Article / Research Article

"Cathelicidin and Vitamin D Serum Levels in Children with Urinary Tract Infection"

Maha Youssif Zein El-Abdin1 , Manal Abd El-Salam1*, Iman Mouhamed El- Bagoury2 , Nouran Abd Al-Magid1 

1 Department of Pediatric, Faculty of Medicine, Al-Azher University, Cairo, Egypt 
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azher University, Cairo, Egypt
 * Corresponding author: Manal Abd El-Salam, Department of Pediatric, Faculty of Medicine, Al-Azher University, Cairo, Egypt. Email: manal24969@gmail.com 
 Received Date: 11 December, 2017; Accepted Date: 22 December, 2017; Published Date: 29 December, 2017

1.    Abstract

1.1.  Background: Urinary Tract Infections (UTI) are the most frequent bacterial infections in infants and children after respiratory tract infection. The risk of having a UTI before the age of 14 years is approximately 1-3% in boys and 3-10% in girls. Cathelicidins are small peptides with amphipathic structures that allow them to disrupt the integrity of the pathogen cell membrane, resulting in its death.

1.2.  Aim: to assess the association between vitamin D and cathelicidin serum levels in children with urinary tract infection.

1.3.  Subjectsand Methods: the study included 25 children with urinary tract infection documented by urine culture and sensitivity,they were selected from children attending the outpatient pediatric clinic and inpatients of the pediatric department, Al-Azhar University hospital. Also, itincluded 25 healthy children age and sex matched as a control group. Serum vitamin D and cathalicidin levels were assessed in both groups.

1.4.  Results: there was significant increasein cathelecidin serumlevel in patients with UTI compared to the controls, it was (52.8 ± 31.59ng|ml) and (27.06 ± 10.43ng|ml) respectively , (P <0.01), on the other hand there was a significant decrease in vitamin Dserumlevel in patients with UTIcompared to the controls ,it was (45.29± 34.17ng|ml) and (48.72 ±17.40ng|ml) respectively( P < 0.05).A positive correlation between serum cathelecidin and vitamin D was detected.

1.5.  Conclusion: vitamin D stores may influence susceptibility to urinary tract infection in children. There was a strong relation between serum vitamin D and cathalicidin levels in those children.

2.      Keywords: Cathalicidin; Vitamin D; UTI

1.      Introduction

Urinary Tract Infection (UTI) is one of the most common bacterial infections of humans (most often E. coli) and a major cause of morbidity. UTI also accounts for 25 to 40% of all nosocomial infections, making these infections an important medical and financial burden on health care systems. UTI usually starts as a bladder infection but can ascend to the kidneys and may result in renal failure[1].                         Antimicrobial Peptides (AMPs) include the gene families of defensins and cathelicidins. AMPs have direct lytic properties against a variety of organisms including bacteria, fungi, and viruses. In addition, it is becoming increasingly appreciated that AMPs are also immunomodulatory[2].Cathelicidins are small peptides with amphipathic structures that allow them to disrupt the integrity of the pathogen cell membrane, resulting in its death. Cathelicidins are expressed by most immune cells or those epithelial cells that are in contact with the environment. Deficiency in these peptides results in increased susceptibility to infection [3], thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract[4].Vitamin D has important roles in addition to its classic effects on calcium and bone homeostasis, as the vitamin D receptors are expressed on immune cells (B cells, T cells, and antigen-presenting cells), and these immunologic cells are all capable of synthesizing the active vitamin D metabolites. Vitamin D can act in an autocrine manner in a local immunologic milieu and modulate the innate and adaptive immune responses[5].Vitamin D could influence cathelicidin production in the urinary tract and thereby help protection from invading microbes. Urinary bladder cells enhance cathelicidin production in response to vitamin D[6].

2.      SubjectsandMethods

This is a case control study included 25 children with urinary tract infection, their ages ranged from 3:12 years, with mean age (8.58±3.41 years),these are the patients, numbers who attended at the pediatricdepartment and the out patients, pediatricclinic at the time of the study after the exclusion criteria. They were 19 females (76%) and 6 male (24%).All cases were presented with symptoms of UTI which was confirmedby: Pyuria (pus cell 10/ HPF) in mid urine sample collected under sterile condition)and +ve urine culture [7],sixty-eight % of the studied cases were found to be with recurrent attack of UTI while 32%were with the first attack. The frequencyof lower UTI and upper UTI in the studied cases were 21 (84%) and 4(16%) respectively. Also 25 healthy children age and sex matched were included in the study as a control group. Patients with:(congenital renal diseases, other acute and chronic infections, chronic illness and childrenon medications e.g. steroids, cytotoxic drugs)were excluded from the study. Informed consent was obtained from the participating patients or their parents in adherence with the guidelines of the ethical committee of AL-Zahraa hospital, AL-Azhar University, Cairo, Egypt.

