Stat-3 Signaling Role in an Experimental Model of Nephropathy Induced by Doxorubicin.

doi:10.21203/rs.3.rs-1303649/v1

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Stat-3 Signaling Role in an Experimental Model of Nephropathy Induced by Doxorubicin.

https://doi.org/10.21203/rs.3.rs-1303649/v1

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Focal Segmental Glomerulosclerosis (FSGS) is one of the most frequent glomerulopathies, and is considered a public health problem worldwide. FSGS is characterized by glomerular loss mainly due to inflammation and collagen fibers deposition. STAT-3 is a transcription factor associated with cell differentiation, migration and proliferation. Its activation occurs in response to several growth factors and cytokines. In renal cells, STAT-3 has been related with disease progression. Considering this perspective, the present study evaluated the involvement of STAT-3 in an experimental model of FSGS induced by Doxorubicin (DOX). First, we described a novel FSGS model in Swiss mice that after DOX administration showed typical signs of kidney dysfunction like higher proteinuria. Since there were no studies of ADM nephropathy model in heterogeneous mice, we were the first to evaluate the model in this lineage. Control animals treated with STAT-3 inhibitor (STATTIC) presented lower levels of albumin/creatinine ratio, glycosuria e proteinuria while DOX-injected mice showed higher levels. After analyzing some molecules involved in the STAT-3 signaling pathway, it was observed that STAT-3 blockade decreased levels of STAT-3, IL-6 and IL-6R, which remained elevated in DOX-administrated mice. Moreover, we detected that SOCS-3 (a regulator of STAT family) was up-regulated only in STATTIC-treated mice. Finally, histopathological analyzes showed that DOX-treated group had a significant increase in a tubulointerstitial fibrosis and tubular necrosis, which were not identified in both control and STATTIC groups. Thus, our results indicate that STAT-3 can have an important role in experimental FSGS induced by DOX and further studies are encouraged.

Focal Segmental Glomerulosclerosis

stat-3

nephropathy and doxorubicin

The Focal Segmental Glomerulosclerosis (FSGS) is one of the main causes of chronic kidney disease (CKD), being considered a public health problem worldwide [1–3]. In Brazil, FSGS correspond to 25% of total CKD cases and furthermore is the most frequent glomerulopathy [3]. This disease is characterized by the obstruction of glomerular capillaries, which is mainly due to collagen fibers deposition [4, 5]. FSGS has several etiologies that are followed by different clinical prognoses [4, 5]. Commonly, FSGS can progress to end stage of renal disease with necessity of renal replacement therapies, such as transplant and/or hemodialysis (HD) [6, 7]. Although HD is widely used as principal therapy, at the same time, it possesses high annual cost for the public health system and is also associated with high morbidity and mortality [6, 7]. In this context, the search for understanding internal molecular mechanisms involved in the FSGS physiopathology is necessary to explore more effective treatment options.

In experimental models, FSGS can be mimicked trough the administration of chemotherapeutic agents like Doxorubicin hydrochloride (DOX), and this model is known as Doxorubicin nephropathy (DN) [4, 8–10]. DN reproduces most sign of FSGS as podocyte foot processes effacement, which is frequently associated with proteinuria triggering the progression of renal lesions through different pathways [2, 4, 5, 11, 12]. One major related cellular mechanism are the induction of tubular chemokines expression and complement system activation, which contribute to fibrosis and infiltration of inflammatory interstitials cells (i.e., macrophages) [13]. In addition, CKD induced proteinuria is close associated with the activation of STAT signaling pathways (signal transducer and activator of transcription) [14–17], where STAT-3 play a pivotal role during inflammatory processes [16, 18–21].

STAT-3 is a transcription factor associated with cell differentiation, migration and proliferation, and its activation occurs in response to several growth factors and cytokines [21–23]. In renal cells, STAT-3 cascade has been related with lesions and progression of some renal diseases [17, 20, 24]. Specifically, the role of STAT-3 in renal diseases has been evaluated in some studies, which have pointed to a possible participation in the progression of CKD through the modulation of interstitial fibrosis [25, 26]. Whilst STAT-3 already have been linked to renal diseases progression, the specific contribution of this pathway for FSGS evolution is poorly elucidated. Considering this perspective, this present study evaluated the involvement of STAT-3 signal in an experimental model of FSGS induced by Doxorubicin.

Animals

Male swiss mice (Mus musculus) with age of 8-10 weeks were obtained from the Biological Sciences Sector of the Federal University of Paraná. The animals were housed in collective boxes, containing five mice per box, with light/dark artificial cycle of 12 hours at a constant ambient temperature of about 20°C and with free water and food supplies. The Ethics Committee of the Federal University of Paraná-Biological Sciences Center (CEUA/BIO-UFPR) approved the procedures for the use of animals under the number 957.

