Increased expression of Toll-like receptors 2, 3, 4 and 7 mRNA in the kidney and intestine of a septic mouse model

Toll-like receptors (TLRs) are the key regulators of innate and adaptive immunity and are highly expressed during sepsis. Thus, studying the expression of TLRs in an animal septic model might indicate their possible association with acute kidney injury in sepsis. Seventy-two male C57BL/6J mice were used for this study. Randomly, these animals were divided into 6 groups (N = 12/group): 3 control and 3 septic groups depending on the euthanasia time (24 h, 48 h, 72 h). Septic groups underwent cecal ligation and puncture (CLP) to induce peritonitis, while control groups had a sham operation. Hematological tests were performed in serum for immune biomarkers; immunohistochemistry, morphometry and qRT-PCR analysis were used on both kidney and intestine tissues to evaluate the expression of TLR 2, 3, 4 and 7 in a septic process. At the end of each experimental period, we found that TLRs 2, 3, 4 and 7 were expressed in both tissues but there were differences between those at various time points. Also, we found that mRNA levels were significantly higher in qRT-PCR evaluation in septic groups than control groups in both kidney and intestinal tissues (p < 0.05); showing a steady increase in the septic groups as the time to euthanasia was prolonged (p < 0.05). Overall, our study provides a suggestion that TLRs 2, 3, 4 and 7 are highly expressed in the kidneys of septic mice and especially that these TLRs are sensitive and specific markers for sepsis. Finally, our study supports the diagnostic importance of TLRs in AKI and provides an insight on the contribution of septic mice models in the study of multi organ dysfunction syndrome in general.

infection in a way that resembles the human response [6][7][8] . Toll-like receptors (TLRs) are germ-line-encoded type I transmembrane proteins expressed in various immune as well as non-immune cells and belong to a family of pattern recognition receptors (PRPs) 9 . TLRs recognize and are activated by certain pathogen-associated molecular patterns (PAMPs) 10 , such as lipopolysaccharides, lipoproteins, peptidoglycans) leading to stimulation of the innate immune system and subsequently to activation of antigen-specific adaptive immunity 11 . The characterization and expression of these receptors is, therefore, essential in understanding the pathophysiology of sepsis and more specifically the related organ dysfunction 12 .
Sepsis is one of the most common causes of acute kidney injury (AKI) 13 . For many years, it was thought that sepsis-related hemodynamic alterations in the macrocirculation resulting in reduced renal perfusion were responsible for this phenomenon. This mechanism has been outdated, and a more complex pathway is suggested; changes in the microcirculation of the kidney along with an exacerbated inflammatory response propose a more accurate, although not completely clarified, theory 14 . This strongly suggests the presence of a common pathway between the initial triggers of tubular cell injury and the inflammatory response in the kidney 15,16 .
Within the kidney different cell types express some of the TLR system proteins. In bacterial infections affecting the kidney, upregulation of TLRs -2, -3 and -4 and subsequent C-C chemokines secretion has been described 17 . Thus, TLR activation may be the common denominator amongst various forms of tubular cell injury and, more specifically, the trigger of the "innate" immune response leading to AKI in a septic state, such as during a CLP mouse model 13,18 .
In the present study, we examined the pathogenic mechanism of AKI in relation to TLR expression. Thus, we used a septic mouse model which is representative of a clinical patient's situation 19 to determine the role of TLRs 2, 3, 4 and 7 in the severity of sepsis, as well as its association with multi-organ dysfunction syndrome triggered by AKI.

Materials and Methods
Animal study and care. Seventy Experimental Design and establishment of CLP Procedure. A clinically relevant mice model of sepsis was created by cecal ligation and puncture (CLP). The protocol of the study has previously been published and the samples referenced in this study derive from the same 60 animals used in the study by Bakopoulos et al. 20 with the addition of 12 new animals (2 new animals/group/each time table). The control group mice underwent a sham surgery receiving a laparotomy without cecal ligation and puncture. All animals were resuscitated with 1 mL isotonic sodium chloride solution administered subcutaneously. The mice of the septic and the control groups were sacrificed 24, 48 and 72 hours following the CLP procedure and the sham operation, respectively, forming 6 groups; 24S, 48S and 72S representing the septic groups, and 24C, 48C, 72C the control ones. At the end of the experimental period for each group, euthanasia was performed. Part of each tissue obtained was formalin fixed and paraffin embedded for histological analysis; the rest was rinsed with distilled (DEPC-treated) water and shock frozen to −140 °C for mRNA analysis. The experimenters were not blind to the surgical procedure but they were blind to all the other experimental analysis.