2.1.  Sampling and Procedure

2.1.1.         Urine Samples: Urine analysis:significant pyuria is defined as >10 leukocytes per mm3 in a fresh uncentrifuged sample[8].Urine culture and sensitivity on a clean catch specimen, more than 10000 colonies in boys suggestlikely infection and 100000 colonies in girls makes the diagnosis of an infection likely[9].

2.1.2.         Blood Samples

·         2 ml were taken on EDTA vacutainer for C.B.C. and ESR.

·         2 ml were taken on plain vacutainer for, urea and creatinine.

·         3 ml were taken on plain vacutainer for:CRP, serum vit D and cathelicidin. samples were separated and stored at -20 °C. till the time of the assay.

Assessment of cathelicidin serum levelusing enzyme-linked immune-sorbent assay (ELISA).

The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to assay the level of human(cathelicidin LL-37) in samples. Add (cathelicidin LL-37) to monoclonal antibody Enzyme well which is pre-coated with human (cathelicidin) monoclonal antibody, incubation; then, add (cathelicidin LL-37) antibodies labeled with biotin, and combined with Streptavidin-HRP to form immune complex; then carry out incubation and washing again to remove the uncombined enzyme Then add chromogenic solution A, B, the color of the liquid changes into the blue, and at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the human substance (cathelicidin LL-37) of sample were positively correlated. The color changes were measured spectrophotometrically at a wave length 450 nm.The concentration of cathelicidin (LL-37) in the sample was determined by comparing the optical density of the samples to the standard curve. The results were expressed as ng/ml[10].

2.2.  Statistical Analysis

Data were collected, revised, coded and entered to the Statistical Package for Social Science (version20). Spearman correlation coefficients were used to assess the relation between two studied parameters inthe same group. P value. P<0.05 was considered statistically significant.

3.      Results

Age and sex distribution among studied groups, it revealed: female was the predominant sex among patients group, male and female was 19 (76%) and 6 (24%) respectively(Table 1).

Comparison between patientsand controls regarding some laboratory parameters, it revealed:significant increase in ESR and CRP levels in children with UTIcompared to the controls and significant decrease in serum creatinine levelin patients with UTI compared to the controls. No significant differences were detected regarding WBCs, and serum urea levels(Table 2).

Comparison between patients and control group regarding vitamin D and cathelicidin serumlevels, it revealed: significant increase in cathelecidin and decrease in vitamin D serumlevels in children with UTIcompared to controls(Table 3).

Organisms causing UTI in children with UTI, it revealed:E. coli is the most common organism causing UTI ,it is detected in 19 cases (76%) followed by Entero-coccus 3 cases (12%), Gm+ve cocci 2 cases (8%) and then Proteus 1 case (4%)(Figure 1).

E.coli remainsthe most common organism causing UTI,in the currentstudy,it is detected in 19 cases (76%) followed by Entero-coccus 3 cases (12%), Gm+ve cocci 2 cases (8%) and then Proteus 1 case (4%).This is in agreement with[11-14]they reported that Escherichia coli was the predominant organism causing UTI in their studies. We reportedsignificant increase in ESR and CRP levels in cases with UTI, elevated Erythrocyte Sedimentation Rate (ESR)and C-Reactive Protein (CRP) are indicators of an acute inflammatory process. These tests do not reliably differentiate between children with cystitis and children with pyelonephritis[15].This study provides evidence that compared to healthy children,serum vitamin D level wasreducedin children with UTI, lower levels of serum vitamin D in UTI cases reflect its importance in immunity and protection against infection. This is in agreement with the study byTekin etal. [17]who reported that a serum 25-hydroxy vitamin D level of <20 ng/mL was associated with UTI in children,in the current study we did not categories' vitamin D to insufficiency or deficiency due to small sample size. Another study done byHacıhamdioğlu etal. [18] reported that frequency of vitamin D insufficiency was significantly higher in children with a UTI than in those in the control group. Also, Yang etal.[19]reportedthat vitamin D deficiency were at an increased for UTI, whereas vitamin D supplementation was associated with a lower UTIfrequency. There is noenough available data concerned with assessment of vitamin D level in children with UTI but there is a lot of data concerned with assessmentof the levelin respiratory tract infections [20-21].

