in renal physiology as well as in innate and adaptive immunity [
7,
8]. An imbalance in the decrease in endothelial repair and increase in endothelial apoptosis-after renal injury causes glomerular lesions (GLs), such as the loss of the glomerular capillaries and glomerulosclerosis [
9]. In diabetic nephropathy, increased blood glucose levels cause capillary injuries [
10]. Furthermore, tubulointerstitial capillaries are involved in the regulation of renal function and hemodynamics [
2]. Capillary endothelial cells have a crucial function in maintaining renal homeostasis and expressing specific chemokines that control compartment-specific T-cell and monocyte recruitment during inflammation [
11,
12]. This functional variability in endothelial cells is probably associated with differential susceptibilities to apoptosis and differential responses to microenvironmental changes or stimuli. Tubulointerstitial capillaries are also injured in diabetic nephropathy, resulting in a reduction in capillary density and progression of tubulointerstitial lesions (TILs) [
13].
Thus, we can distinguish between the renal pathogenesis of GLs and TLs. Earlier drug-induced or spontaneous rodent models, and autoimmune disease models such as MRL/MpJ-Fas
lpr/lpr
and BXSB/MpJ-
Yaa (Yaa), in particular, have been widely used, and they manifest severe glomerulonephritis caused by immune-complex deposition [
14,
15]. Male Yaa mice show more severe glomerulonephritis than females because of a Y-linked autoimmune acceleration (Yaa) mutation on the Y chromosome; however, we previously clarified that the BXSB/MpJ (BXSB)-genetic background also contributes to the progression of autoimmune disease-mediated glomerular damage [
14]. In Yaa mice, GLs appeared earlier and were more severe than TILs, which appeared at a late stage in glomerulonephritis [
16]. For TILs, unilateral ureteral obstruction (UUO) is usually performed to induce tubulointerstitial fibrosis in mice. UUO results in hydronephrosis, interstitial infiltration of inflammatory cells, tubular cell death from hypoxia, and collagen deposition in the tubulointerstitium, followed by the development of tubulointerstitial fibrosis [
17]. A previous study identified TILs, rather than GLs, as the main cause of progressive and end stage kidney disease [
18]. However, other researchers have shown the involvement of microvasculature in the advance of human and experimental animal glomerulonephritis [
19‐
21].
Nevertheless, there are no reports clarifying the correlations between altered capillary structures and renal histopathology or renal function in GLs and TILs. The present study investigated the morphological, quantitative, and ultrastructural alterations in glomerular and tubulointerstitial capillaries using two mouse models, namely the spontaneous glomerulonephritis model and UUO model. In addition, based on an analysis of these models, we clarified the correlations between the number of local capillaries in GLs and TILs and kidney diseases severity.
Methods
Animals
The authors adhered to the
Guide for the Care and Use of Laboratory Animals of Hokkaido University, Graduate School of Veterinary Medicine (approved by the Association for Assessment and Accreditation of Laboratory Animal Care International). Animal experimentation was approved by the Institutional Animal Care and Use Committee of the Graduate School of Veterinary Medicine, Hokkaido University (approval no. 13–0032, 16–0124). Experimental mice were purchased from Japan SLC Inc. (Shizuka, Japan). A maximum of five mice were held in one cage containing wood chip bedding material in a pathogen-free animal house. Food and water are provided
ad libitum to the animals. Light and dark conditions were maintained at 1:1 ratio. Strain and number of mice used in different disease models and protocols are shown details in Additional file
1. Six month-old male Yaa mice (
N = 12) were used for the GL model, and same-aged male BXSB mice (
N = 12) were used as healthy controls. To create the TIL model, 8-week-old male C57BL/6 mice (
N = 12) were subjected to UUO for 7 days. The kidney paired with the UUO kidney in the same mouse was used as a normal control. Briefly, mice were deeply anesthesized with a mixture of 0.3 mg/kg medetomidine (Kyoritsu Seiyaku, Japan), 4 mg/kg midazolam (Astellas Pharma, Japan), and 5 mg/kg butorphanol (Meiji Seika Pharma, Japan), and laparotomy in the sublumbar region was performed to ligate the right ureter tightly with silk thread at the renal hilus. Buphrenorphine hydrochloride (Otsuka Pharmaceuticals, Japan) was injected intraperitoneally at a dose rate of 0.3 mg/kg as an analgesic. Recovery from anesthesia was facilitated by intraperitoneal administration of 0.3 mg/kg atipamezole (Zenoaq, Japan).
