Renal Fibrosis, Immune Cell Infiltration and Changes of TRPC Channel Expression After Unilateral Ureteral Obstruction in Trpc6-/- Mice
Weiying Konga,b,c,d Timo Nicolas Haschlera,d,e Bernd Nürnbergb,c
Stephanie Krämerd,f Maik Gollascha,g Lajos Markóa
aExperimental and Clinical Research Center (ECRC), Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine, Berlin, Germany, bDepartment of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, Tübingen, Germany, cInterfaculty Center of Pharmacogenomics and Drug Research, University of Tübingen, Tübingen, Germany, dMax-Rubner-Laboratory, German Institute of Human Nutrition Potsdam-Rehbrücke, Nuthetal, Germany, eCentre for Nephrology, UCL Medical School, Royal Free Campus, London, United Kingdom, fDepartment of Laboratory Animal Science and Animal Welfare, Justus-Liebig University Giessen, Germany, gCharité Campus Virchow, Nephrology/Intensive Care, Berlin, Germany
Key Words
Trpc channels • TRPC6 • UUO • Renal fibrosis • Inflammatory infiltration
Abstract
Background/Aims: The transient receptor potential cation channel subfamily C member 6 (TRPC6) is a Ca2+-permeable nonselective cation channel and has received recent attention because of its capability to promote chronic kidney disease (CKD). The aims of this study were (i) to examine whether deletion of TRPC6 impacts on renal fibrosis and inflammatory cell infiltration in an early CKD model of unilateral ureter obstruction (UUO) in mice; and (ii) whether TRPC6-deficiency as well as UUO affect the regulation of TRPC expression in murine kidneys. Methods: Wild-type (WT), Trpc6-knockout (Trpc6-/-) and New Zealand obese (NZO) mice underwent sham operation or unilateral ureteral obstruction (UUO). The kidneys were harvested 7 days after surgery. We examined renal fibrosis and inflammatory cell infiltration by histological and immunohistochemical staining. The mRNA expression of TRPC members and markers of fibrosis and inflammation in kidney were assessed by using real-time quantitative reverse transcription PCR. Results: Histological and immunohistochemical analyses revealed less inflammatory cell infiltration (F4/80 and CD3) in UUO kidneys of Trpc6-/- mice compared to UUO kidneys of WT mice as well as less fibrosis. Genomic deletion of TRPC6 also affected the expression of pro-fibrotic genes in UUO Trpc6-/- kidneys compared to UUO WT kidneys while the expression of pro-inflammatory genes did not differ. UUO caused marked up-regulation of Trpc6 and down-regulation of Trpc1 mRNA in kidneys of WT and NZO mice. Trpc3 mRNA expression was significantly elevated in kidneys of Trpc6-/- mice underwent UUO while the levels did not change in kidneys of neither WT nor in NZO mice underwent UUO. Conclusion: TRPC6 contributes to renal fibrosis and immune cell infiltration in the UUO mouse model. Therefore, inhibition of TRPC6 emerges as a promising novel therapeutic strategy for treatment of chronic kidney failure in chronic obstructive nephropathy. However, confounding genomic and non-genomic effects of other TRPC channels should be taken into consideration to fully comprehend the renoprotective potential of targeting TRPC6 therapeutically under chronic kidney damaging conditions.
Introduction
Renal fibrosis is the inevitable consequence of an excessive accumulation of extracellular matrix that occurs in virtually every type of chronic kidney disease (CKD) irrespective of the underlying etiology [1]. Renal fibrosis is the principal process underlying the progression of CKD to end-stage renal disease. It is characterized by glomerulosclerosis and tubulointerstitial fibrosis, and by deposition of excess matrix in the interstitial space surrounding tubules and peritubular capillaries, coupled with the appearance of interstitial fibroblasts [2]. The initiation and progression of renal fibrosis appear to involve a complex, so far incompletely characterized, interaction between injured tubules, pericytes, fibroblasts, endothelial cells and inflammatory cells [2, 3]. Considerable efforts are being made to identify novel drug targets to prevent or halt renal fibrosis, the final common pathway of a wide variety of CKD.
The unilateral ureteral obstruction (UUO) model is a standard and widely used experimental model of renal interstitial fibrosis (for review see [4]). Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, in conjunction with an infiltration of macrophages and other inflammatory cells into the renal interstitium. Proliferation of fibroblasts and transformed myofibroblasts produce excessive extracellular matrix leading to accelerated renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process [4].