Experimental design

In order to induce the renal injury model in heterogenous mice, it was used the chemotherapeutic agent Doxorubicin hydrochloride (DOX), popular known as Adriamycin (Adriblastina®, Pfizer™, Inc. New York, USA). 25 mg/kg of DOX was injected in the caudal vein following the protocol of Jeansson et al [27]. To inhibit the STAT-3 molecular signal, the STATTIC (Abcam ™, Inc., Cambridge, MA) was administered intravenously at 2 mg/kg daily during all experiments. The animals were divided into four groups: 1) control group; 2) DOX group (treated with DOX only); 3) DOX + STATTIC group (treated with DOX and STATTIC); 4) STATTIC group (treated with STATTIC only). After five days mice were euthanized and biological samples (serum and kidney) were collected (Figure 1).

Estimative of albuminuria

The albuminuria was estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 10% with correction by urine creatinine levels [28]. The method consists in the migration of charged particles under the influence of an electric field in a viscous matrix. Then, the urine was diluted and mixed with a buffer solution (with mercaptoethanol as reducing agent). After, the gel was stained with Coomassie blue and albuminuria quantification was performed on a General Electric® Amersham Imager 600 photo documenter.

Urinalysis with reagent strips

For uranalysis the UroAction 10® uranalysis kit from Labtest (Labtest Diagnostics S.A. MG, Brazil) was used. This reagent consists in strips for rapid and semi-quantitative determination of glucose, bilirubin, ketone bodies (ketoacetic acid), density, blood, pH, protein, urobilinogen, nitrite and leukocytes in urine by colorimetric method.

Histological analysis

Histological analyzes were performed using double-blind tests. Kidneys fragments were stained with hematoxylin and eosin (H&E) for quantification of glomerular, tubular lesions and interstitial fibrosis under an optical microscope. Twelve random fields of each slide were analyzed using a photographic camera coupled to the computer containing the Zeiss® AxionVision software (Carl Zeiss, Oberkochen, Germany). For each field a semi-quantitative score was considered: 0, when there were no alterations; mild when had present alterations about 10%; moderate for alterations from 10–50%; and intense when changes were more than 50%. The pathological parameters evaluated were acute tubular necrosis, cellular and intracellular vacuolization, tubular collapse and distension and presence of inflammatory cells. Tubulo-interstitial fibrosis was characterized by expansion of the cellular matrix with distortion, collapse and thickening of the basement membrane. Alterations were considered positive when detected in more than five cells per analyzed field.

Real time PCR

For quantitative real-time PCR (qPCR), total RNA was extracted from frozen kidneys (-80ºC) using PureZOL® (Bio Rad, California, USA). The cDNA was synthesized using the reverse transcriptase enzyme (Bio Rad, California, USA) and the qPCR was performed using SYBR Green® kit (Bio Rad, California, USA). We use HPRT as housekeeping gene and the results were analyzed by the relative quantification method according to the formula 10,000/2^Δct [29]. The primers to quantify the mRNAs for STAT-3, IL-6, IL-6R and SOCS-3 were synthesized using Primer Express software (Applied Biosystems, USA) based on the known sequence of nitrogen bases described in GenBank. The sequences of primers are as follows:

Stat3 forward 5’- GGATCGCTGAGGTACAACCC- 3’;
Stat3 reverse 5’- GTCAGGGGTCTCGACTGTCT- 3’;
IL-6 forward 5’- CCTCTGGTCTTCTGGAGTACC- 3’
IL-6 reverse 5’- ACTCCTTCTGTCACTCCAGC- 3’
IL-6 R forward 5’- AAGAGTGACTTCCAGGTGCC- 3’
IL-6 R reverse 5’- GGTATCGAAGCTGGAACTGC- 3’
SOCS3 forward 5’- GGGTGGCAAAGAAAAGAAG- 3’
SOCS3 reverse 5’- GTTGAGCGTCAAGACCCAGT- 3’

Statistical analyzes

Data are reported as means (SEM). All data were initially tested for normality by the Kolmogorov–Smirnov test. For parametric variables, differences between 2 groups were evaluated by the unpaired t-student test and for non-parametric variables, the Mann-Whitney test was utilized. In the case of more than two groups, the one-way analysis of variance (ANOVA) analysis was performed. For samples that did not present normal distribution, was used analysis of variance of single-point ANOVA, by the Kruskal-Wallis test. In the case of difference, the groups were compared with Student-Newman-Keuls correction for parametric variables and Dunn's correction for non-parametric variables. Differences were considered significant if the p value was < 0.05. Statistical analyzes were performed using GraphPad Prism^® 6 software (GraphPad Software, San Diego, California - USA).

