MMP9 overexpression is associated with good surgical outcome in children with UPJO: Preliminary results

Patient selection

After obtaining institutional ethic board approval, UPJ specimens were obtained from 16 children (11 boys, 5 girls) at the time of dismembered pyeloplasty due to intrinsic UPJ stenosis (median age, 4.0 years, range, 1–16 years). All cases of UPJ obstruction were confirmed on the basis of radiological, scintigraphic, and operative findings.

We first analyzed MMP9, TIMP1 and RECK expression levels in fresh tissue specimens from the 16 children using quantitative real-time polymerase chain reaction (qRT-PCR). Tissue sample was taken at the time of the surgery and a small fragment of 1 by 1 cm from the transition between ureter and pelvis were analysed.

Then correlated the expression levels of the genes with important clinical parameters (Grade and DTPA) and surgical outcome in children with UPJO. According to grade study population was divided in 2 categories according to Society of Fetal Urology classification, grade 3 (moderate) and 4 (severe). For DTPA we subdivided the childrens in 2 groups, obstructive (T 1/2 more than 20 min) and partial obstructive (T 1/2 between 10 and 20 min) and success in a surgery is defined as decrease in T 1/2 to less than 20 min, absence of symptoms, improving renal function and decreasing dilatation on successive exams.

RNA isolation and cDNA synthesis

All tissue samples were obtained from surgical specimens and immediately frozen at −170 °C in liquid nitrogen. Total RNA was isolated with an RNA aqueous Kit (Applied Biosystems, CA, USA) according to the manufacturer’s instructions. RNA concentration was determined by 260/280 nM absorbance using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). cDNA was generated using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). The reactions were incubated at 25 °C for 10 min, followed by 37 °C for 120 min and 85 °C for 5 min. The cDNA was stored at −20 °C until further use.

Quantitative real-time PCR and gene expression

Statistical analysis

We used the ΔΔCT method to calculate the relative expression of the three target genes using the formula ΔΔCT. The fold change in gene expression was calculated as 2^-ΔΔCT. To compare the clinical characteristics of children with UPJ, we used the Mann–Whitney test. Statistical analysis was performed using SPSS 19.0 for Windows using a significance of p ≤0.05.

Discussion

UPJ obstruction is mostly considered as a functional obstruction originating from abnormalities in the smooth muscle of the pelvis and ureter [14]. Although surgery in UPJ obstruction is efficient to protect the patient against renal function lost, results obtained in both experimental and human-studies suggest that UPJ obstruction induces permanent modifications of the renal parenchyma.

The nature of the abnormalities at the UPJ in children with congenital intrinsic UPJ obstruction remains controversial. Many studies revealed that UPJ obstruction is associated with a significant difference in the collagen and smooth muscle structural components [15]. The finding of the increased tissue matrix ratio was believed to decrease the ureteral distensibility resulting in damage to muscle cells influencing the contractility [16]. Furthermore, a variety of intrarenal factors lead to progressive interstitial and renal parenchyma fibrosis in patients with Congenital anomalies of the kidney and urinary tract, like UPJ, including growth factors, cytokines, chemokines and adhesion molecules, which are produced by the hydronephrotic kidney. An altered renal expression of growth factors and cytokines modulates cell death by apoptosis or phenotypic transition of glomerular, tubular, and vascular cells. Mediators of cellular injury include hypoxia, ischemia, and reactive oxygen species, while fibroblasts undergo myofibroblast transformation with increased deposition of extracellular matrix [17].

The present study is the first to investigate the expression of MMP9 and its negative controllers in obstructed UPJ tissue. The change in expression of ECM components could be an alternative mechanism leading to UPJ obstruction together with the reduction of interstitial cells of Cajal. The reduction in peristalsis, result of the reduced number of Cajal’s cells associated to reduction in distensibility, result of alteration in ECM components could be the physiopathology of the disease [18]. MMPs have many important functions in wound healing processes and angiogenesis. In the case of deregulation of their production, matrix degradation and turnover are the consequences [19] and It has been shown that an increase in ECM turnover influences the neuronal network within the ureteral wall. Also, some MMPs have been proved to be neurotoxic degrading ECM proteins like collagen type 1, which are normally able to protect cultured neurons from cytotoxic cell death [20].

Defective collagen production from smooth muscle cells has been held responsible for this pathology and decreased neural cells have been thought to play an important role, especially in intrinsic type obstructions [21]. There are other studies showing that this disintegration in the configuration of smooth muscle and overdeposition of collagen may be an etiologic factor, and collagen to smooth muscle ratio may have a prognostic value [15].

There are some limitation that should be pointed out. Our small number of cases would interfere in our findings and may affect in our subgroups analysis (successful against failure and intrinsic against crossing vessel group). Since it refers to a low incidence disease with decreasing surgical indication it may be of difficult to increase sample size in a small period of time. Also the lack of control group because of the absence normal tissue also may impact in our findings. At last, there is an intentional bias in selection, including a higher number of failure cases does not reflects the overall success in the surgical approach.