Transient receptor potential (TRP) channels have been implicated in renal fibrosis. The classical or canonical TRP channels (TRPCs) are the subfamily most closely related to the founding member of the TRP family, the Drosophila TRP channel [5, 6]. TRPCs are Ca2+-permeable nonselective cation channels encoded by Trpc1-7. While most TRPC subunits can form functional homomeric channels, heteromerization of TRPC channel subunits of either the same subfamily or different subfamilies has been widely observed to extend functional diversity [7-12].
TRPC6 is widely expressed in renal tissues, including glomerular podocytes, mesangial cells, endothelial cells, tubulointerstitial vascular and epithelial cells, as well as renal blood vessels [13]. TRPC6 is one of the podocyte slit-diaphragm proteins associated with proteinuria, which is mainly mediated by Ca2+ influx [14]. TRPC6, nephrin, podocin and CD2-associated protein directly or indirectly interact with α-actinin-4 to maintain the integrity of the glomerular filtration barrier [14, 15]. Podocyte TRPC6 channels play a role in inherited focal segmental glomerulosclerosis (FSGS) [6]. Moreover, angiotensin II (Ang II), known as an important causal driver of chronic kidney disease, can rapidly activate and upregulate the expression of TRPC6 in podocytes. As a result, the intracellular Ca2+ concentration increases and eventually leads to podocyte apoptosis and progressive kidney failure [16, 17].
Interestingly, up-regulation of both TRPC3 and TRPC6 expression has been reported in UUO kidneys [18]. It is suggested that up- or downregulation of other TRPCs could possibly play critical roles in kidneys. For another example, diabetic kidneys show reduced TRPC1 expression [19], which together with increased TRPC6 activity in podocytes could contribute to glomerulopathy [20-22]. Given that the TRPCs have the ability to form homo- and heteromers, it is very likely that other TRPC family members additionally contribute to the formation of nephropathy in mouse and man.
The metabolic syndrome characterized by hypertension, hypoglycemia and lipedema is a complex disease leading to chronic kidney disease and fibrosis, as well. The New Zealand Obese (NZO) mouse represents one of the most thoroughly investigated polygenic models for the human metabolic syndrome and type 2 diabetes. It presents the main characteristics of the disease complex, including early-onset obesity, insulin resistance, dyslipidemia, and hypertension [23-25]. To the best of our knowledge, no studies have been reported on renal expression of TRPC channels in this model and their putative role in CKD progression.
We tested the hypothesis that up-regulation of renal TRPC6 is a common feature in renal fibrosis and immune cell infiltration in mice. We used the UUO model to induce renal fibrosis, immune cell infiltration and analyzed the expression of TRPC channels in the kidneys of wild-type (WT) and Trpc6-knockout (Trpc6-/-) mice. Further we used the NZO mouse model [23-26] to evaluate UUO nephropathy as well as the regulation of TRPC channel expression in an inbred obese mouse strain carrying susceptibility genes for diabetes and hypertension.
Materials and Methods
Animals
Male Trpc6-/-
mice (24.51±2.73 g body weight (b.w.), n=31, 9-12 weeks old) and age-matched WT
controls (25.69±1.79 g b.w., n=32, p>0.05) were used. The Trpc6-/-
mice have been generated on C57BL/6J:129/Sv genetic background and
characterized previously [27]. Since 129Sv and C57BL/6J mice display similar
renal damage in the UUO model [28], we chose C57BL/6J mice as control Trpc6+/+
(WT) mice. Age-matched male NZO mice from our own colony were used, which were
obese
(39.90±4.11
g b.w., n=15, p<0.05 vs. both WT and Trpc6-/- mice)
and carry
susceptibility
genes (from the NZO/BomHIDife genetic background) for obesity, diabetes and
hypertension [23-26]. The mice were housed in groups of three to five and
single housing was applied 7 days before and after the UUO surgery to assure
uniformity. The mice were reared under specific pathogen free (SPF)-conditions
in individually ventilated cages (IVC) with a diurnal 12 h light and dark cycle
(lights on at 06:00 h) at a temperature of 21±1°C. All animals had free access
to water and food. The mice were housed and handled in unity with good animal
practice as defined by the Federation of European Laboratory Animal Science
Associations (FELASA) [29] and the national welfare body GV-SOLAS [30]. Animal
care followed American Physiological Society guidelines [31]. All animal
protocols were approved by local authorities (Landesamt für Umwelt, Gesundheit
und Verbraucherschutz (LUGV) Brandenburg, Germany; Permit-Number: 2347-7-2016).
