|Year : 2015 | Volume
| Issue : 5 | Page : 947-952
|Renal inflammatory response to urinary tract infection in rat neonates
M Zarepour1, H Moradpoor1, F Emamghorashi2, SM Owji3, M Roodaki4, M Khamoushi4
1 Jahrom University of Medical Sciences, Jahrom, Iran
2 Department of Pediatrics, Jahrom University of Medical Sciences, Jahrom, Iran
3 Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran
4 School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
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|Date of Web Publication||7-Sep-2015|
| Abstract|| |
Urinary tract infection (UTI) is one of the most common bacterial infections. Maternal UTI is a risk factor for neonatal UTI. The aim of the present study was to determine the severity of renal inflammation in neonate rats born from mothers with induced UTI. Twelve pregnant rats (Sprague-Dawley) were included in study. The rats were divided into two groups (six rats in each group). In the first group, pyelonephritis was induced in the third trimester of pregnancy and the second group was used as a control group. After delivery, the neonates were divided into three groups based on days after birth (the 1 st, 3 rd and 7 th days after birth). In each group, two neonates of each mother were killed and a midline abdominal incision was made and both kidneys were aseptically removed. On the 7 th day, rat mothers were killed and their kidneys were removed. The preparations were evaluated with a bright field microscope for inflammatory response. Renal pathology showed inflammation in all UTI-induced mothers, but only two cases of neonates (2.1%) showed inflammation in the renal parenchyma. There was no relation between the positive renal culture and the pathological changes. We conclude that neonates with UTI born to UTI-induced mothers showed a lesser inflammatory response.
|How to cite this article:|
Zarepour M, Moradpoor H, Emamghorashi F, Owji S M, Roodaki M, Khamoushi M. Renal inflammatory response to urinary tract infection in rat neonates. Saudi J Kidney Dis Transpl 2015;26:947-52
|How to cite this URL:|
Zarepour M, Moradpoor H, Emamghorashi F, Owji S M, Roodaki M, Khamoushi M. Renal inflammatory response to urinary tract infection in rat neonates. Saudi J Kidney Dis Transpl [serial online] 2015 [cited 2019 Mar 20];26:947-52. Available from: http://www.sjkdt.org/text.asp?2015/26/5/947/164575
| Introduction|| |
Urinary tract infection (UTI) is one of the most common bacterial infections. It affects 17.9-26% of pregnant women. There is also a high incidence of infection in the third trimester of pregnancy. , The exact rate of UTI in newborns is not known, but studies have found that from about 1 in 1000 to 1 in 100 in full-term infants, and up to 1 in 10 premature infants, will have a UTI during the first month of life, and higher incidence of UTI in the Neonatal Intensive Care Unit has been detected in 15.05%.  A study showed that 5% of neonates with sepsis developed UTI.  Our previous work found maternal UTI as a risk factor for neonatal UTI. 
Host factors play a vital role in protection from UTI. Natural host defense factors include unidirectional flow of urine from the kidney to the bladder, secretion of glycosamines by bladder transitional cells, Tamm-Horsfall protein and defensins. Once the bacteria come into contact with urothelial cells, an innate immune response is started.  Neonatal inflammatory response has been considered to be intrinsically hyporesponsive. , A study showed that an immature compensatory anti-inflammatory response syndrome (CARS) in pre-term infants predisposes them to the harmful effects of the pro-inflammatory cytokines, resulting in severe organ damage during infection.  Neonates and infants are at a higher risk for UTI during their first few months of life. This susceptibility has been attributed to an incomepletely developed immune system.  It is suggested that renal damage in adequately treated infants is probably due to a reflux-associated, pre-existing, congenital renal parenchymal pathology and not to the inflammatory process. 
The aim of the present study was the determination of severity of renal inflammation in neonate rats born to mothers with induced UTI.
| Materials and Methods|| |
We studied 12 Sprague-Dawley pregnant rats weighing 244 ± 41.0 g obtained from the Laboratory Animal House of the Shiraz University of Medical Sciences, Shiraz, Iran. The experiment was performed in the Laboratory Animal House of the Jahrom University of Medical Sciences with cooperation of the Laboratory Animal House of the Shiraz University of Medical Sciences during the summer of 2012. The rats had free access to food and water throughout the experiment. They were maintained in terms of 12-h light, 12-h dark, temperature 24°C and humidity of 40-60% based on standard guidelines. They were divided into two groups (six rats in each group). In the first group, pyelonephritis was induced during the third trimester of pregnancy and the second group without infection was used as the control group.
The bacterial strain used to induce pyelonephritis was Escherichia coli (E. coli) (H2O6, pap+); it was obtained from a patient with pyelonephritis. The animals were anesthetized before induction of pyelonephritis. The side of the animals was shaved and rendered aseptic by an intraperitoneal injection of ketamine and xalazine. Then, a small incision was made at the level of the kidney. The left kidney was exposed and 0.05 mL of an inoculum containing 10 9 bacteria was injected through the upper pole of the left kidney. This technique, described previously by Kaye, produces pyelonephritis. 
After the induction of pyelonephritis, the animals were locked up in separate cages. After delivery, the neonates were divided into three groups based on days after birth (1 st , 3 rd and 7 th days after birth). In each group, two neonates from each mother were killed and a midline abdominal incision was made, and both kidneys were aseptically removed. Each kidney was divided into two parts by a sagittal incision and one half was sent in a sterile container for microbiologic study. In positive cases of kidney culture, types of infectioncausing organisms were defined. Other halves were fixed in 10% neutral formalin solution and stained with hematoxylin and eosin (HE). The preparations were evaluated with a bright field microscope and were photographed. Microscopic renal lesions were scored on plastic sections at a magnification of ×400. Each slide was coded such that identification of the groups was not possible for the observer. Slices came from three different pieces of renal cortex for each rat. The cortex and medulla, including glomerulus, tubules, vessels and interstitium, were evaluated. The percentage of cellular infiltration in each section was considered as severity of inflammation [interstitial inflammatory cells (IIC)].