Sample preparation
Mice without external abnormalities were used in this experiment. The average weights of GLs and TILs model were 25.02 and 20.60 g, respectively. Urine was collected from anesthetized mice. Mice were euthanized by exsanguination from the carotid artery, and blood and kidneys were collected for serological analysis and histological examination, respectively. The kidneys were fixed with neutral buffer formalin (NBF), 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4) or 2.5% glutaraldehyde (GTA) in 0.1 M PB.
Serological and urinary examination
Serum levels of antibody against anti-double stranded DNA (dsDNA ab) were measured with a Mouse Anti-dsDNA Ig’s (Total A + G + M) ELISA Kit (Alpha Diagnostic International, San Antonio, TX, USA). Serum blood urea nitrogen (sBUN) and creatinine (sCr) levels in all mice were measured using a Fuji Drichem 7000v (Fujifilm, Tokyo, Japan). Urinary albumin creatinine ratios (uACR) were determined using Albuwell M and Creatinine Companion Kits (Exocell, Philadelphia, PA, USA).
Histopathological examination
Paraffin sections of kidneys fixed with NBF or PFA were cut at a thickness of 2 μm and stained with periodic acid Schiff-hematoxylin (PAS-H) or Masson’s trichrome (MT). Immunostaining for alpha smooth muscle actin (αSMA), B220, CD3, CD34, Iba1, and interleukin 1 family, member 6 (IL-1F6/IL-36α) [
22] was performed to detect myofibroblasts, B-cells, T-cells, capillary endothelial cells, macrophages, and damaged renal tubules, respectively. Staining conditions are listed in Table
1. Briefly, after deparaffinization, kidney sections were subjected to antigen retrieval. Then, slides were submerged in methanol containing 3% H
2O
2 for 20 min at room temperature. After blocking, sections were incubated with primary antibody overnight at 4 °C. After washing in phosphate-buffered saline (PBS), sections were incubated with secondary antibody for 30 min at room temperature, then washed and incubated with streptavidin-biotin complex (SABPO kit, Nichirei, Tokyo, Japan) for 30 min. All sections were then incubated with 3,3-diaminobenzidine tetrahydrochloride-H
2O
2 solution. Finally, the sections were counterstained with hematoxylin and dehydrated with an ascending series of alcohols.
Table 1
Summary of immunostaining conditions
Antigen retrieval | CB 105 °C, 20 min | CB 105 °C, 20 min | TB 105 °C, 20 min | CB 105 °C, 20 min | 0.1% pepsin 37 °C, 5 min | CB 105 °C, 20 min |
Blocking | 10% NGS | 10% NGS | 10% NGS | 10% NGS | 10% NGS | 5% NDS |
Primary antibody | Rabbit polyclonal antibodies (Abcam, Cambridge, UK) 1:3000 | Rat polyclonal antibodies (Cedarlane, Ontario, Canada) 1:1000 | Rabbit polyclonal antibodies (Nichirei, Tokyo, Japan) 1:200 | Rabbit polyclonal antibodies (Abcam, Cambridge, UK) 1:400 | Rabbit polyclonal antibodies (Wako, Tokyo, Japan) 1:2000 | Goat polyclonal antibodies (R&D Systems, Minnesota, USA) 1:400 |
Biotinylated secondary antibody | Goat anti-rabbit (SABPO kit, Nichirei, Tokyo, Japan) 1:100 | Rat anti-goat IgG (Caltag Medsystems, Buckingham, UK) 1:100 | Goat anti-rabbit (SABPO kit, Nichirei, Tokyo, Japan) 1:100 | Rat anti-goat IgG (Caltag Medsystems, Buckingham, UK) 1:100 | Goat anti-rabbit IgG (SABPO kit, Nichirei, Tokyo, Japan) 1:100 | Donkey anti-goat IgG (Santa Cruz, California, USA) 1:100 |
Histoplanimetry
Digital images of over 30 glomeruli or over 30 tubulointerstitial areas randomly selected from each mouse were obtained at 400× magnification using an All-in-One Fluorescence Microscope BZ-X710 (Keyence, Osaka, Japan). The size and number of total cells in each glomerulus were determined using PAS-stained sections. The number of B220