UUO model
All surgical procedures were performed under aseptic conditions. The mice were anesthetized with 2% isoflurane and placed on a heating pad to prevent hypothermia. After the depth of anaesthesia was confirmed by a loss of reflexes (toe pinch) the anterior abdominal skin was shaved, wiped off with 70% ethanol and 500 mg/kg metamizole (500 mg/ml, WDT) was injected intraperitoneally (i.p.). Eventually a midline laparotomy was conducted via an incision of the avascular linea alba and the left ureter was exposed. The ureter was then ligated twice close to the renal pelvis using a 5-0 polyglycolic acid (PGA) suture wire (Resorba®), and subsequently, 0.05 ml of a 10% enrofloxacine solution (Baytril, Bayer) was applied in the abdominal cavity. Sham operation was performed without ureteral ligation. The linea alba and skin were closed separately. The wound was sanitized with a silver aluminium spray (Henry Schein®) and 1 ml of warm (37°C) isotonic sodium chloride solution (Berlin-Chemie Menarini) was injected subcutaneously (s.c.). Subsequently, each mouse was placed in a cage in front of an infrared (IR) lamp and monitored until they recovered consciousness. For the following two days mice received metamizole (500 mg/ml, Lichtenstein) in their drinking water with a final concentration of 1.33 mg/ml. The tissue harvest occurred 7 days after the surgeries (UUO and sham). For this, mice where sacrificed with an overdose of isoflurane (1 ml/ml, CP-Pharma) and death was confirmed via a lack of reflex formation upon targeted provocation. Left kidneys were excised and decapsulated for further analysis. The kidneys were transversely divided into two portions. Half of the kidney was immersed in a 4% phosphate-buffered paraformaldehyde (PFA) (Sigma) solution for histology, and the other half was snap-frozen in liquid nitrogen for RNA preparation.
Histology, immunohistochemistry and histological analyses
Paraffin-embedded kidneys were cut in three micrometer thick sections followed by deparaffination in sequential steps of xylene, ethanol solutions (100%, 96%, 70%) and a final rehydration step in water. For the morphological and histological analyses, Sirius red (SR) stains and Periodic acid Schiff (PAS) reactions were performed according to the manufacturer’s protocols (Sigma). SR specifically stains collagen type 1 and 3 fibrils and allows a quantification of interstitial fibrosis. The PAS reaction visualizes the basement membranes of the capillary loops of the glomeruli through which the glomerular damage can be evaluated.
For the immunohistochemical (IHC) analysis and detection of proteins of interest the following procedure was conducted: Antigen retrieval was achieved by immersing the samples in boiling sodium citrate buffer (MW-buffer, pH 6.0, S2031, DAKO) in a microwave. This included two 4 min steps and a 5 min step at room temperature in between. In order to block the endogenous peroxidase activity, the tissues were incubated in 3% H2O2 in purified water for 10 min. Furthermore, the tissue was perforated using Tris-buffered saline with tween 20 (TBST) buffer for 15 min and potential unspecific antibody binding was prevented via a 10 min blocking step with DAKO antibody diluent (S3022, DAKO). In between each of these steps, the samples were washed with a TBST buffer for 5 min and a final washing step with phosphate-buffered saline (PBS) before the incubation with the following primary antibodies: anti-F4/80 (rat monoclonal, 1:8000, MCA497GA, Serotec), anti-CD3 (rabbit polyclonal, 1:250, ab5690, Abcam), anti-alpha smooth muscle actin (αSMA) (rabbit polyclonal, 1:500, ab5694, Abcam), anti-vimentin (rabbit monoclonal, 1:2000, ab92547, Abcam), collagen type 4, alpha 1 (Col4α1) (rabbit polyclonal, 1:2000, ab6586, Abcam), anti-proliferating cell nuclear antigen (PCNA) (rabbit polyclonal, 1:1000, ab18197, Abcam) and anti-cleaved-caspase 3 (cCasp3) (rabbit polyclonal, 1:280, 9661S, Cell Signaling). The specificity of the antibodies was ensured by negative and positive controls, as well as secondary antibody controls. All primary antibodies were diluted in DAKO antibody diluent and incubated over night at 4 °C – except for the anti-CD3 antibody whose incubation occurred at room temperature for 1 hour (h). After antibody incubation, the samples were washed three times with PBS (5 min each) and Histofine® Simple Stain™ MAX PO, which uses the 3, 3’-diaminobenzidine (DAB) chromogen, was applied according to the manufacturer’s protocol (Nichirei). This allowed the visualization of the proteins of relevance. Eventually, the tissue samples were counterstained with haematoxylin (Roth) and dehydrated in a sequence of ethanol solutions followed by a final xylene dehydration step. The slides were then sealed with mounting medium (Histokitt, 1025/500, Hecht) and stored until they were imaged.