Measurement of serum biomarkers and inflammatory mediators.
Different serum biomarkers were measured to evaluate the kidney injury after CLP. More specifically, blood samples were drawn with intra-cardiac aspiration after each experimental period. Those samples were used for the assessment of several biochemical and immunological markers to evaluate the kidney injury. Serum urea, creatinine, total and direct bilirubin were measured in Chemical Awareness analyzer (Awareness Tech, USA) with the corresponding commercial kit (Human, Wiesbaden, Germany). Elisa kits were used to determine mouse serum interleukin 18 (IL18) (MBL International, Woburn, USA) and neutrophil gelatinase associated-lipocalin (NGAL) (R&D systems, Abingdon, UK) according to the manufacturers' instructions.
Histological examination. Five μm serial paraffin kidney sections collected from all experimental animals were cleared in xylene and hydrated with different grades of absolute alcohols. Subsequently, those renal sections were hematoxylin and eosin stained for the study and evaluation of tissue injury.

Statistical Analysis.
The assumption of normality in our data was checked using the Shapiro-Wilk test.
Differences in blood test results and TLR expression between the septic and control groups were assessed with one-way Anova followed by Tukey's test analysis (mean ± SD). Every difference with p < 0.05 was considered of statistical significance. All statistical analysis was performed using Graph Pad Prism v 4.03 for Windows.

Results
Serum biomarkers during experimental sepsis. Renal dysfunction is an established side effect of sepsis secondary to peritonitis, among other organs such as the lungs and the liver. To evaluate the onset and extent of AKI, several blood serum biomarkers were investigated in our study. Standard parameters, such as urea and creatinine, although slightly outdated but still reliable 23 were measured in both control and septic groups at different time points. During the experimental period, statistical differences were found between CLP and sham groups. There was a significant increase in each serum marker highlighted among the septic groups, in a time dependent manner, with statistical significance being more profound between S24 and S72 groups (p < 0.05) ( Table 1). Moreover, to investigate renal inflammation, NGAL and IL18 markers were measured and their serum levels were monitored after CLP and sham operation. IL18 and NGAL also rose similarly as blood biomarkers to a peak at 72 hours after CLP (Table 1), proving as potentially related biomarkers for sepsis-induced AKI 24 .
Renal histopathology and Immunofluorescence. Toll-like receptors (TLRs) are the first identified family of pattern recognition receptors. They have been best studied and it is common knowledge that they are expressed in immunocytes, as well as widely distributed in various cell types, including renal cells. Hence, they contribute significantly to various pathologies of the kidney 25 . They respond to remote signals and increase their expression, evidence found in both histopathology and immunofluorescence.
In our study, as far as histological changes are concerned, they were more prominent in tubular dilation in septic groups rather than in the control groups; additionally, more extensive renal damage was observed in a time dependent manner in septic groups (Fig. 1). Furthermore, immunofluorescence analysis revealed that cells around kidney tubules expressed TLR 2, 3, 4 and 7 in all septic groups (Fig. 2a). As indicated in our experimental www.nature.com/scientificreports www.nature.com/scientificreports/ data results (Table 2), there was a significant increase of TLR 2, 3, 4 and 7 in septic mice over time (Fig. 2b) (p < 0.05). In contrast, control mice demonstrated no detectable expression of these TLRs.
CLP enhances TLR signaling molecule expression in kidney and intestine tissue. The expression of TLR 2, 3, 4 and 7 receptor genes was quantified by RT-PCR after 24, 48 and 72 hours of CLP induction in both, kidneys (Table 3) and intestine (Table 4). Each time point in the graphs (Fig. 3; kidney and Fig. 4; intestine)  www.nature.com/scientificreports www.nature.com/scientificreports/ represents the fold change in expression, resulting from septic groups being compared with control groups, using the 2 −ΔΔCT method. No significant changes in expression were demonstrated between control groups at any time point (data not shown). On the contrary, increased levels of TLR expression were observed after 72 hours of CLP induction in both kidney and intestine tissues. A statistically significant increase concerning each TLR was demonstrated at the prolonged septic process (p < 0.05).
In Fig. 5, the difference of each TLR expression between the kidney and the intestine tissues of the septic mice is depicted. Comparisons were made between septic groups of the respective euthanasia time. TLR 2 and TLR 4 gene expression was found higher in the kidney compared to intestine tissue during sepsis. At 72 h, the levels of the TLR 3 gene were significantly higher in intestine compared to kidney tissue. In contrast, at 24 h and 48 h TLR 3 gene expression was higher in the kidney tissue. Last, but not least, at 24 h sepsis, TLR 7 gene expression was significantly higher in the intestine tissue.

