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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 5  |  Issue : 1  |  Page : 32-36

Comparison of renal interstitial fibrosis induced by different animal models of urinary tract infection


Department of Nephrology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China

Date of Web Publication26-Mar-2018

Correspondence Address:
Dr. Yi Wang
Department of Nephrology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jina.jina_35_17

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  Abstract 


Objectives: To identify a better animal model that more closely mimics early renal interstitial fibrosis induced by long-term recurrent urinary tract infection (UTI), as occurs in humans, three different murine models were compared. Methods: Three different murine models of upper UTI were established, including direct injection of bacteria into renal tissues, ascending urinary infection with partial unilateral ureteric obstruction, and repeated infusion of bacteria into the bladder. The histopathology of the kidneys was assessed by hematoxylin and eosin staining. Masson's trichrome staining and immunohistochemistry for α-smooth muscle actin (α-SMA) expression were used for the detection of fibrosis. Results: All three models developed different levels of inflammation in the kidney. However, in contrast to the severe renal interstitial fibrosis in the other two models, the model of repeated infusion of bacteria into the bladder demonstrated early renal interstitial fibrosis by Masson's trichrome staining and immunohistochemistry for α-SMA. Conclusions: The model of repeated infusion of bacteria into the bladder developed low levels of renal interstitial fibrosis, which resembles the early tissue damage in the kidney induced by recurrent UTI s in humans. This model may therefore offer a better way to study the early therapeutic intervention of renal interstitial fibrosis caused by inflammation.

Keywords: Animal model, inflammation, renal interstitial fibrosis, urinary tract infection


How to cite this article:
Gu X, Han S, Xu Z, Cen J, Chen M, Wang Y. Comparison of renal interstitial fibrosis induced by different animal models of urinary tract infection. J Integr Nephrol Androl 2018;5:32-6

How to cite this URL:
Gu X, Han S, Xu Z, Cen J, Chen M, Wang Y. Comparison of renal interstitial fibrosis induced by different animal models of urinary tract infection. J Integr Nephrol Androl [serial online] 2018 [cited 2022 Aug 14];5:32-6. Available from: http://www.journal-ina.com/text.asp?2018/5/1/32/228498




  Introduction Top


Known as one of the most common bacterial infections in humans, urinary tract infection (UTI) is also a major cause of renal tissue damages and renal failure, mainly characterized by renal interstitial fibrosis and glomerulosclerosis.[1],[2],[3] However, the study of renal interstitial fibrosis induced by recurrent UTIs is impeded by the lack of a relevant model system. There have been many attempts to establish a murine model resembling the disease that occurs in humans. Direct injection of bacteria into the renal cortex has long been used as an established model of UTI.[4] However, this model is definitely not similar to the natural development of UTI, as it combines infection with trauma. Furthermore, it has been known for many years that, in young children, congenital obstructive uropathy is responsible for pyelonephritis and leads to dysplasia and renal failure.[5],[6],[7] Therefore, to recapitulate an actual vesicoureteral reflux-mediated UTI, ureteral obstruction is performed before the injection of bacteria into the bladder.[8] Several studies have shown that this model can impair renal function and facilitate renal interstitial fibrosis.[9],[10],[11] Interestingly, anatomic malformations are not a prerequisite for renal scarring. Women are known to be at the highest risk of UTI, owing to physical structure and hormonal changes.[12],[13],[14]{Benador, 1994 #6} However, most women diagnosed with UTIs do not have severe forms of reflux. Thus, the existing animal models do not mimic well the development of renal interstitial fibrosis induced by recurrent UTI. This study examined a novel murine model established by repeated intravesical instillations of bacteria into the bladder, without surgical manipulation, in order to offer a better murine model that more closely resembles natural disease progression in humans.


  Materials and Methods Top


Bacteria

 Escherichia More Details, coli strain 0111B4 (CMCC No. 44155).

Animal models

A 12-week-old female Sprague–Dawley rats weighing 180–200 g (Shanghai, China) were randomized into six groups (n = 10/group). Rats had access to typical rat chow and tap water ad libitum and were housed at an optimal temperature and humidity with a 12-h light/dark cycle. Animal usage and all experimental procedures were approved by the Ethics Committees of the Shanghai University of Traditional Chinese Medicine and followed the guidelines for the Care and Use of Laboratory Animals of the National Research Council.