The kidneys of all animals in each group were analyzed in a blinded manner to minimize the observer bias. Images were taken with an Axio Imager.A1 (Zeiss) microscope at either 20x (all IHC stains) or 40x (PAS and SR). The cortex of each kidney sample was screened thoroughly starting at one end of the tissue and ending at the other. In order to achieve a concise quantification of the area of interest in a semiautomatic manner we used an NIH ImageJ plug-in, which has previously been generated by Gabriel Landini (University of Birmingham). This plug-in allows a colour threshold to be set and moreover, the generation of macros that can be individually applied to all stored images of each stain to guarantee consistency. The mesangial expansion and thus glomerular damage could be visualized with the PAS stain. For this, the PAS+ area of at least 20 glomeruli in each animal was analysed. This was done by assessing the glomerular perimeter and normalizing the positively stained area to the glomerular capillary tuft area. The SR stain allowed the quantification of fibrosis in the renal cortex. At least 15 pictures of each SR-stained kidney were taken and the total positively stained area of each visual field was quantified. The F4/80, vimentin, αSMA, Col4α1 positive areas were measured in the same manner whereas CD3+ as well as PCNA+ cells were counted individually using the ImageJ threshold plug-in as previously mentioned.
Quantitative Real-Time (qRT)-PCR
qRT-PCR was performed as described earlier [32]. Briefly, total RNA was isolated from snap-frozen kidney cortex after homogenization with a Precellys 24 homogenizer (Peqlab) using RNeasy RNA isolation kit (Qiagen). RNA quality and concentration were determined by a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific). Two micrograms of total renal RNA were transcribed to cDNA (Applied Biosystems). Quantitative analysis of target mRNA expression was calculated using the relative standard curve method. TaqMan and SYBR green analysis was conducted using an Applied Biosystems 7500 Sequence Detector (Applied Biosystems). The expression levels were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and eEF1α1 (eukaryotic translation elongation factor 1 alpha 1) and mean Ct values of each groups are reported (Supplementary Fig. 1). Primer sequences are provided in Supplementary Table 1 - for all supplemental material see www.cellphysiolbiochem.com.
Statistical analyzes
Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). All data are presented as mean ± SD and p-values of <0.05 were considered as statistically significant. The p-values in the figures are denoted as follows: ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Data were analyzed by regular two-way ANOVA with Bonferroni’s multiple comparisons test. Data with two groups were tested by two-sided unpaired t-test (data with normal distribution).
Results
UUO induces renal damage and apoptosis
Urinary tract obstruction led to hydronephrosis, caused by urine stasis in the renal pelvis or calyces in the kidneys (Supplementary Fig. 2). The glomerular and tubular basement membrane as well as the brush border of the proximal tubules were visualized by the periodic acid Schiff (PAS) staining. The increases in mesangial matrix deposition in the glomeruli in both WT UUO and Trpc6-/- UUO versus sham kidneys implicates renal damage induced by UUO (Fig. 1A and B). Besides, there were no differences in glomerular injury in WT versus Trpc6-/- kidneys upon UUO indicating similar glomerular damage in both genotypes (Fig. 1A and B). Next, we stained sections of the kidneys with antibodies against proliferating cell nuclear antigen (PCNA) and programmed cell death marker cleaved-caspase 3 (cCasp3). UUO-induced increases in PCNA positive (PCNA+) cells and cCasp3 positive (cCasp3+) cells were clearly visible in the kidneys of both WT and Trpc6-/- mice (Fig. 2A, C). Although there was no difference in the number of PCNA+ cells between UUO WT and Trpc6-/- kidneys (Fig. 2B), less cCasp3 positive cells were found in Trpc6-/- UUO kidneys compared to WT UUO kidneys, indicating that ureteral obstructed Trpc6-/- kidneys show less apoptosis compared to obstructed WT kidneys (Fig. 2D).
Role of TRPC6-deficiency in renal inflammation
UUO induces immune responses such as infiltration of blood cells leading to renal inflammation [4]). Therefore, we determined whether Trpc6-deletion can affect renal immune infiltration as well as mRNA expression of pro-inflammatory markers in the kidneys. Antibodies against the macrophage marker F4/80 and the T-cell marker CD3 were applied in IHC analyses. UUO caused immune cell infiltration in WT and Trpc6-/- kidneys, as assessed by F4/80 positive (F4/80+) areas and the numbers of CD3 positive (CD3+) cells in the respective kidney sections (Fig. 3A-D). However, cellular infiltration was smaller in Trcp6-/- UUO kidneys compared to WT UUO kidneys (Fig. 3A-D) which indicates a protective effect when TRPC6 is absent.
