Shuai Guan, Chuan Hao , Cheng-Yong Li

Department of Urology, Second Hospital of Shanxi Medical University

Keywords:Proteus mirabilis Vanillic acid Acetohydroxamic acid Stone Infection Pathology

ABSTRACT Objective: The pathological changes of urinary tract were observed after the treatment of infectious stone by vanillic acid. Methods: 40 male Wistar rats were randomly divided into blank control group, infectious stone group, vanillic acid group and acetohydroxamic acid group. Blank control group: 4 male Wistar rats were fed without special treatment. Infectious Stone Group: 12 male Wistar rats were implanted with 4 mm bacteria bearing foreign body in the front of the polyurethane vein indwelling needle infected with Proteus mirabilis into the bladder to induce the formation of infection stone. In the vanillic acid group, after the model were establishmented, 12 male Wistar rats were treated with vanillic acid at 50mg/kg·d for 3 weeks. Acetohydroxamic acid group: after the model were establishmented，12 male Wistar rats were treated with acetohydroxamic acid at 12.5mg/kg·d for 3 weeks. The rats were killed 21 days later.The kidney, ureter and bladder were fixed in 10% paraformaldehyde solution for 24 hours. The specimens were embedded in paraffin, and the pathological analysis was carried out after HE and immunohistochemical staining. Results: In addition to the blank control group, the other three groups of rats had calculus formation in the bladder. No obvious pathological changes were found in the kidney, ureter and bladder in the blank control group, and inflammatory reactions were found in the other three groups: severe urinary tract inflammation was found in the infectious stone group, followed by acetylhydroxamic acid group. In the vanillic acid group, the pathological changes of urinary tract were the lightest, with a small amount of inflammatory cell infiltration and mucosal hyperplasia, but no serious pathological changes such as mucosal loss. The expression of Tamm-Horsfall Protein（THP） in the ascending branch of loop of Henle and the proximal part of distal tubule in vanillic acid group was not significant compared with that in the infectious stone group and acetohydroxamic acid group. Conclusion: Proteus mirabilis and its infective stones can lead to the damage of urinary epithelium. Vanillic acid can reduce the inflammatory lesions of urinary tissue.?Corresponding author: Hao Chuan.

1. Introduction

Infectious stones account for about 15% of urinary calculi. Proteus mirabilis is the most common pathogenic bacteria. The widespread use of urinary catheter in clinic has increased the incidence of infectious stones[1].Proteus mirabilis can secrete urease to its environment. Urease decomposes urea in urine to produce ammonia and carbon dioxide. Meanwhile, it alkalizes urine to form stones quickly[1-3].Infection and stone formation are mutually causal factors that aggravate the pathological damage of

urothelium[4].In the artificial urine, vanillic acid has a stronger inhibitory activity on bacterial growth and crystal formation than traditional urease inhibitors (acetohydroxamic acid), and has an inhibitory effect on all strains used in the experiment[5].

It was also proved that vanillic acid has a significant effect on inhibiting the development of stones in rats in the early experiment[6].The purpose of this study is to further observe the pathological changes of urinary tract in rats with infectious stones and the pathological effects of vanillic acid on urinary tract.

2. Materials and methods

2.1 Experimental materials

Male Wistar rats，weight（300±10）g，Provided by experimental animal center of Shanxi Medical University( License No: SCXK 2015-0001).Proteus mirabilis was provided by Beijing Beina Chuanglian Biotechnology Research Institute（Batch number：BNCC336669）.Vanillic acid product number（V104428 Aladdin reagent company）；Acetohydroxamic acid：Product number：（A106239 Aladdin reagent company）.Tamm-Horsfall protein Rabbit antibody(Reagent Company).

