Background
Unilateral ureteral obstruction (UUO), a commonly used experimental model of chronic kidney injury is characterized by tubular atrophy, inflammation and interstitial fibrosis [
1,
2]. In UUO, the initiating damage is increased ureteral pressure transmitted retrograde to the kidney that causes secondary renal vasoconstriction and resultant reduced glomerular blood flow [
3]. If the pressure is not relived, the renal vascular resistance remains increased, causing ischemia (reviewed in [
4]). The resultant tubulointerstitial fibrosis in UUO is multi-factorial, including interstitial macrophages producing pro-inflammatory cytokines, tubular cells undergoing apoptosis, and resident renal cells transitioning to collagen-producing cells [
1]. The origin of the collagen producing cells has been attributed to the perivascular cell population, namely pericytes and perivascular fibroblasts. Following UUO, perivascular cells become activated, and detach from the underlying vessels. The consequences of pericyte detachment include that endothelial cells are deprived of survival factors [
5], vascular tubes become unstable and more permeable leading to microvascular rarefaction [
6], and migrating perivascular cells can de-differentiate into myofibroblasts, thereby becoming a source of collagen [
7,
8].
Less is understood the reparative, or attempted reparative processes, if any, in UUO. Cells of renin lineage (CoRL) refer to all possible cellular derivatives originating from renin expressing cells at time captured by reporting. Fate tracking studies showed that in development, CoRL give rise to juxtaglomerular (JG) cells producing renin and to non-renin-producing cells such as smooth muscle cells, mesangial cells, tubular cells and extrarenal cells [
9]. Recently, the lineage relationship between Ren
+ and Foxd1
+stromal cells was clarified when it was revealed that all mural cells, including renin cells, are derived from Foxd1 stromal cells [
10]. Studying CoRL (Ren
+ progenitors) provides an opportunity to characterize in depth a subpopulation of stromal cells. It is important because we need better understanding of different subpopulation of stromal cells to design specific therapeutic interventions [
11].
Pericytes have gained recognition in the kidney for their role in pathogenesis of fibrosis [
7,
8]. Interestingly, CoRL have been acclaimed as a candidate progenitor of the kidney. Several studies showed that CoRL can regenerate mesangial cells [
12,
13], podocytes [
14], parietal epithelial cells [
14], and erythropoietin-producing cells [
15]. The most recent study from our lab demonstrates that in glomerular injury and remnant kidney models, CoRL migrate to the interstitium and regenerate into pericytes [
16]. The current study was designed to further explore the role of CoRL in progressive tubulointerstitial injury, the experimental model of unilateral ureteral obstruction.
Methods
Animals
To study the fate of renin lineage cells in chronic kidney disease, we used RenCreER (Ren1cCreERxRs-tdTomato) transgenic mice on a mixed C57 BL10/C3H background [
16]. In RenCreER mice, cells of renin lineage are only labeled permanently in inducible manner with tdTomato red protein within temporal windows defined by the administration of tamoxifen. 8–9 week-old mice were given Tamoxifen (100 mg/kg) by IP injection for 6 days on alternate days, as we have previously reported [
16,
17]. We waited at least 7 weeks between giving tamoxifen and inducing the disease model, to allow significant washout of tamoxifen to exclude possibility of recombination in other cell types. Animal protocols were approved by the University of Washington Institutional Animal Care and Use Committee (2968-04).
Experimental model of kidney fibrosis
Unilateral ureteral obstruction (UUO) was performed in adult female mice, as previously described [
7,
18,
19]. Briefly, mice were anesthetized with isofluorane (1 %, inhaled). UUO was induced by left ureteral ligation using a 4-0 silk tie suture at two points. Sham operated mice underwent the same procedure except that left ureter was only exposed by flank incision, served as controls (
n = 6). Kidneys were harvested on d3 (
n = 6), d7 (
n = 17), and d14 (
n = 6).
Assessment of kidney fibrosis
Histological analysis of fibrosis was performed on fixed renal tissue, embedded in paraffin, and sectioned at a thickness of 4 μm. Connective tissue deposition was examined with Picrosirius Red Stain Kit (Polysciences, Inc, Warrington, PA, USA) and collagen I (1:100, Millipore, Billerica, MA, USA), staining, as we have previously described [
20‐
22]. Additionally, co-staining of CoRL and a myofibroblast marker alpha smooth muscle actin (αSMA, 1:10.000; Sigma, Saint Louis, MI, USA) were performed to determine whether CoRL become myofibroblasts in kidney fibrosis.