Next, we applied qRT-PCR to analyse mRNA expression of the pro-inflammatory markers interleukin 1 beta (IL1β), interleukin 6 (IL6) and tumor necrosis factor alpha (TNFα). We found that all mentioned markers were significantly increased upon UUO in the renal cortex of both WT and Trpc6-/- kidneys (Fig. 4). Surprisingly no differences between Trpc6-/- UUO and WT UUO kidneys were seen (Fig. 4A-C).
Role of TRPC6-deficiency in renal fibrosis
To examine a possible role of TRPC6-deficiency in renal fibrosis, we performed histological analysis including IHC and measured gene expression by qRT-PCR. A Sirius red (SR) stain was performed and SR positive (SR+) areas were quantified (Fig. 5A, B) to assess the degree of collagen deposition in the kidneys. Sham-treated kidneys of either genotype exhibited only small areas of SR+ areas (Fig. 5A). In contrast, WT UUO kidneys displayed a 6-fold increase in SR+ areas compared to controls. This increase in collagen deposition was smaller in Trpc6-/- UUO kidneys compared to WT UUO kidneys (Fig. 5B). Next, IHC studies were performed using antibodies against collagen type 4 alpha 1 (Col4α1) to determine the level of fibrosis, the mesenchymal marker vimentin to identify epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells and alpha smooth muscle actin (αSMA) to detect myofibroblasts and mesangial cells as indicators of cell types involved in fibrosis (Fig. 5C-H). We observed a significant increase in Col4α1 positive (Col4α1+) area in UUO kidneys compared to sham kidneys of both genotypes (Fig. 5C, D). However, this increase in Col4α1 positive (Col4α1+) area was smaller in Trpc6-/- UUO kidneys compared to WT UUO kidneys (Fig. 5C, D). Furthermore, a marked increase in vimentin positive (vimentin+) area (Fig. 5E, F) and αSMA positive (αSMA+) area (Fig. 5G, H) was observed in UUO kidneys of both genotypes. Again, the increase in vimentin+ mesenchymal cells and αSMA+ myofibroblasts was smaller in Trpc6-/- UUO kidneys compared to WT UUO kidneys (Fig. 5E-H). Taken together, these data suggest a protective effect in renal fibrosis when TRPC6 is absent.
By using qRT-PCR, we analysed the mRNA expression of pro-fibrotic markers in renal cortex, including collagen type 1 alpha 1 (Col1α1), collagen type 3 alpha 1 (Col3α1), collagen type 4 alpha 1 (Col4α1), transforming growth factor beta 1 (TGFβ1) and αSMA (Fig. 6). Whereas UUO caused increased mRNA expression of all pro-fibrotic markers in the renal cortex of WT mice (Fig. 6A-E), Trpc6-deficiency UUO kidneys displayed only a significant increase in Col3α1, Col4α1 and TGFβ1 but not αSMA mRNA levels (Fig. 6B, C, D). When we compared the mRNA expression of the aforementioned markers within the UUO-treated groups, we found that all pro-fibrotic markers except for TGFβ1 were reduced in Trcp6-/- UUO kidneys relative to WT UUO kidneys (Fig. 6A-E) supporting the idea that Trpc6-deficiency is protective in this kidney disease model.
Expression profile of TRPC channels in renal cortex
The genomic absence of one TRPC channel may affect the expression of the remaining which could contribute to the renal outcome. We performed qRT-PCR to first identify the relative expression of TRPC family members (TRPC1-7) in renal cortices of WT and Trpc6-deficient mice. In sham-operated kidneys we found that deletion of Trpc6 was associated with reduced Trpc2, Trpc3 and Trpc4 mRNA expression compared to WT kidneys (Fig. 7A-D). Conversely, Trpc6-deficiency increased Trpc1 mRNA expression. Similarly, Trpc5 mRNA was present in Trpc6-/- kidneys but below detection limit in WT kidneys (Fig. 7E). As reported earlier [18], whereas Trpc6 mRNA expression could be determined in WT kidneys it was below the detection levels in Trpc6-/- kidneys (Fig. 7F). Trpc7 mRNA was not detectable in both WT and Trpc6-/- kidneys, but was present in WT brain tissue, which served as positive control (Supplementary Fig. 3B).