STAT-3 signaling pathway inhibition prevent kidney disfunction

To validate our experimental model of focal segmental glomerulosclerosis (FSGS), we firstly injected doxorubicin hydrochloride (DOX) in animals and observed typical renal parameters of chronic kidney diseases (CKD). After 5 days, an intense increase in albuminuria and significant weight loss were observed in the group treated with DOX than control group (Figure 2). After to assess the role of STAT3 signaling pathway in DOX-induced nephropathy, we treated animals with STAT-3 inhibitor, STATTIC (known to inhibit STAT-3 phosphorylation by blocking dimers formation and consequent STAT-3 activation). We observed a significant albuminuria index in DOX treated-groups when compared to control group (Figure 3-A). STATTIC treatment slightly attenuated the levels of albumin/creatinine ratio (Figure 3A). The urinalysis also showed lower levels of glycosuria and proteinuria in the group control and mice treated with STATTIC in comparison with DOX-injected animals (Figure 3B and 3C). STATTIC treatment alone did not had substantial effect for all analysis.

STAT-3 blockade regulates renal inflammatory cytokines expression

In order to understand the mechanism behind of renal protection during STAT-3 inhibition, we evaluated major renal pro-inflammatory cytokines which are predicted to be involved in FSGS progression [22, 24–26, 30, 31]. Initially, we verified that STATTIC administration promoted a reduction in STAT-3 mRNA expression (Figure 4A). After, we observed in STATTIC-treated group a down regulation of IL-6R mRNAs, despite the fact IL-6 mRNA was t affected by STATTIC administration, suggesting a not activation of an inflammation cascade usually reported in chronic kidney diseases [8, 9, 32] (Figure 4B and 4C). In contrast, only for DOX group, the expression of both IL-6 and its receptor remained elevated (Figure 4B and 4C).

Moreover, we analyzed SOCS-3 expression (a pivotal regulatory molecule) during this kidney inflammatory process induced by DOX. The result indicated that treatment with STAT-3 inhibitor up-regulated the SOCS-3 mRNA expression, considering that control and DOX group demonstrated reduced values (Figure 4D). Thus, it is possible suggests that STATTIC inhibit the STAT-3 signaling pathway by two independent mechanisms: i) direct blocking of STAT-3 signal and ii) by activation of SOCS3 which also attenuate STAT-3 cascade.

Inhibition of STAT-3 cascade protect kidney from tissue injury

The histopathological analyzes of the kidneys showed that DOX-treated group had a significant increase in a tubulointerstitial fibrosis and tubular necrosis. The same alterations did not were identified in both control and STATTIC groups (Figure 5 and 6). These findings indicate that treatment with DOX effectively induces a severe nephropathy with dramatic alteration in renal architecture. On the other hand, the treatment with STAT-3 inhibitor significantly decreased or prevented the tubule-interstitial fibrosis and tubular necrosis. Finally, as consequence of FSGS progression is expected an exacerbated infiltrating process with classical inflammatory cells presence in renal tissues. This event only was observed in DOX-injected animals and STATTIC administration blocked this process (Figure 5). Since these histological parameters are important in the progression of DOX-induced nephropathy, the results reported here point up that inhibition of STAT-3 attenuates renal injury which is often present in this experimental model.

The focal segmental glomerulosclerosis (FSGS) is a complex renal disease characterized by different kind of lesions with distinct progression and responses to treatments. The search for a representative experimental model is fundamental to deep understand the course of disease. Balb/c mice is the classic strain used for FSGS experimental model, however others strain as C57/black 6 was also adapted presenting similar dysfunctions to classic model [27]. Here, we were innovative and by first time, it was adapted a heterogeneous mouse strain for experimental FSGS, Swiss line, that importantly mimic the heterogenicity of FSGS patients, providing tools for more effective investigations [2, 4–6, 11, 12, 32–36]. We observed in our novel experimental model that treatment with DOX caused typical FSGS signs (higher proteinuria and weight loss) in mice representing a new FSGS model in the literature, since the model of DOX nephropathy has not been yet developed in this lineage yet.