+ B-cells, CD3
+ T-cells, Iba1
+ macrophages, and CD34
+ capillaries observed in the digital images of glomeruli were counted using immunohistochemical sections and a BZ-X Analyzer (Keyence). Further, glomerular damage was semi-quantitatively scored according to methods described by Ichii et al. [
23]. For TILs, the numbers of B220
+ B-cells, CD3
+ T-cells and IL-1F6/IL-36α
+ damaged tubules throughout the cortex were counted. Additionally, CD34
+ capillaries, Iba1
+ macrophages, and αSMA
+ reaction areas in the tubulointerstitium were counted using immunohistochemical sections and a BZ-X Analyzer (Keyence), based on digital images of the renal cortex.
Ultrastructural examination
Kidneys were pre-fixed with 2.5% GTA in 0.1 M PB for 4 h at 4 °C, post-fixed with 1% osmium tetroxide in 0.1 M PB for 2 h at 4 °C, and then dehydrated in graded alcohol and embedded in epoxy resin (Quetol 812 mixtures; Nisshin EM, Tokyo, Japan). Ultrathin sections (60 nm) were stained with uranyl acetate and lead citrate. Mounted samples were observed under a JEOL transmission electron microscope (TEM, JEM-1210; JEOL, Tokyo, Japan).
Visualization of the renal vasculature
Vascular corrosion casts of kidney were prepared according a method described by Verli et al. [
24]. Mice other than those used for the histological analysis (
N = 4) were used for vascular casts of mice in both the control and disease groups. Briefly, after euthanasia, PBS containing EDTA, followed by a mixture of resin and catalyst (Mercox II cast, Ladd Research, Williston, USA) was perfused through left ventricles of heart. Dissected kidneys were held in a water bath at 37 °C overnight to allow polymerization with resin. Tissue surrounding the vascular bed was corroded by applying 15% potassium hydroxide for 30 h at room temperature. After washing with distilled water, casts that emerged in
t-butyl alcohol were subjected to freeze-drying in a vacuum freeze dryer (ES-2030, Hitachi, Tokyo, Japan). The dried specimens were mounted on a specimen stub, sputter-coated using a Hitachi E-1030 ion sputter coater (Hitachi, Tokyo, Japan), and then examined by scanning electron microscopy (SEM, S-4100; Hitachi, Tokyo, Japan) with an accelerating voltage of 4 kV.
In addition, after euthanasia, rubber (Microfil, Flow Tech, Inc. Massachusetts, USA) was perfused through left ventricles according to a method described by Walker et al. [
25]. Then, dissected kidneys were fixed with 4% PFA in 0.1 M PB overnight at 4 °C. Fixed kidneys were cut into 200-μm-thick sections using a microslicer (DSK Microslicer DTK-3000, Ted Pella, Inc. Redding, USA) and hydrated with an ascending series of alcohols. Finally, thick sections were cleared with methyl salicylate and examined under a BZ-X Analyzer of an All-in-One Fluorescence Microscope BZ-X710 (Keyence, Osaka, Japan) to obtain Z-stack images.
Statistical analysis
Results are expressed as the mean ± standard error (SE). For comparisons between healthy controls and experimental mice, a nonparametric Mann–Whitney U test (P < 0.05) was utilized. The Kruskal-Wallis test was used to compare three or more populations, and multiple comparisons were performed using Scheffe’s method when significant differences were observed (P < 0.05). Correlations between the CD34+ renal capillary number and renal function or histopathological indices were analyzed using Spearman’s rank correlation coefficient (P < 0.05).