Next, we determined in WT animals whether the relative expression of TRPC isoforms is affected by UUO. Interestingly, ureter obstruction did not affect the relative mRNA expression levels of most TRPC family members including Trpc2, Trpc3, Trpc4, and Trpc5 (Fig. 7B,C,D,E). In contrast UUO provoked an approximately 50% downregulation of Trpc1 and notably a 60% upregulation of Trpc6 mRNA expression in WT kidneys (Fig. 7A and F) relative to sham-operated WT animals.
On the other hand, Trpc6-deficiency provoked a highly significant decrease of Trpc2 and Trpc4 transcript levels in both sham and UUO kidneys (Fig. 7B,D) whereas Trpc1 and Trpc3 mRNA levels showed significant differences under sham conditions compared to WT (Fig. 7A,C), in UUO kidneys their level was similar. In addition, Trpc5 transcript levels were below detection levels in the presence and absence of Trpc6 (Fig. 7E), and as expected in Trpc6-/- kidneys lacking Trpc6 mRNA (Fig. 7F).
UUO in NZO mice
We next used the complex disease NZO mouse model to evaluate UUO nephropathy and regulation of TRPC channel expression in an inbred obese mouse strain carrying susceptibility genes for diabetes and hypertension. In NZO mice, UUO caused also significant damage to the kidney. Histological analyses showed that UUO induced increases in PAS+ area, PCNA+ cells, SR+ area, F4/80+ area and CD3+ cells (Fig. 8). Furthermore, we detected an UUO-induced increase in mRNA expression of the pro-fibrotic markers Col1α1, Col3α1, Col4α1 and TGFβ1 (Fig. 9A) and the pro-inflammatory markers IL1β, IL6, TNFα, ICAM1, VCAM1 and MCP1 relative to the sham-operated group (Fig. 9B).
We also examined the relative expression profile of TRPC genes in kidneys of NZO mice in sham and UUO kidneys by qRT-PCR. NZO sham kidneys expressed mRNA of six TRPC genes in renal cortex (Fig. 10). UUO caused, similarly to the previous findings in WT mice, up-regulation of Trpc6 mRNA expression while Trpc1 and Trpc5 mRNA expression was down-regulated. There was no change in Trpc2 and Trpc3 mRNA expression in NZO kidneys upon UUO surgery (Fig. 10). Noteworthy, Trpc7 mRNA was not detectable in NZO kidneys without and in response to UUO (Fig. 10), which is in line with our findings in WT and Trpc6-/- kidneys (Supplementary Fig. 3).
Discussion
Renal fibrosis and inflammation
Mechanisms that promote kidney disease progression include renal atrophy, fibrosis and increased leukocyte infiltration into the kidneys [33, 34]. Renal fibrosis and inflammation are two histological hallmarks of progressive kidney disease and specific antibody staining is a common tool to estimate renal damage [35, 36]. UUO is an established model for renal fibrosis and chronic kidney failure [4]. TRPC6, which is regarded a major slit diaphragm-associated channel, can be increasingly detected in various kidney diseases such as FSGS [37, 38], diabetic nephropathy [22, 39-41], minimal change nephrosis [38, 42] and membranous nephropathy [42-44]. In previous studies, TRPC6 was reported to contribute to fibroblast-to-myofibroblast transdifferentiation (FMT) and is thought to promote tissue scarring [45-47]. Some study suggest that TRPC6 as a non-selective cation channel can be responsible for increased Ca2+ flux in myo-/fibroblasts [48]. Recent studies show that TRPC6 is involved in the pathogenesis of kidney fibrosis and genomic inhibition of the channel can reduce rat kidney fibroblast proliferation and myofibroblast differentiation in vitro [49]. As our study was undergoing, Wu, et al [18]. reported that inhibition of TRPC6 can ameliorate renal fibrosis in the mice based on UUO model. In the present study, we used Trpc6-/- mice and equally carried out UUO surgery to induce early stage CKD. We found similarly to Wu et al. that Trpc6 loss reduces renal fibrosis in the murine UUO model. In addition, we were able to show that TRPC6-deficiency caused a decreased mRNA expression of multiple pro-fibrotic genes. Our results therefore confirm the previous finding that Trpc6 deletion attenuates UUO-induced kidney fibrosis in mice [18].