Discussion
Sepsis, a commonly encountered complication in an intensive care unit (ICU), often leads to multi-organ dysfunction and the kidney is one of the organs frequently afflicted. Despite considerable research during the last decades, the pathophysiology of sepsis-induced-AKI remains incompletely understood.
In the present model of sepsis induced AKI (CLP septic model), serum creatinine, urea, total and direct bilirubin, NGAL and IL18 levels were significantly increased during the experimental period. This phenotype of renal injury is acting in the same way with AKI in patients 26 .
Our study followed the same concept, principle and protocol to two other already published articles in literature. Aravanis        www.nature.com/scientificreports www.nature.com/scientificreports/ and intestinal tissues after CLP induced peritonitis 27 , whereas Bakopoulos et al. concentrated on the expression of hematological and biochemical markers related to respiratory failure as well as TLR expression in the lung and intestine after the same experimental procedure 20 . We, on the other hand, measured AKI biochemical and specific immune biomarkers, in addition to TLR expression in the kidney and the intestine after CLP. Liver function tests, AKI biochemical and immune biomarkers as well as respiratory biochemical markers and monocytes were increased similarly in septic groups versus control groups in a time dependent manner. On the contrary, hematological markers were decreased in the septic groups of sepsis-induced ARDS gradually over time. All TLRs displayed increased expression in septic versus control groups over time in all remote organs (liver, lung, kidney) secondary to CLP induced intra-abdominal peritonitis. TLRs have been found to be associated with sepsis-related dysfunction of various organs such as the intestine, the lung and the liver 20,28 . Analysis of TLR 2, 3, 4 and 7 expression in the intestine tissues of septic mice groups, showed a statistically significant increase in each TLR expression, progressively from 24 h to 72 h euthanasia time. Comparing control groups with time equivalent septic mice produced statistically significant differences for every TLR in each different euthanasia time.
The expression of TLR 2, 3, 4 and 7 was also found to be upregulated in the kidneys of septic mice, whilst it was only negligible in the kidney of control groups. These results were of statistical significance for each single TLR between time-equivalent groups (S24 vs C24, S48 vs C48, S72 vs C72) in qRT-PCR analysis. This data also suggests that, potentially, bacteria released from an injured intestine mediate inflammatory responses via TLR 2, 3, 4 and 7 within a different organ than the gut, such as the kidney. Destruction of Gram-negative bacteria results in the release of LPS into the bloodstream where binds to the LPS-binding protein (LBP). The LPS-LBP complex could bind to monocytes, macrophages and neutrophils 29 , through the co-receptor CD14 and interactions with the cell surface Toll-like receptor 4-MD-2 complex. This complex also binds to other cells, including renal tubular epithelial cells 30 , which are then stimulated to produce cytokines through a myeloid differentiation primary response gene (MyD88)-dependent and an MyD88-independent pathway 31 . The septic AKI has been shown to be dependent mainly on MyD88b 32 .
In the present study, a CLP septic model was used to examine the progression of sepsis, since it remains the most reliable animal model to resemble human conditions 6 . Various biomarkers expressed in organ failure were analyzed to examine the effect of TLR activation and expression in sepsis and eventually, MODS.
Additionally, serum NGAL as well as IL18 were similarly increased to the other serum biomarkers. This highlights the fact that systemic inflammation responds to high levels of NGAL. Previous studies suggest that neutrophil gelatinase associated lipocalin (NGAL) activity is a very sensitive marker of AKI, and it is produced by injured tubular epithelial cells 33,34 . Alteration of NGAL serum levels expressed in patients with AKI, identify the organ pathology 35,36 . Overall from our study we show that targeting NGAL levels provides kidney injury during sepsis and is associated with the inflammatory stress that is enhanced in the presence of IL18, the critical mediator of sepsis.