2.2 Experimental methods

2.2.1 According to the previous literature, the bacterial solution and foreign matters were prepared[7].

40 Wistar male rats were randomly divided into 4 groups. Blank control group(n = 4): No foreign bodies were implanted into the bladder.Infectious Stone Group( n = 12) : 10% chloral hydrate 0.3ml/100g was injected intraperitoneally for anesthesia. After shaving, the abdomen was upward and the sacrococcygeal part was slightly raised. All the limbs were taped. After disinfection with Iodophor, sterile operation sheet was placed and aseptic ultrasonic gel was applied to the lower abdomen. Ultrasound probe placed on the upper edge of pubic symphysis to locate bladder，The needle was inserted about 0.5cm from the upper edge of pubic symphysis in the middle of the lower abdomen to 30 °toward the bladder，The needle feeding process is conducted under the guidance of ultrasound；After confirming that the puncture needle enters the bladder, fix the puncture needle with the left hand and pull out the core with the right hand，Using the needle core as the push rod, push the polyurethanee venous indwelling needle with Proteus into the bladder. Remove the puncture needle after the foreign body enters the bladder；6 hours after the operation, food and water were restored. Acetohydroxamic acid group(n=12):The method of model construction was the same as that of infectious stone group.，After the operation, the rats were fed with acetohydroxamic acid (12.5mg / kg·d). Vanillic acid group(n=12). The method of making model is the same as that of infectious stone group. The rats were treated with vanillic acid (50mg / kg·d).

2.2.2 Specimen acquisition, tissue section, HE staining and immunohistochemical staining

On the 21st day after the operation, all rats were killed by overdose anesthesia.Lower abdominal incision into abdominal cavity，To separate the kidney, ureter and bladder from the tissue，Lift the bladder bottom and separate it to the proximal urethra，After the kidney, ureter and bladder were removed, the size and texture of the organs and the formation of calculus in the bladder were observed with naked eyes.Urinary tissue samples were fixed in 10% formalin solution for 24 hours，Paraffin-embedded tissue specimens were sectioned at a thickness of 2 m.HE staining and immunohistochemical staining were performe.

2.2.3 Observation

HE stained sections were directly observed under light microscopy.One immunohistochemical section was taken from each rat's kidneys.Each slice was photographed in three positive fields randomly selected in the high power field of view.Image-Pro.Plus (IPP) 6.0 software analysis system was used to measure the mean optical density (OD) of the positive visual field,Statistical analysis was then performed.

3. Results

Under naked eye observation, the urinary tract tissues of rats in the blank control group showed no special changes, and there was no stone formation in the bladder.The quality of intravesical stones was the highest in rats with infectious stones（91±7）mg，The quality of bladder stones in acetohydroxamic acid group was next（54±3）mg，The quality of intravesical stones was the smallest in the vanillic acid group.（32±6）mg（Fig. 1）.The above stones were mainly composed of magnesium ammonium phosphate and calcium phosphate stones by infrared spectroscopy.There was only slight congestion and edema of bladder in vanillic acid group.The volume of bilateral kidneys of rats in the infectious stone group and the acetohydroxamic acid group increased significantly, the renal pelvis mucosa was hyperemic and edematous, and the ureter wall and bladder wall were significantly thickened.Abscess formation on renal surface and a small amount of purulent secretion in renal pelvis were observed in the kidneys of rats in the infected stone group. HE staining：No significant pathological changes were found in the kidney, ureter and bladder of rats in the blank control group.Hyperemia and edema of bilateral renal pelvic mucosa were found in rats of infectious stone group and acetohydroxamic acid group, and pelvic mucosal loss was seen in some specimens, thickening of ureter wall and bladder wall is obvious, finger-like hyperplasia of bladder mucosa is obvious, scattered ulcer formation can be seen. At high magnification: local renal tubular epithelial and interstitial edema was observed in rats with infectious stones group and acetohydroxamic acid group, ureteral epithelial hyperplasia, a large number of neutrophils can be seen in the lumen, a large number of inflammatory cells infiltrate the submucosa of bladder hyperplasia, the mucosal edema and partial loss of bladder mucosa can be seen.（Fig. 2,Fig. 3）. In vanillic acid group, the infiltration of inflammatory cells in kidney was not obvious, hyperplasia of ureter and bladder mucosa. However, the mucosal margin was clear without mucosal loss and a small amount of eosinophils infiltrated under the mucosa (Fig. 4). Immunohistochemistry of kidney：Tamm-Horsfall protein (THP) in kidney of rats in the infectious stone group and acetohydroxamic acid group was significantly higher than that in the vanillic acid group (Fig. 5, table 1).

Fig. 1 From left to right: infective calculi group, acetohydroxamic acid group, vanillic acid group rats bladder calculi (black arrow) and polyurethane catheter end (white arrow).

