STAT-3 signaling role in an experimental model of nephropathy induced by doxorubicin

The focal segmental glomerulosclerosis (FSGS) is one of the most frequent glomerulopathy in the world, being considered a significative public health problem worldwide. The disease is characterized by glomerular loss mainly due to inflammation process and collagen fibers deposition. STAT-3 is a transcription factor associated with cell differentiation, migration and proliferation and in renal cells it has been related with fibrosis, acting on the progression of the lesion. Considering this perspective, the present study evaluated the involvement of STAT-3 molecule in an experimental model of FSGS induced by Doxorubicin (DOX). DOX mimics primary FSGS by causing both glomerular and tubular lesions and the inhibition of the STAT3 pathway leads to a decrease in fibrosis and attenuation of kidney damage. We described here a novel FSGS experimental model in a strain of genetically heterogeneous mice which resembles the reality of FSGS patients. DOX-injected mice presented elevated indices of albuminuria and glycosuria, that were significantly reduced in animals treated with a STAT-3 inhibitor (STATTIC), in addition with a decrease of some inflammatory molecules. 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 pathway possess an important role in experimental FSGS induced by DOX and may be an important molecule to be further investigated.


Introduction
The focal segmental glomerulosclerosis (FSGS) is one of the main causes of chronic kidney disease (CKD) with an steady increase in its prevalence worldwide over the past few decades, having become the most frequent primary glomerular disease that reaches end stages of CKD in the USA [1][2][3][4]. However, the proportion between primary and secondary forms has remained stable for the last 20 years [5]. The estimated incidence of FSGS varies from 1.4 to 21 cases per million population, and in Brazil correspond to 25% of total CKD cases and furthermore is the most frequent glomerulopathy [3,4]. FSGS can occur at any age, being in adults more common in males compared to female, with 5 times higher incidence in black patients when compared to white patients [4]. This disease is characterized by the obstruction of glomerular capillaries, which is mainly due to collagen fibers 1 3 deposition [6,7]. FSGS has several etiologies that are followed by different clinical prognoses that result in different therapeutic approaches [6,7]. Commonly, FSGS can progress to end stage of renal disease with necessity of renal replacement therapies, such as transplant and/or hemodialysis (HD) [8,9] however, the disease has a post-transplant recurrence rate of 30% [10]. 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 [8,9]. 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) [6,[11][12][13]. DN reproduces most sign of FSGS as podocyte foot processes effacement, which is frequently associated with proteinuria as well as the characteristic fibrosis triggering the progression of renal lesions through different pathways [2,6,7,14,15]. Although the DN is well established in the literature, animals of genetically homogeneous lineage are commonly used. The FSGS, however, has a heterogeneous character [16]. Thus, to develop an experimental model compatible with the reality of patients with FSGS, with regard to genetic variability, in this work we used Swiss mice, which are genetically heterogeneous.
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 [25][26][27]. In renal cells, STAT-3 cascade has been related with lesions and progression of some renal diseases [21,24,28]. 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 [29,30]. Whilst STAT-3 already have been linked to renal diseases progression, the specific contribution of this pathway for FSGS evolution is poorly elucidated. Also, in view of the difficulty in treating FSGS, associated with its high rate of mortality and morbidity, the STAT-3 pathway appears as a potential therapeutic target. Therefore, it is extremely important to study in depth the role of this signaling pathway in the progression of FSGS.

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 h 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 accession process 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. [31]. 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). The experiment had duration of 5 days and STATTIC was injected daily until day 5 where blood and renal tissues were collected for several analyses.