Detection of tdTomato reporter
In order to visualize tdTomato reporter labeling used to label CoRL in RenCreER mice, kidneys were fixed in 10 % buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm. Kidney sections underwent deparaffinization, heat-mediated antigen retrieval in citrate buffer pH 6.0, and blocking unspecific background (Accurate, San Jose, CA, USA). Immunofluorescent staining for the tdTomato reporter was performed with DyeLight 594-conjugated RFP (Red Fluorescent Protein) rabbit antibody (1:100, Rockland Immunochemicals for Research, Gilbertsville, PA, USA) at room temperature for 1.5 h. tdTomato fluorescent signal is discernable in non-paraffin- embedded tissue only.
Identification of cell proliferation
BrdU (5-Bromo-2-deoxyridine) incorporation assay was used to quantitate cell cycle entry and proliferation. 10 μl of BrdU (Amersham Cell Proliferation Labeling Reagent, GE Healthcare Life Sciences, Little Chalfont, UK) per gram body weight was administered via IP on alternate days following UUO. Double immunostaining was performed for BrdU and tdTomato. Antibody paraffin embedded tissue was prepared and blocked as described above. Avidin/biotin blocking (Vector Laboratories, Burlingame, CA, USA) was performed to block endogenous biotin and prevent unspecific staining while using biotin-streptavidin labeling system. Tissue was incubated overnight at 4 °C with a primary mouse anti-BrdU (1:200, Amersham, GE Life Sciences, Buckinghamshire, UK). This was followed with a biotinylated goat anti-mouse antibody (1:500, Jackson Immunoresearch Laboratories, Inc, West Grove, PA, USA) incubated at room temperature for 1 h. The signal was amplified by incubation with streptavidin-conjugated with Alexa Fluor 488 (1:100; Invitrogen, Grand Island, NY, USA) for 45 min. Negative control staining was performed by omitting primary antibody staining.
Assessment of vascular changes
Endothelial marker staining CD31 (PECAM-1) was performed to examine changes in microvascular density, and to demonstrate any perivascular location of CoRL and pericytes. Rat anti-mouse CD31 (1:100; Dianova, Hamburg, Germany) was incubated overnight at 4 °C following incubation with secondary anti-rat antibody conjugated with Alexa Fluor 647 (1:100 Invitrogen).
Pericytes were identified by the expression of NG2 and PDGFRß. To identify pericytes derived from CoRL, triple imunostaining was performed on frozen tissue sections as follows. Rabbit anti-NG2 antibody (1:100; Millipore, Billerica, MA, USA) was incubated overnight at 4 °C, followed by biotinylated anti-rabbit antibody (1:500; Vector) incubation at room temperature for 1 h, and streptavidin conjugated with Alexa Fluor 647 (1:100; Invitrogen, Grand Island, NY, USA) for 45 min. To prevent non-specific staining for the primary antibodies from the same species pre-incubation with anti-rabbit IgG Fab (1:25; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was followed by rabbit IgG Fab incubation (1:25; Jackson ImmunoResearch Laboratories). Rabbit anti-PDGFRß antibody (1:100; Abcam, Cambridge, MA, USA) was incubated with tissue sections overnight at 4 °C. Secondary donkey anti-rabbit antibody conjugated with Alexa Fluor 488 (1:100, Invitrogen) was incubated for 1 h at room temperature. Finally, anti-tdTomato antibody was applied.
To examine hypoxia-activated locations in UUO, HIF-2α (hypoxia inducible factor-2 α) staining was performed together with tdTomato reporter staining on frozen tissue sections. Rabbit anti-HIF-2α antibody (1:200; Novus Biological, Littleton, CO, USA) was incubated overnight at 4 °C, followed by biotinylated anti-rabbit antibody (1:500; Vector) incubation at room temperature for 1 h, and streptavidin conjugated with Alexa Fluor 647 (1:100; Invitrogen) for 45 min. Positive staining was assessed based on nuclear HIF-2α localization confirmed by DAPI staining.