Fig. 2 Bladder of rats with infective calculi group (HE×200)Edema of bladder mucosa and hyperplasia of mucosa (green arrow), indistinct margin of bladder mucosa (blue arrow), infiltration of inflammatory cells such as neutrophils can be seen under proliferative mucosa (red arrow)

Fig. 3 Renal papilla of rats in infectious Stone Group (HE×100) Dropsy of renal pelvis mucosa, loss of renal pelvis mucosa (blue arrow), infiltration of a large number of inflammatory cells under the mucosa (red arrow)

Fig. 4 Bladder of rats in vanillic acid (HE×200) Hyperplasia of bladder mucosa, clear margin, no mucosal loss (green arrow), a small amount of inflammatory cell infiltration under mucosa(red arrow)

Fig. 5 Expression of THP in kidney of rats group with infectious calculus (IHC×400) Expression of THP protein in Henley's loop(yellow arrow) and the glomerulus (purple arrow)

Table 1 The OD value of THP in positive field of kidney in each group

4. Discussion

In this experiment, we found that Proteus mirabilis infection can produce infectious stones and cause severe pathological damage to urinary tract tissues in a well-established rat model of infectious stones. The intervention of vanillic acid and acetohydroxamic acid group acid can inhibit the formation of stones, and vanillic acid can reduce the damage of urinary tract tissues in a large extent[7].

At present, the mechanism of urothelial cell injury caused by urease-positive bacteria is still unclear.Urease-producing bacteria are mainly Proteus mirabilis, which is mainly found in the intestinal tract.Bacteria migrate from the intestine to the perineal area, and in patients with indwelling catheters, bacteria grow into elongated flagella by deformation.Bacteria move along the surface of the catheter and into the bladder in the form of flagellar movements, which eventually migrate to the kidney, leading to pyelonephritis[8].Urothelial damage caused by this bacterial infection plays an important role in the formation of infectious stones:the mucosal necrosis and exfoliation of the renal pelvis or bladder expose the local basement membrane, creating conditions for the spread of bacterial infection and stone formation.Magnesium ammonium phosphate crystals and apatite carbonate crystals formed by bacterial infection, which together with local inflammatory necrotic substances form the core of infectious stones, make stone removal alone or the use of antibiotics have limited therapeutic effects.

Acetohydroxamic acid is currently the only recommended urease inhibitor, which is a non-reversible, non-competitive inhibitor，it can inhibit urea decomposition, acidify urine, and prevent the formation and recurrence of infectious stones[1]，but the toxic and side effects of nerve, skin, blood and so on limit its clinical application[9-10].

Vanillic acid widely exists in nature, among which the highest content is the root of Angelica sinensis，it has many effects, such as antimicrobial, inhibiting fibrosis,lowering blood pressure and myocardial protection[11-12]，the inhibitory effect of vanillic acid on calculi was also confirmed in artificial urine and previous animal experiments[5].

Tamm-Horsfall protein (THP) is the most abundant protein in urine, which is mainly expressed and secreted by the epithelial cells in the thick segment of ascending branch and proximal segment of distal convoluted tubule of Henry loop of renal tubule and released into urine.The formation of urinary tract infectious stones and the inflammatory response caused by bacterial infection are closely related to Tamm-Horsfall protein.Mutations in the human THP gene have been found to be associated with the onset of MCKD/FJHN (renal medullary cystic nephropathy/familial juvenile hyperuricemic nephropathy),deletion of THP is prone to urinary tract infection in MCKD/FJHN[13]，other studies have found that THP knockout mice lack resistance to Proteus mirabilis[14]。The presence of THP plays an important role in inhibiting bacterial colonization and infection of urinary tract, protecting kidney and bladder tissues, and inhibiting stone formation[15-16].

The histopathology of urinary tract in this experiment suggested that the infiltration of inflammatory cells in the kidney tissue of rats in the vanillic acid group was not obvious, the edges of ureter and bladder mucosa were clear, and no mucosal loss was observed.The THP protein in the kidney of rats in the vanillic acid group was significantly lower than that in the infectious stone group, which verified that vanillic acid inhibited infectious stones and alleviated inflammatory lesions in urinary tract tissues under the intervention of vanillic acid.

Bacterial infection, urease and stone formation are important factors leading to renal and urothelial damage.The mechanism of vanillic acid in alleviating urothelial inflammatory lesions remains unclear.Whether vanillic acid can alleviate inflammation by inhibiting bacterial colonization, proliferation or deformation？This still needs to be verified by bacterial experiments.Whether vanillic acid inhibits stone formation and inflammation by inhibiting urea decomposition, even if whether it can even be used as a new urease inhibitor？Further studies in biochemistry and toxicology are needed.