Discussion
For the GL model using Yaa mice, the number of glomeruli with cells positive for CD34, a representative marker of endothelial cells, significantly and negatively correlated with serum levels of autoantibody and deterioration in renal function, as well as increased numbers of infiltrating cells in the glomeruli. TILs in GL models showed an increase in cell infiltration and damaged renal tubules, but not myofibroblasts; however, the number of CD34
+ capillaries in TILs was comparable between BXSB and Yaa mice and did not correlate with any renal pathological parameters. Further, no significant GLs were noted in UUO models. The Yaa mouse is a model of spontaneous autoimmune-mediated membranoproliferative glomerulonephritis, with hyperproliferation of pathogenic B-cells resulting from a Yaa mutation that contributes to autoantibody production, activation of pathogenic T-cells, and secretion of pro-inflammatory cytokines that contribute to the development of GLs [
16,
22,
26‐
28]. Therefore, GL events appear earlier and were more severe than TILs in Yaa mice, whereas severe TILs, with fibrotic features characterized by an increase in myofibroblasts, were prominent end stage kidney disease in Yaa mice. However, because glomerular efferent arterioles directly connect to branches of peritubular/tubulointerstitial capillaries, a reduction in blood supply to the glomerulus because of GLs might affect TILs. Collectively, the study data indicate that a decrease in CD34
+ cells in glomerular capillaries correlates with the progression of GLs in autoimmune disease-prone Yaa mice.
In GL models, glomerular capillaries had narrow lumens, thickened endothelial cytoplasms with vacuolation, loss of endothelial fenestration, and detached luminal endothelia in capillary lumens. Although renal tubulointerstitial capillary injury, particularly capillary basement membrane multilamination, is prominent feature in chronic microvascular injury in renal allograft rejection [
29,
30], we did not find such features in the kidneys of autimmene-disease prone Yaa mice. These results indicate that CD34
+ capillaries decreased with endothelial cell injury, evident by their morphological changes. These capillary endothelial cells seemed to play crucial roles in the progression of glomerulonephritis and renal dysfunction. Briefly, several studies have shown an imbalance in endothelial cell proliferation and death that is associated with a decrease in renal capillaries leads to the progression of kidney disease [
19,
20,
31‐
33]. Importantly, hypoxia promotes GLs, similar to mesangial cell proliferation in patients with lupus nephritis and in MRL/MpJ-Fas
lpr/lpr
mice [
34]. Therefore, the loss of CD34
+ glomerular capillaries and their morphological changes in Yaa mice occur after the progression of GLs, including the development of membranoproliferative lesions and inflammation.
Furthermore, podocytes are involved in the maintenance of healthy intracapillary environments through crosstalk with glomerular endothelial cells and as a source of VEGF in glomeruli [
35,
36]. Podocyte injury is a critical event that causes albumin hyperfiltration from glomerular capillaries [
37]. Indeed, our ultrastructural study showed podocyte foot process effacement in GL model mouse kidneys, but not in those of the TIL model mouse. Thus, pathological changes in the glomerular microenvironment resulting from injury of podocytes and capillary endothelia coordinately aggravate GLs and lead to an elevation in uACR.
Capillaries in the tubulointerstitium are essential for renal oxygen supply and maintenance of kidney tubulointerstitial hemodynamics [
38,
39]. TILs activate the endothelium, which may correlate with enhanced inflammation and activation of coagulation that favors further capillary and interstitial injury [
40,
41]. Eventually, persistent TILs cause a loss of capillaries in the tubulointerstitium [
42]. In the present study, TIL models were created by UUO. The advantage of UUO was that diseased and control kidneys could be obtained from the same mice. TILs model mice clearly showed a decrease in CD34
+ capillaries in the tubulointerstitium with the progression of TILs, characterized by an increase in infiltrating cells and myofibroblasts, as well as damaged tubules in the tubulointerstitium. Furthermore, the ultrastructural study revealed capillary injuries in TILs in detail. These injuries were characterized by thickened and stratified endothelial cytoplasms with vacuolation, loss of fenestration, detaching endothelia with subendothelial vacuolation, and accumulation of collagen fibers beneath the capillary basement membrane. Thus, our results clearly indicate pathological correlations between TILs and capillary injury and/or loss in tubulointerstitium in UUO-based TIL models.