Image analysis and statistical analysis
Reporter positive-, BrdU- stainings were quantified on 20 images of kidney cortex/medulla using 200x total magnification. Fluorescent imaging was performed using EVOS®FL Cell Imaging System (Life Technologies). One-way ANOVA with Bonferroni post hoc test was used to compare groups with P ≤ 0.05 as a criterion for statistical significance. Data were presented as means ± SEM. All data were analyzed in GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).
Discussion
UUO is characterized by hypoxia, interstitial vascular loss and progressive kidney fibrosis. The compensatory mechanisms that might attempt to counter these events are not well understood. We demonstrated that following UUO, the number of medullary cells of renin lineage (CoRL) number increases due to cell proliferation and migration. Although CoRL initially appear to be reparative of interstitial microvessels following UUO by transdifferentiating in to pericyte-like cells, they are likely ultimately injurious by transdifferentiating into myofibroblast-like cells. These changes are preceded by activation of HIF2a in CoRL in the juxta-glomerular compartment following UUO.
The first major finding from these studies was that the number of CoRL increases following UUO. We used inducible Ren1cCreER xRs-tdTomato-R reporter mice to fate map a subset of CoRL that were permanently labeled specifically during the period of tamoxifen induction. The distribution of cells-expressing renin is very different during development compared to adults [
9]. Therefore, labeling mice at 7–8 weeks of age focuses on adult CoRL that derive from the juxtaglomerular (JG) compartment.
We next asked how might CoRL number increase following UUO? A first consideration was migration, from their original location in the JG to the medulla. Several lines of evidence help support this. First, using an inducible reporter system, labeled CoRL in the cortex were restricted to the JG. Thus, the presence of labeled CoRL in the intracapillary loops of some glomeruli, and in the cortical interstitium is in keeping with them migrating. Second, many CoRL in the cortex and medulla had an elongated shape, reminiscent of a migrating cell. Third, several interstitial vessels contained labeled CoRL within their lumens. Proliferation is another mechanism that might explain the twofold increase in medullary CoRL post UUO. To this end, we frequently performed BrdU pulse chases to maximally capture cell proliferation. BrdU staining was detected in a subset of CoRL in the cortex and medulla, but not in the JG. Moreover, several of CoRL with elongated shapes stained for BrdU. These data suggest that once CoRL had moved from the JG, a subset proliferated. However, we cannot exclude the possibility that native CoRL in the medulla did not proliferate, not migrate. Taken together, the increase in medullary CoRL post UUO is likely due to a combination of proliferation in CoRL that had migrated away from the JG, migration of non-proliferating CoRL and proliferation of pre-exisiting CoRLs in the medulla.
The second major finding in these studies was the transdifferentiation of medullary CoRL in to pericytes or myofibroblasts. Several lines of evidence show that CoRL have marked plasticity under certain conditions [
13‐
16]. Accordingly, we next asked what, if any, cell type did CoRL transdifferentiate into in the medulla following UUO. The first clue that they likely were transdifferentiating was the loss of their endocrine function by virtue that they no longer expressed the renin protein. Second, because at d7 labeled CoRL were largely confined to surrounding interstitial vessels, we explored the possibility that a subset were transdifferentiating in to pericytes. Indeed, non-renin expressing labeled CoRL co-expressed the pericyte markers NG2 and PDGFRß. Indeed, CoRL have been reported to transdifferentiate into pericytes during development [
9] and in glomerular disease [
16]. We can only speculate that the transdifferentiation of a subset of CoRL to pericytes early in disease is an attempt to maintain or even replace native pericytes, and thus the interstitial vasculature.
Detached pericytes can differentiate into collagen-producing myofibroblasts [
7,
19]. Here, we report that a subset of CoRL later underwent further changes to that more consistent with a myofibroblast. At day 14 post UUO, the majority of labeled interstitial CoRL were away from any blood vessels. Because these areas were typified by interstitial fibrosis, we asked if CoRL were transdifferentiating into myofibroblasts. Indeed, a subset of CoRL did begin to express aSMA, suggesting that they acquired a pro-fibrotic phenotype. It is not clear which of the following scenarios occurred first: a decrease in interstitial vessels (i.e. reduced CD31 staining) forced pericyte-like CoRL to detach, and/or if the detachment of pericyte-like CoRL lead to unhealthy underlying vessels, followed by rarefaction. Regardless, it is likely that similar to other native cells in the interstitium that acquire myofibroblast-like features, the subset of CoRL doing so also likely contribute to the increased fibrosis later in UUO. Finally, our data are in agreement with the current view that Foxd1 lineage cells contribute to fibrosis [
35] especially that CoRL have been recently shown to derive from Foxd1
+ cells [
10].