Model 1

The model was established as previously described.[15] Under anesthesia induced by 0.35 L/kg 10% chloral hydrate injected intraperitoneally, a right longitudinal flank incision was made and the right ureter was exposed. The upper third of the right ureter was embedded in the psoas muscle by two sutures of 9-0 silk, causing partial unilateral ureteric obstruction, according to the technique as previously described. The abdomen was closed in two layers. After surgery, 200 μl of E. coli with a concentration of 1 × 109 cfu/mL was infused into the bladder of each rat using a sterile 24-gauge fluoroethylene propylene catheter through the urethra. After the catheter was removed, the rats were returned to their cages. Rats in the control group were infused with normal saline.

Model 2

The model was established as previously described.[16] Rats were anesthetized intraperitoneally with chloral hydrate as above, a midline abdominal incision was used to expose the right kidney, then 200 μl of 1 × 109 cfu/ml E. coli suspension was directly injected into the right renal cortex of rats using a 24-gauge needle. Rats in the control group were injected with normal saline only.

Model 3

200 μl of E. coli with a concentration of 1 × 109 cfu/mL was instilled into the bladder of rats using a sterile 24-gauge fluoroethylene propylene catheter after being anesthetized with chloral hydrate, as above. The intravesical instillation of E. coli was repeated twice a week for 6 weeks. Rats in the control group were infused with normal saline.

Kidney histology

Six weeks after infection, all rats were killed for histological examination. The kidneys were removed and fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4 μm sections. Hematoxylin and eosin (H and E) and Masson's trichrome staining were then performed. Assessment of interstitial fibrosis and inflammation was performed in a blinded manner, under × 400 magnification.

Immunohistochemistry

α-smooth muscle actin (α-SMA) immunohistochemical staining was performed as previously described.[17] Briefly, 4 μm sections of renal tissue, prepared from paraffin-embedded tissues, were deparaffinized, and endogenous peroxidase was inactivated with H2O2. Sections were then blocked with 2% normal goat serum in phosphate-buffered saline (PBS) for 10 min at room temperature and incubated with a rabbit anti-rat α-SMA antibody (1:200, Santa Cruz, Dallas, TX, USA) at 4°C overnight. The sections were washed with PBS and incubated with secondary antibody (Goat anti-rabbit, 1:1000, Life Technologies, Grand Island, NY, USA) for 1 h. Positive staining was revealed by peroxidase-labeled streptavidin and diaminobenzidine substrate. Negative controls for immunochemistry staining were performed by substitution of primary antibody with PBS.


  Results Top


The histopathology of three animal models of urinary tract infection

In model 1, the right kidney was small with severe renal damage consisting of tubular atrophy and dilatation with a variable degree of interstitial infiltrate composed of lymphocytes and monocytes. In model 2, the right kidney exhibited a wedge-shaped cortical interstitial scar at the E. coli injection site filled with inflammatory cells, with progressive renal tissue destruction with almost no normal structure left. In model 3, the structure of the kidney was almost normal with diffuse monocyte infiltration of the renal interstitium [Figure 1].
Figure 1: Histological appearance of the kidneys after initiation of an upper urinary tract infection. Model 1 demonstrated focal tubulointerstitial scarring, tubular atrophy and loss, and an area of resolving tubulointerstitial inflammation. Model 2 displayed infiltration of numerous neutrophils accompanied by fibrosis, replacing tubules in extensive areas of the medulla and cortex. Model 3 showed normal kidney structure with diffuse monocyte infiltration (×200)

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Fibrosis level as assessed by Masson's trichrome staining

Masson's trichrome staining highlighted diffuse fibrosis in the right kidney in model 1, with fibrosis replacing tubules in extensive areas of the medulla and cortex. In contrast to model 2, interstitial fibrosis only developed at the injection site, with the production and accumulation of extracellular matrix. Notably in model 3, in contrast to the other two models, the kidney only demonstrated slight focal interstitial fibrosis [Figure 2].
Figure 2: Fibrosis level of kidneys as assessed by Masson's trichrome staining. Marked interstitial fibrosis was observed in Model 1. Interstitial fibrosis was present in the area injected with  Escherichia coli Scientific Name Search  2. Small areas of fibroses were present in the kidneys of Model 3 (×200)

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α-smooth muscle actin expression in the three animal models of urinary tract infection