For the microbiology study, half of each kidney was homogenized in 3 mL of sterile saline at 4°C. Appropriate dilutions of homogenized kidneys were made and 10-μL samples were placed in triplicate on MacConkey agar. The numbers of colony-forming units (CFUs) of E. coli in the kidneys were determined after an incubation of 18 h at 37°C (the CFU per milliliter of homogenate was transformed into CFU per gram of tissue).
The design of this experiment was approved by the ethic committee of the Jahrom University of Medical Sciences.
| Statistical Analyses|| |
All statistical analyses were performed with SPSS version 11.5. The data were analyzed by ANOVA to determine the statistical significance of the difference between groups. A Pvalue less than 0.05 (P <0.05) was considered to be significant.
| Results|| |
One hundred and eight kidney samples were obtained from mother rats and their neonates. [Table 1] shows the results of cultures and pathology of the kidneys. Cultures of eight kidney samples of 12 mothers were positive; 50% of the samples showed a positive culture. Overall, 56 (51.9%) of the kidney cultures were positive. [Table 2] shows the relationship between positive kidney cultures in mothers and their neonates. The kidney cultures were positive in all UTI-induced mother rats. Two mother rats in the control group also had a positive kidney culture, which was not related to the induction of infection.
|Table 1: Frequency of positive kidney cultures in mother rats and their neonates.|
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E. coli was the causal agent in the UTIinduced group and non-hemolytic streptococci was the causal agent in one case of the control group. There was a correlation between the positive kidney cultures in mothers and neonates (58.8% vs. 40%, P = 0.05). However, there was no difference in the incidence of UTI in the neonates according to days after birth.
The renal pathology showed inflammation in all the UTI-induced mothers, but only 2/96 (2.1%) of neonate kidney cultures showed inflammation in the renal parenchyma; one of these two neonates belonged to mothers without infection and another neonate had a mother with a positive kidney culture and inflammation. There was no correlation between positive renal culture and inflammatory response in renal pathology. Our data showed that neonates have reduced inflammation in the kidney compared with adults during UTI, even though both adult and neonatal kidneys had a similar bacterial load [Figure 1].
| Discussion|| |
In this study, we developed a neonatal model of UTI. In spite of positive kidney culture in neonates of UTI-induced mother, the pathology showed a low inflammatory response in neonates with UTI. Neonatal inflammatory response has been considered to be intrinsically hyporesponsive. , Infiltration of neutrophils into the uro-epithelium and bladder lumen of mice has been observed as early as 6 h after inoculation with uropathogenic E. coli (UPEC).  Neutrophils are essential for bacterial clearance from the urinary tract,  and defects in neutrophil recruitment or function increase susceptibility to UTI. Uropathogenic E. coli suppress the host inflammatory response.  It has been suggested that the inflammatory process causes irreversible renal parenchymal scarring. The study could not confirm that younger children are at a greater risk for developing renal sequelae following pyelonephritis. 
A central element of innate immunity to bacterial infection is the neutrophil-a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against gram-negative bacteria is believed to depend, to a significant degree, on the presence of bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. This study showed that the neutrophils of newborns are selectively deficient in BPI. BPI deficiency correlates with decreased antibacterial activity of the newborn neutrophil extracts against serum-resistant E. coli and could contribute to the increased incidence of gram-negative infection. , Clinical evidence of the pre-term neonates' immune incompetence includes the frequent development of neutropenia in response to bacterial sepsis. , The immediate fall in neutrophil count that accompanies neonatal sepsis may be due to more than an inability to increase the proliferation of early progenitors. Studies in newborn rats have demonstrated that their absolute neutrophil cell mass per gram of body weight is only onequarter of that of adult animals. After birth, the neutrophil cell mass per gram of body weight expands to reach adult levels after about four weeks.  Neonatal neutrophils display less interaction with endothelial monolayers in conditions of flow than adult cells. Rolling adhesion is diminished, fewer cells attach to the activated endothelium and fewer cells migrate to the sub-endothelial tissue. 
The inflammatory response in the urinary tract presents as pyuria in urinalysis. Less inflammatory response results in less pyuria in neonates with UTI. However, pyuria is not a useful marker for the diagnosis of cultureproven UTI in neonates. 
Variability in inflammatory response in neonates has been observed in other infections. Neonatal mice developed similar SeV titers and cleared the virus with similar efficiency despite developing a dramatically lower degree of pulmonary inflammation compared with adults.  Pulmonary virus of the mouse (PVM) infection presented in an atypical fashion in neonatal mice; chemokine production is minimal in the lung tissue of neonatal mice and recruitment of pro-inflammatory leukocytes is diminished when compared with older mice.  A whole blood lipopolysaccharide stimulation assay showed significantly lower tumor necrosis factor-α values in preterm neonates who subsequently developed sepsis, indicating a degree of immunoparalysis. 
We conclude from our study that neonate rats with UTI showed less inflammatory response than adult rats. Further studies are warranted to evaluate whether intrauterine-acquired UTI in neonates can cause renal injury or scar.
Conflict of interest: None.
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Pediatric Nephrology, Motahary Hospital, Jahrom
[Table 1], [Table 2]
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