In particular, the number of infiltrating CD3
+ cells strongly correlated with the number of CD34
+ capillaries in TILs of UUO models. Therefore, we presumed that interstitial T-cells mediate inflammation and the accumulation of macrophages in the tubulointerstitium. Importantly, an increase in Iba-1
+macrophages significantly correlated with a decrease in CD34
+ capillaries in the present study. Another study showed that apoptosis of endothelial cells triggered capillary regression by blocking blood flow to the site of apoptosis in macrophage-dependent cell death [
43]. Renal fibrosis results from reduced endothelial proliferation following alterations in local expression of both angiogenic and antiangiogenic factors, and this imbalance is mediated by macrophage-associated cytokines, such as interleukin 1 beta, and vasoactive mediators [
38]. Based on these findings, we considered tubulointerstitial inflammation, especially macrophage infiltration, to underlie injury of capillary endothelial cells and the subsequent net loss of capillaries.
The present study revealed a significant correlation between the number of CD34
+ capillaries and the numbers of IL-1F6/IL-36α
+ damaged renal tubules and αSMA
+ myofibroblasts. Evidently, loss of tubulointerstitial capillaries caused TILs because of local hypoxia, because tubulointerstitial capillaries and renal tubules show functional crosstalk to maintain normal renal interstitial structure and function, including preserving the blood supply to maintain tubular epithelial cells. This speculation is strongly supported by previous study indicating that renal ischemia caused by vascular obliteration is a major contributor to renal fibrosis [
3]. Moreover, the tubular epithelium is a source of VEGF in the tubulointerstitium [
44]. Therefore, we concluded that injury and/or loss of tubulointerstitial capillaries contribute to the progression of TILs, and fibrosis and tubular damage in particular, in UUO kidneys.
In this study, we examined the decrease in CD34
+ renal capillaries associated with the progression of kidney disease using murine GLs and TILs models. The quantitative changes in CD34
+ capillaries could represent two possible pathological events: 1) a decrease in capillary number, or 2) a decrease in CD34
+ cell number. As shown in the figures, the numbers of local capillaries decreased in GLs and TILs. However, interestingly, CD34
−capillaries were found in TILs of UUO models, but not in GLs of Yaa mice. CD34 is a single-pass transmembrane sialomucin protein that is expressed on hematopoietic stem cells and in vascular-associated tissue [
45]. It is an important adhesion molecule required for lymphocytes to enter lymph nodes [
46]. Kusano et al. suggested that the loss of CD34
+ capillaries was a result of the disappearance of cell markers in phenotypically altered endothelium [
21]. Therefore, a decrease in CD34
+ cells in kidneys indicates a decrease in renal capillaries, as well as altered functions of endothelial cells resulting from decreased CD34 due to capillary injury. In a future study, we will compare the loss of renal capillaries and decreased expression of CD34 as contributors to kidney disease progression. Results of this study indicate the importance of morphology and density of local capillaries in human and animal kidney diseases. Blood vessel has been caught the attention recently as it contribute to stem cell niches in various organs, signifying that vascular system may serve a preserved concerned role for stem cells throughout the body. Moreover, the kidney is a highly vascularized organ, and this study shows that GLs and TILs are associated with reduced number of glomerular capillaries and tubulointerstitial capillaries, respectively. Specially, there is a strong correlations between the number of local capillaries in GLs and TILs and the severity of kidney diseases. Pathological changes might also affect vasculature-associated stem cell niche in the kidney. In the future, we will investigate these possibilities.