Estimative of albuminuria
The albuminuria was estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 10% with correction by urine creatinine levels [32]. 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 Masson's trichrome stain for quantification of 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 to 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 (− 80 °C) 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 [33]. 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:

Statistical analysis
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 (0.01 vs 0.005) albumin/creatinine ratio, n = 10) and significant weight loss (− 5 vs + 3.3 g, n = 10) were observed in the group treated with DOX than control group. 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 (Fig. 1a). STATTIC treatment slightly attenuated the levels of albumin/creatinine ratio (Fig. 1a). The urinalysis also showed lower levels of glycosuria and proteinuria in the group control and mice treated with STAT-TIC in comparison with DOX-injected animals (Fig. 1b and  c). STATTIC treatment alone did not have 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 [26, 28-30, 34, 35].
Initially, we verified that STATTIC administration promoted a reduction in STAT-3 mRNA expression (Fig. 2a). After, we observed in STATTIC-treated group a down regulation of IL-6R mRNAs, despite the fact IL-6 mRNA was also affected by STATIC administration, suggesting a not activation of an inflammation cascade usually reported in chronic kidney diseases [11,12,16] (Fig. 2b and c). In contrast, only for DOX group, the expression of both IL-6 and its receptor remained elevated (Fig. 2b and c).
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 (Fig. 2d). 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 were not identified in both control and STATTIC groups (Figs. 3 and 4). 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 (Fig. 3). Since these histological parameters are important in the progression of DOXinduced nephropathy, the results reported here point up that inhibition of STAT-3 attenuates renal injury which is often present in this experimental model. In order to analyze the fibrosis induced by DOX injection we also evaluate the positive staining for Masson's trichrome (Fig. 5), this analysis showed that DOX-induced a high percentage of Masson's staining which was attenuated by ST injection as we could see in DOX + ST group.

Discussion
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 [31]. Here, we were innovative and by first time, it was adapted a heterogeneous mouse strain (Swiss line) for experimental FSGS that importantly mimic the heterogenicity of FSGS patients, providing tools for more effective investigations [2, 6-8, 14-16, 36-39]. 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 Through urinalysis significant albuminuria differences were detected between the DOX group and the control group and between the DOX and DOX + STATTIC groups. The DOX group had significantly higher urinary glucose and protein concentration than the other groups (p < 0.05). n = 10 animals per study group, p < 0.05 Another novelty presented in this work is the study of the STAT-3 signaling pathway specifically in the experimental model of FSGS. Our results demonstrate the relevance of the STAT-3 pathway in the development of typical FSGS kidney lesions and that its inhibition may have great therapeutic potential in the treatment of the pathology. 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.
In rodent FSGS model as in human, we can observe glomerular and tubular lesions and podocyte foot process effacement. Thus, animals treated with DOX present glomerular sclerosis, obstruction and tubular dilatation by protein clusters and interstitial fibrosis with monocyte infiltration, characteristics which mirror that can be seen in human CKD due to primary focal segmental glomerulosclerosis [9], on the other hand in humans, DOX nephrotoxicity is rare [40].
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 [11,12,41]. 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 [42].
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 [43]. 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 [44]. Moreover, Hunter and Jones [45] 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  [46], beyond other nephropathies, such as IgA nephropathy [47]. 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 nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway), blocking binding to the substrate [48]. 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 [49]. 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 were not observed in control animals e STATTIC-treated group. Tao et al. [50] 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 and positive staining for Masson's trichrome, while the STAT-3 inactivation causes decrease in tubulointerstitial fibrosis and Masson's trichrome staining. 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.

Conclusion
Our study documents a new adapted experimental model of focal segmental glomerulosclerosis (FSGS) utilizing a heterogeneous mouse strain, that provide 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 Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by TCOS, GP. The first draft of the manuscript was written by TCOS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding Thabata Caroline de Oliveira Santos was supported by a grant of CAPES (Coordenação de aperfeiçoamento de pessoal de nível superior) -Brazil.

Data availability
The data analyzed during the study is not publically available but can be available on request to corresponding author.

Declarations
Competing interests The authors have no relevant financial or nonfinancial interests to disclose.
Ethical approval All experimental procedures were carried out following the ethical principles established by the Experimental Brazilian Council (COBEA) and approved by the local Animal Ethics Committee (protocol n°957).