Deficiency in vitamin D is associated with an increased susceptibility to UTI, When the bladder cells were infected, a significant increase in cathelicidin expression after vitamin D supplementation was observed[6].Furthermore, vitamin D has a regulatory role on innate and adaptive immune responses. Vitamin D has been shown to promote antimicrobial responses through the production of antibacterial peptides. The epithelial cells of the urinary tract up regulate the production of the human antimicrobial peptide LL-37 upon infection with uropathogenicE. coli. Thus, the cathelicidin LL-37 plays an important role in the protection against infections of the urinary tract [22]. Antimicrobial peptides such as cathelicidin constitute an integral part of the innate immune response to a variety of infections especially at barrier sites[23].Our study demonstratedhigh level of serum cathalicidin in children with UTI compared to the controls. Cathelicidin expression and secretion were increased during E. coli urinary tract colonization in children with cystitis or pyelonephritis [24]. We observed significant positive correlation between serum cathelecidin and vitamin D, therapy with vitamin D in animal models of sepsis modulates levels of systemic inflammatory cytokines including TNF-α and IL-6.Furthermore, Vitamin D can enhance the induction of the antimicrobial peptides cathelicidinand β-defensin which are found on mucosal and epithelial surfaces and act as the body's first line of defense against viral and bacterial pathogens,so we hypothesized that vitamin D may be involved in the defense against UTI, and can be mediated by cathelicidin. In the same line with our results a study done byHacıhamdioğlu etal. [18]who reported that urine cathelicidin level was significantly unregulated in children with UTI and sufficient vitamin D status. In contrast, urine cathelicidin levels did not increase significantly during a UTI in children who had vitamin D insufficiency.

4.      Conclusion

We concludeda strongpositive relation between serum vitamin D and cathalicidin levels in children with urinary tract infection. Our research suggests that vitamin D enhances immune system and increases antibacterial defense, thus vitamin D can be used as an adjuvant drug in treatment of UTI and may solve the antibiotic microbial resistance.

 


Figure 1: Urine cultureresults in children with UTI.



Figure 2: Positive correlation between serum cathelicidin and vitamin D levels.


Variable

Cases (no=25)

Controls (no=25)

P-value

Age (years)

 8.58±3.41(3-12)

 9.18±2.74 (3- 12)

0.537

Gender

 

 

0.496

Male

6 (24.0%)

9 (36.0%)

Female

19 (76.0%)

16 (64.0%) 

Table 1: Age and sex distribution in the study groups.

 

Parameters

 Groups

t/z#

P-value

Cases group

Control group

no=25

no=25

Mean ± SD

Mean ± SD

WBCs (x10^3\ul)

8.11± 3.61

8.20 ±2.93

-0.101

0.92

ESR(normal<10)

21.20±19.61

6.76 ±1.81

-5.015#

0.01**

Urea (mg\dl)

24.32 ±7.28

24.00 ± 4.25

0.19

0.85

Create (mg\dl)

0.38 ± 0.13

0.54 ± 0.05

-5.553

0.01

CRP (normal<6)

17.2 ± 14.3

5 ± 2

-12.2#

0.01

# Mann-Whitney test 

Table 2:Comparison between patients and controls regardinglaboratory investigations.

 

Parameters

Groups

 

Z

P-value

Cases group

Control group

 no=25

 no=25

Mean ±SD

Mean ±SD

Serum vitamin D(ng/ml)

45.29 ±34.17

48.72 ± 17.40

-2.397

0.017*

Serum cathelecidin(ng/ml)

52.84± 31.59

27.06± 10.43

3.774

0.01** 

Table 3: comparison between patients group and controls regarding vitamin D and cathelecidin serum levels.

 

 

1.       Vejborg RM, Hancock V,Schembri MA, Klemm P (2011) comparative genomics of Escherichia coli strains causing urinary tract infections. Applied and Environmental Microbiology77:3268-3278.

2.       Kin NW, Chen Y, Emily K, Stefanov EK, Gallo RL, Kearney JF (2011) Cathelin-related antimicrobial peptide differentially regulates T and B-cell function Eur J Immunol41: 3006-3016.

3.       Gombart AF (2009) The Vitamin D-antimicrobial peptide pathway and its role in protection againstinfection. Future Microbiology4: 1151-1165.

4.       Chromek M, Slamova Z, Bergmanet P, Kovács L, Podracká L, et al. (2008) The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterialinfection. NatureMedicine12.