Hypoxia is a common pathway for chronic kidney disease, including UUO [
36,
37]. During kidney obstruction, renal blood flow is reduced as a consequence of pre-glomerular vessel constriction that in turn impairs post-glomerular/peritubular perfusion [
38]. HIF-2α is activated in interstitial and endothelial cells in hypoxia [
32,
33]. Extracellular matrix deposition further propagates hypoxia to the tubulointerstitium since it increases the distance between capillaries and tubules, decreasing oxygen diffusion [
39]. In general, HIF-1 and -2 are both activated in hypoxia, but in a different manner: HIF-2 is activated in mild hypoxia <5 % O
2 for many hours (still upregulated after 72 h), whereas, HIF-1 is rapidly induced at 1 % O
2 to mediate acute responses, and declines to the low levels within 72 h [
40]. This suggests that HIF-2α has a role in the adaptation to chronic hypoxia, where it is the main regulator of erythropoietin production [
41], vascular tumorigenesis [
42], cell proliferation [
43], and vessel remodeling in disease [
44,
45]. Hypoxia is also associated with stem cell phenotype, pluripotent stem cell culture at hypoxic conditions (5 % O
2) stabilize HIF-2α, increase proliferation and stem cell marker expression [
46].
The third major finding was that HIF-2α, a hypoxia-activated factor, is induced in CoRL in the JG and afferent arterioles following UUO. The HIF-2α staining pattern shifting from a cytoplasmic subcellular location before UUO to a nuclear location HIF-2α on day 3 post UUO is very suggestive of HIF-2α changing from its inactive form to an active form. Although this phenomenon preceded the increase in CoRL number in the intersitium, activated HIF2a persisted in a subset of CoRL at all time points studied. There is precedence for HIF in CoRL. Kurtz et al. mimicked chronic hypoxia in CoRL by the deletion of Hippel-Lindau protein, and showed increased HIF-2α expression along the afferent arterioles, glomerular vascular poles, and intraglomerular cells [
15]. Also perhaps relevant to the current studies is that the chronic activation of HIF-2α transforms a subset of cells in the JG compartment into fibroblasts-like cells [
47]. Taken together, we propose that a likely mechanism that underlies the transdifferentiation of CoRL in the JG following UUO is the activation of HIF-2α. Further studies are needed to prove if HIF-2α favors a pericyte-like and/or myofibroblast-like transdifferentiation of CoRL in UUO.
This study has some limitations. First, the study is largely descriptive as we focus on association between HIF-2α and CoRL activation. However there is sufficient literature to support our proposed claim that HIF-2α may be a causative factor leading to CoRL migration and proliferation. JG area has been shown to display marked plasticity in disease settings [
13‐
16]. Also, developmental studies demonstrate that arterioles are the source of pericyte recruitment [
48]. Second, UUO model does not provide functional data [
49,
50]. One may argue that only a small number of CoRL is involved in vascular remodelling in UUO, however in an inducible fate-mapping approach only a fraction of cells is labelled (as we observe glomeruli without labelled CoRL). Importantly, the strength of this approach is that it allows to faithfully track the subpopulation of CoRL that were permanently labeled during tamoxifen induction. Finally, it adds to the pool of known pro-fibrotic progenitors arteriolar-derived myofibroblasts. Third limitation of the study is a gender bias since we have only used female mice. Most of the studies use male mice and rats and the pro-injury effect of androgens in renal injury in males is well documented [
51,
52]. Nevertheless, in our UUO model females demonstrated the classical features of renal injury.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SS, JP, AS: study design, AS: collection and analysis of data, and drafting of the manuscript, SS, KG, JD &JP: critical revision of the article for important intellectual content and final approval of the article. DE&NK: critical roles in immunostaining and generating transgenic mice. All authors read and approve the final manuscript.