Considering that FSGS was adapted in our Swiss mice, we decided to investigate the participation of STAT-3 in our model of nephropathy induced by DOX. Proteinuria, especially albuminuria, is one of the main signs found in the model of DOX-induced nephropathy. Albuminuria is due to alterations in the glomerular filtration barrier and oxidative damage in the podocytes caused by DOX [8] [9, 37]. In our study, significant albuminuria was observed in animals injected with DOX and treatment with STATTIC significantly decreased not only albuminuria but also glycosuria in these animals. In addition, we detected that STATTIC modulated STAT-3 levels and was possible to pointed out the STAT-3 signal molecule as an important marker for experimental FSGS progression [38].

In order to evaluated the dynamic of STAT-3 signaling, we searched for pro-inflammatory cytokines present in this pathway and that could be altered in our model. Initially, we verified elevated IL-6 levels in DOX-treated animals. In contrast, the inhibition of STAT-3 signal caused a dramatic reduction in its expression. Interleukin-6 has a pleiotropic action on several biological functions and can be secreted by many cell types through stimuli such as infection, inflammation or cancer [39]. For instance, The secretion of IL-6 by monocytes and macrophages following the activation of Toll-like receptors is especially important in cases of inflammation [40]. Moreover, Hunter & Jones [41] emphasized that there is extensive literature showing that IL-6 modulates various aspects of the innate immune system, including hematopoiesis and neutrophil accumulation at sites of infection or trauma. Thus, it is possible to infer that DOX causes inflammation, with a possible infiltration of macrophages, which in our study can be represented by elevated index of inflammatory cells in renal tissues. These cells are responsible to increase of IL-6 mRNA expression that is frequently observed in patients with primary FSGS associated with cutaneous and systemic plasmacytosis [42], beyond other nephropathies, such as IgA nephropathy [43]. In line with this, it was also detected significant differences in IL-6 receptor gene (IL-6R) expression between the DOX-injected and STATTIC-treated animals. The blocked of IL-6/IL-6R axis by inhibition of STAT-3 suggests a possible modulation of inflammation with consequent preservation of renal tissue and function. Furthermore, we observed that the treatment with STAT-3 inhibitor resulted in great increase of cytokine signaling suppressor type 3 (SOCS-3). SOCS-3 down regulates the JAK/STAT signal by binding simultaneously to glycoprotein Gp130 (one subunit of the type I cytokine receptor within the IL-6 receptor family) and JAK (a non-receptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway), blocking binding to the substrate [44]. Thus, these findings can be interpreted as a cellular response for modulate inflammation caused by STAT-3 activation.

Finally, is reported that renal inflammation can trigger renal fibrosis cascade and impair kidney function [45]. Then is expected that DOX-mediated inflammation may promote fibrosis in FSGS experimental model. According to this we observed that DOX-administrated mice demonstrated consistent signs of tubulointerstitial fibrosis and tubular necrosis which did not were observed in control animals e STATTIC-treated group. Tao et al [46] using microscopic and transcriptomic evidences also identified glomerular and tubulointerstitial STAT-3 activation in renal biopsy of FSGS patients. Additionally, Bienaimè [25] yet demonstrated an important role for STAT-3 in tubulointerstitial communication during chronic kidney disease. The authors reported that STAT-3 activation promotes accumulation of interstitial matrix and fibroblasts, leading to increasing of tubulointerstitial fibrosis, while the STAT-3 inactivation causes decrease in tubulointerstitial fibrosis. Thus, our results indicate that STAT-3 pathway possess an important role in experimental FSGS induced by DOX and may be an important molecule to be investigated in further studies.

In summary our study documents a new adapted experimental model of focal segmental glomerulosclerosis (FSGS) providing a new platform for study of chronic nephropathy. All together our findings also suggest that STAT-3 signaling pathway participate actively of FSGS progression and its inhibition protects mice from kidney disfunction and injury. Although more precise mechanism should be investigated in relation to central role of STAT-3 in FSGS progression (i.e., up/down stream associated molecules), we believe that STAT-3 may be a predicted therapeutic target with great potential in the treatment of experimental FSGS.

Funding

Thabata Caroline de Oliveira Santos was supported by a grant of CAPES (Coordenação de aperfeiçoamento de pessoal de nível superior) - Brazil.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the development of the study.

Data availability

The manuscript has no associated data

Ethics approval

The Ethics Committee of the Federal University of Paraná-Biological Sciences Center (CEUA/BIO-UFPR) approved the procedures for the use of animals under the number 957.

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You are reading this latest preprint version

Stat-3 Signaling Role in an Experimental Model of Nephropathy Induced by Doxorubicin.

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals

Experimental design

Estimative of albuminuria

Urinalysis with reagent strips

Histological analysis

Real time PCR

Statistical analyzes

Results

Discussion

Conclusion

Declarations

References

Status:

Version 1