5.       Aranow C (2011) Vitamin D and the Immune System. JInvestig Med 59: 881-886.

6.       HerttingO,Holm A, Lüthje P, Brauner H, Dyrdak R,et al. (2011) Vitamin D induction of the human antimicrobial peptide cathelicidin in the urinary bladderPlos One5: e15580.

7.       HabibS (2012) Highlights for Management of a Child with a Urinary Tract Infection. Int J Pediatr2012.

8.       Saadeh SA and MattooTK (2011) Managing urinary tract infections, PediatrNephrol26:1967-1976.

9.       Chishti AS, Maul EC,Nazario R J, Bennett JS, Kiessling SG(2010) A guideline for the inpatient care of children with pyelonephritis. Ann Saudi Med30: 341-349.

10.    Gambichler, Demetriou, Terras et al. (2011) "Dermatology. Basel, Switzerland2011.

11.    Gurgoze M, Akarsu S, Yilmaz E, Gödekmerdan A, Akça Z, et al. (2005) Proinflammatory cytokines and procalcitonin in children with acute pyelonephritis. Pediatric Nephrol20: 1445-1448.

12.    Cheng CH, Tsai MH, Huang YC, Su LH, Tsau YK, et al.(2008) Antibiotic resistance patterns of community acquired urinary tract infections in children with vesicoureteral reflux receiving prophylactic antibiotic therapy. Pediatrics122:1212.

13.    Navidinia M, Karimi A, Rahbar M, Fallah F,AhsaniRR, et al. (2012) Study prevalence of verotoxigenic E.coli isolated from urinary tract infections (UTIs) in an Iranian children hospital. The open microbiology journal 6: 1-4.

14.    Neuhaus TJ, Berger C, Buechner K, Parvex P, Bischoff G, et al.(2008) Randomized trial of oral versus sequential intravenous/oral cephalosporin's in children with pyelonephritis. Eur J Pediatr167:1037-1047.

15.    Yamshchikov AV, Kurbatova EV, Kumari M, Blumberg HM, Ziegler TR, et al. (2010) Vitamin D status and antimicrobial peptide cathelicidin (LL-37) concentrations in patients with active pulmonary tuberculosis. J ClinNutr92:603-611.

16.    Watkins R, Yamshchikov AV, Lemonovich TL, Salata RA (2011) The role of vitamin D deficiency in sepsis and potential therapeutic implications. Journal of infection321: 326.

17.    Tekin M, Konca C, Celik V, Almis H, Kahramaner Z, et al. (2015) The Association between Vitamin D Levels and Urinary Tract Infection in Children. Horm Res Paediatr83:198-203.

18.    Hacıhamdioğlu DO, Altun D, BülentHacıhamdioğlu B, Çekmez F, Aydemir G, et al. (2016) The Association between Serum 25-Hydroxy Vitamin D Level and Urine Cathelicidin in Children with a Urinary Tract Infection Clin Res Pediatr Endocrinol8: 325-329.

19.    Yang j, Chen G, Wang D, Chen M, Xing C, et al. (2016) Low serum 25-hydroxyvitamin D level and risk of urinary tract infection in infants. Medicine 95: 27.

20.    Bikle DD (2008) Vitamin D and the immune system: role in protection against bacterial infection. CurrOpinNephrolHypertens 17:348-352.

21.    Karatekin G, Kaya A, Salihoglu O, Balci H, Nuhoğlu A (2009) Association of subclinical vitamin D deficiency in newborns with acute lower respiratory infection and their mothers. Eur J ClinNutr63:473-437.

22.    Kai-Larsen Y,thje P L, Chromek M, Peters V, Wang X, et al. (2010) Uropathogenic Escherichia coli Modulates Immune Responses and Its Curli Fimbriae Interact with the Antimicrobial Peptide LL-37. PLoSPathog6.

23.    Kamen DL and Tangpricha V (2011) Vitamin D and molecular actions on the immune system: modulation of innate and autoimmunity. J Mol Med88:441-445.

Chromek M, Slamová Z, Bergman P, Kovács L, Podracká L, et al. (2006) The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Nat Med12:636-641.

Citation: Abd El-Salam M, Zein El-Abdin MY, Mouhamed El- Bagoury I, Abd Al-Magid N (2017) Cathelicidin and Vitamin D Serum Levels in Children with Urinary Tract Infection. J Urol Ren Dis: JURD-172. DOI: 10.29011/2575-7903. 000072