The renal damage occurring in CKD is, at least in part, promoted by the immune system [50]. Most kidney diseases involve an accumulation of immune cells of the innate (e.g. macrophages and neutrophils) and adaptive (e.g. T-cells) immune system. In particular, tubulointerstitial monocyte immune infiltration can mediate tissue remodeling that ultimately promotes fibrogenesis, renal atrophy and thus, kidney failure [51-53]. Our results revealed a less infiltration of CD3 cells and F4/80+ area in UUO Trpc6-/- kidneys compared to those in UUO WT kidneys. We have therefore performed qPCR analysis in our model as well to quantify inflammatory cytokine levels. Whereas, UUO provoked an increase in mRNA expression of the pro-inflammatory markers in both Trpc6-/- and WT kidneys we did not find a significant beneficial effect of lacking Trpc6 on the expression of pro-inflammatory markers at the mRNA level upon UUO surgery. This might be because we collected tissues only at day 7 after induction of UUO and therefore we might have missed the time-point where lacking Trpc6 influence pro-inflammatory gene expression. On the other side it lacking Trpc6 can influence immune cell function and therefore pro-inflammatory cytokine expression [54]. Taken together, our data point to a complex (in-)direct contribution of TRPC6 to renal inflammation in the UUO model.
To estimate renal apoptosis as well as regenerative potential, kidney sections were stained with antibodies against the apoptosis marker cleaved-caspase 3 (cCasp3) and the mitosis marker proliferating cell nuclear antigen (PCNA). Since cCasp3 and PCNA levels correlate positively with renal injury, they are commonly used to determine the degree of renal damage under chronic damaging conditions [55-57]. It is known that chronic activation of TRPC6 is positively correlated with the rate of apoptosis in cells such as podocytes [58, 59] and endothelial cells [60]. Indeed, we found less cCasp3 positive cells in kidneys of Trpc6-/- mice underwent UUO. It might however reflect the less inflammatory cell infiltration, since cCasp positive cells were count in the whole kidney section irrespective of the signal’s origin. PCNA is known as a probe for the detection of cell proliferative activity [61]. In our study, PCNA+ cells were found to be increased after UUO in both WT and Trpc6-/- kidneys. Nevertheless, we did not find significant difference in PCNA+ cells between WT and Trpc6-/- mice. Correlated with the result of cCasp3 cell infiltration, it might be due to an imbalance between cell apoptosis and proliferative activity, or it might be because PCNA is also regulated by other factors, e.g. p53[62] and DNA damage binding protein 2 (DDB2)[63]. In summary, the results indicate that TRPC6-deficiency protects renal cells from UUO-induced apoptosis.
In summary – based on our key findings of reduced renal fibrosis and inflammation after UUO in the Trpc6-/- mice, we believe that these changes are associated with improved kidney function, which ultimately should delay CKD progression to end stage renal disease. Therefore, we suggest that non-cell specific TRPC6 inhibition seems to be meaningful therapeutic approach to improve the outcome of CKD. However, further studies are needed to evaluate this approach in other experimental models of CKD.
TRPC6 and other TRPCs
We further revealed that TRPC6-deficiency per se impacts on renal TRPC channel expression in mice as indicated by reduced mRNA expression of TRPC2, TRPC3 and TRPC4 and increased mRNA expression of TRPC1. Moreover, we found that UUO caused reduced Trpc1 mRNA and increased Trpc6 mRNA expression without changes of Trpc3 expression in WT kidneys. Collectively, the data suggest that counterbalanced or increased expression of TRPC1 and TRPC6 could play a role in UUO-induced kidney damage. Since genomic deletion of Trpc6 improved renal fibrosis and was associated with an increase of Trpc1 mRNA expression and a reduction of Trpc2, Trpc3 and Trpc4 mRNA expression in sham-operated kidneys to levels similar or greater than those observed in UUO kidneys, we conclude that TRPC6 could drive renal fibrosis with or without modifying effects of TRPC1/2/3/4. Our studies revealed an unrecognized renal counter-regulation of TRPC5 and TRPC7 gene expression. This conclusion is supported by findings that TRPC5 and TRPC7 is not expressed in normal kidneys of mice [64].