After immunostaining, renal fibrosis was analyzed using Image J software (NIH, Bethesda, MA, USA) to measure the percentage area staining positive for α-SMA. We found that α-SMA expression was increased in all three animal models, with the staining most present in the renal interstitium. Furthermore, compared with models 1 and 2, the model of repeated infusion of bacteria into the bladder (model 3) was more representative of the early stages of renal interstitial fibrosis induced by recurrent UTI [Figure 3].
Figure 3: Immunohistochemical staining of a-smooth muscle actin in kidneys with upper urinary tract infections. In Model 1, extensive a-smooth muscle actin staining was found in the renal interstitium. Model 2 demonstrated a-smooth muscle actin staining in the Escherichia coli injected area. Model 3 showed small areas of a-smooth muscle actin staining in the renal interstitium (×200). Data are expressed as the positive staining area of a-smooth muscle actin per high-power field in the interstitium area. Values are expressed as mean standard error of the mean. *P < 0.05, **P < 0.01 n = 10. The positive areas are indicated by black arrows

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Comment

UTI is an important health problem with a high incidence and prevalence.[18] Owing to physical structure and hormonal changes, it is well known that women are at higher risk of UTIs than men, especially women at menopause and postmenopause.[12],[13],[14] Recurrent UTIs can affect mental health and quality of life throughout the lifespan of women. Moreover, it is often accompanied by vesicoureteral and renal reflux, leading to chronic pyelonephritis, which can cause severe inflammation, deterioration of renal function, and renal scarring, and accounts for 19.8%–21.2% cases of ESRD. Therefore, it is important to establish a stable animal model that closely resembles the process of renal fibrosis induced by recurrent UTI to better study the progression and development of UTIs and potential drug interventions.

Previous studies have shown that in a number of animal models, most species are resistant to UTI and it has been necessary to use surgical manipulation or other invasive procedures that increase the susceptibility of the urinary tract. Thus, the frequently used animal models that mimic upper UTI can be categorized into two types: direct injection of E. coli or lipopolysaccharide (LPS) into the kidney and instillation of E. coli into the bladder after unilateral ureteric obstruction. The first model, direct injection of E. coli or LPS into the parenchyma, was initially demonstrated by Miller and Phillips [4] It causes serious inflammation and fibrosis and is regarded as a classical model of pyelonephritis and has been used in a number of studies. However, the inflammation and fibrosis are localized to the injection area, and it is totally different from the natural process of pyelonephritis initiation, namely being due to surgical procedures. As for the latter model, several studies have used unilateral ureteral obstruction to trigger ascending UTI in animal models. Brooks et al.[9] developed a model with transcutaneous loop around the left ureter in rats and general ureteral obstruction after instillation of E. coli into the bladder. Bitz et al.[10] used the complete obstruction of the left ureter for 24 h in rats, followed by bacterial inoculation immediately after the release of ureteral obstruction. Another approach developed by Zeidan et al. consisted of a right partial ureteral obstruction and transurethral inoculation with E. col i.[15] All these animal models demonstrated the effect of preexisting ureteral obstruction and UTI on the occurrence of renal damage. Sometimes, however, it was found that the fibrosis induced was too severe in this animal model with UUO to be intervened in by drugs. Furthermore, vesicoureteric reflux may not explain varied differences in tissue responsiveness and inflammation. Therefore, Svensson et al.[19] used mIL-8Rh-/- mice in a UTI model of intravesical injection of E. coli, and acute bacteremic pyelonephritis and renal scarring developed owing to a dysfunctional neutrophil response. Renal scarring developed in the mIL-8Rh-/- mouse following mucosal, noninvasive infection, and without obstruction or prior tissue damage.[19] However, the major defect of this model is that it is not easily accessible.


  Conclusions Top


To establish a better animal model of upper UTI, we developed a method of repeated instillation of E. coli into the bladder twice a week, for a total of 6 weeks. Results confirmed that this animal model is also capable of causing inflammation in the kidney. In addition, fibrosis was also detected by Masson's trichrome staining and α-SMA staining by immunohistochemistry. Interestingly, the fibrosis level was not as severe as in the other two models tested. Therefore, we think that it is a novel and useful animal model to study the early therapeutic intervention of renal interstitial fibrosis caused by inflammation.

Acknowledgment

This work was supported by funds from National Natural Science Foundation (81774106 to YW), Shanghai university of T. C. M. Gaofeng and Gaoyuan clinical grant to XG, and the 3-year plan of action for the development of traditional Chinese medicine in Shanghai (ZY3-JSFC-2-2018 to MC).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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