There are two phases of immune responses secondary to infection involved in the pathogenesis of sepsis 37,38 -both of which are displayed in a CLP model: systemic inflammatory response (SIRS) and compensatory anti-inflammatory response (CARS). Acute-phase proteins (CRP, procalcitonin), as well as pro-inflammatory cytokines (TNFα, IL1β, IL6, IL18) are common biomarkers for SIRS and significantly increase early in the onset of the septic response 39,40 . Single or multiple organ failure may be the result of the "cytokine" storm, which refers to these pro-inflammatory cytokines reaching their highest levels 41,42 . The second phase of the immune response is mediated by the secretion of anti-inflammatory cytokines (IL-4, IL-10). A moderate increase of these cytokines in the blood stream can play a protective role against the organ dysfunction and subsequently promote an antimicrobial immune response 43 . Thus, it is important to study and measure all mediators participating in this cascade and a serum cytokine profile could be used as a prognostic biomarker to predict the severity of the disease.
Furthermore, current studies suggest the potential role of IL18 is multifactorial including increases in septic mice during time. IL18 is an important pro inflammatory cytokine found in intestinal and immune system cells 44 . Circulating IL18 increases during sepsis and is strongly correlated with the severity of the disease 45 . Currently, our results enhance those previous studies. We observed elevation of the circulating IL18 in this CLP model in septic groups at all experimental points, indicating involvement in organ failure, since it is believed that high levels of IL18 are harmful in sepsis. More specifically, IL18 increased dramatically in S24 and S48 groups, representing an early onset of the cytokine storm in the setting of sepsis.
Also, biochemical markers are associated with the alterations in IL18 levels during sepsis. Renal function is mainly monitored by serum creatinine and secondarily urea, the levels of which along with the urine output and other markers define AKI 46 . We analyzed the differences in the expression of both creatinine and urea in septic and control mice at all euthanasia times (24 h, 48 h and 72 h). These two markers were at normal and unchanging levels in the control groups. On the other hand, there was a constant and significant increase among the septic groups as the time of euthanasia was prolonged, which also lead to significant differences between the time-adjusted control and septic groups. In our results, an alike pattern of expression alterations was denoted for total and direct bilirubin, as well, indicating a possible dysfunction of other organs such as the liver or the lung 47 .
So far, eleven human and thirteen mouse TLRs have been identified. These type I transmembrane proteins recognize pathogen-associated molecular patterns and trigger immune responses and inflammatory cascades 9 . Not all TLRs act upon the same targets; TLR-2 recognizes gram (+) bacteria, TLR-4 responds to gram (-) bacteria, whilst TLRs -3 and -7 counter viruses 48 . TLRs -1, -2, -4, -6 are expressed in interstitial and glomerular macrophages; tubular cells and mesangial cells express the same proteins with the addition of TLR 3, while dendritic cells within the kidney express TLR -4, -7, -8 and -9 49 . The upregulation of these TLRs in AKI of septic etiology, which for some TLRs is shown in other literature studies as well, might describe the involvement of these particular proteins in this pathology 13,49,50 .
One of the interesting results of our study is the significant difference in TLR 7 expression levels in kidney tissues of all septic groups when compared to the control ones and the statistically significant increase among the