Of note, the data in the present study shows that the deficiency for TRPC6 is associated with reduced Trpc3 mRNA expression in Trpc6-/- kidneys compared to WT kidneys, which disappears in obstructive nephropathy. At a first glance, this is surprising as we observed in our previous studies on Trpc6-/- mice that loss of TRPC6 is associated with up-regulation of constitutively active TRPC3-type channels in some arteries [27]. However, to our knowledge, TRPC channels (TRPC1-7) expression and resulting putative (functional) counterbalancing effects have not been investigated in experimental obstructive nephropathy. This is of particular interest for TRPC3, which is capable of contributing to fibrogenesis and tissue inflammation [65-69]. Additionally, Wu et al. reported that TRPC3 mRNA expression is upregulated in UUO-induced renal fibrosis [18]. However, we could not detect an increase in TRPC3 expression level in WT kidneys (neither in our NZO mouse model) which argues against an important role in progression of fibrosis in obstructive nephropathy. Based on our findings, it is unlikely that increased TRPC3 expression upon UUO accounts for the beneficial role of TRPC6-deficiency in this model of kidney failure. These mechanisms might be particularity relevant for inherited FSGS caused by mutations in TRPC6 [6, 70]. This conclusion is supported by recent findings demonstrating that knockout of both TRPC3 and TRPC6 did not diminish UUO-induced fibrosis more than deletion of TRPC6 alone [18]. BTP2, an inhibitor of several TRPC channels, including TRPC3 and TRPC6, had an effect similar to that of Trpc6 knockout on fibrosis, but also attenuated the up-regulation of TRPC6 expression, suggesting that TRPC6 channel activity may induce its own gene expression in the UUO model [2] and promote fibrosis in this model. Nevertheless, future studies using more specific TRPC3/6 blockers are necessary to clarify the contribution of TRPC3 in the absence and presence of functional TRPC6 channels. Recently, novel TRPC6 blockers have been developed [71] [72] [73], which could represent important tools to evaluate the role of TRPC6 in these conditions.
Diabetic nephropathy
Today, 25-40% of diabetic patients suffer from diabetic nephropathy [74, 75]. Thus, diabetic patients can be considered as high-risk patients for the development of chronic kidney disease. Diabetic nephropathy shows pronounced glomerular changes that are present in patients with long-standing diabetes often even before the detection of albumin in the urine. Several mechanisms that can lead to diabetic nephropathy are under discussion. We evaluated whether UUO-operated NZO mouse model could represent a model of progressive nephropathy in co-morbid conditions of obesity, hypertension and type 2 diabetes [24], which had not been reported before. Our study shows that UUO can successfully induce renal fibrosis and inflammatory infiltration in the NZO mouse model (NZO/HIBomDife) carrying susceptibility genes for obesity, diabetes and hypertension.
More importantly, our study also suggests that Trpc1-4 and Trpc6 expression in NZO mice underwent UUO/sham-operation showed similar tendency to those observed in WT mice, which verified the regulation of TRPC channels in a second mouse model. Interestingly, we found that Trpc5 expression was detected in the kidneys of NZO mice, which indicated that TRPC5 could be specifically involved in this process in NZO mice. Our results suggest that the UUO-operated NZO mouse model might represent a valuable mouse model to be used to evaluate the specific contribution of TRPC6 and other TRPC channel subfamilies in the renal injury upon obesity, hypertension and diabetes. This approach could take advance of testing better, i.e. TRPC subfamily specific blockers and agonists. In spite of recent progress in this field [71-73, 76], we are optimistic that those drugs are becoming available in the near future for in vivo testing.
Conclusion
Our study observed that inhibition of TRPC6 is associated with a decrease in inflammatory cell infiltration in murine UUO model for the first time. Also, the findings are in line with the concept that up-regulation of TRPC6 contributes to fibrosis in UUO kidneys. The results support the view that increased TRPC6 expression can play an important pathophysiological role in the development of progressive kidney disease [14, 38, 77, 78]. Moreover, the present results imply that the genomic loss of TRPC6 is associated with dysregulation of multiple TRPC channels (TRPC1-4) under chronic kidney damaging conditions, which could represent additional drivers of renal fibrosis and chronic kidney disease. Our results show that UUO causes also up-regulation of TRPC6 in kidneys of NZO mice, which implies that inhibition of TRPC6 is also a promising therapeutic strategy for treatment of renal fibrosis and immune cell infiltration in polygenic models for the human metabolic syndrome.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (B.N. and M.G.). We thank Elisabeth Meyer, May-Britt Köhler, Juliane Anders and Jana Czychi for help and expert technical assistance. M. Gollasch and L. Markó jointly supervised this work.
All animal protocols were locally approved by Landesamt für Umwelt, Gesundheit und Verbraucherschutz (LUGV) Brandenburg, Germany. (Permit-Number: 2347-7-2016).
All authors planned and designed experimental studies. W.K. and T.H. performed the experiments and analyzed the data. M.G., L.M. and S.K. contributed reagents/materials/analysis tools. W.K. and M.G. drafted the manuscript and all authors contributed to its completion. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charité – Universitätsmedizin Berlin.
Disclosure Statement
The authors have no conflicts of interest to declare.
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