Pyeloureteral Junction Obstruction: Underdevelopment or Fibrosis?

Introduction: congenital ureteral stenosis is one of the leading causes of impaired urinary drainage and subsequent dilatation of urinary collecting system, known as hydronephrosis or ureterohy-dronephrosis. Mechanism that leads to obstruction is not clearly known. Multiple studies in rat models have shown increased angiotensin 11 and TGFβ levels in obstructed ureteral tissue. The aim of the study is to investigate expression of fibrosis related genes in obstructive and normal ureteral tissue. Material and methods: it is a monocentric study in which nineteen patients were selected prospec-tively. 17 patients of which underwent Hynes-Anderson pyeloplasty due to the PUJO, 2 patients underwent ureteroneocystostomy due to ureterovesical junction obstruction (UVJO) and 6 patients were chosen for control group: 5 underwent nephrectomies due to the kidney tumor and 1- upper pole heminephrectomy due to the duplex kidney with normal pyeloureteric junctions in all. Tissue RNA was chemically extracted after freezing the biopsy samples in liquid nitrogen, with cDNA synthesis performed immediately after nucleic acid isolation. qPCR to evaluate the relative expres-sion of Tgfb1, Mmp1, Timp1, Pai1, Ctgf and Vegfa was performed. Expression levels of Gapdh and Gpi genes (geometric average) were used to calculate the relative expression of the investigated genes. Outliers were removed prior to calculating confidence intervals for the experimental groups, and Wilcoxon rank-sum test was performed to determine statistical significance of the differences. Results: significant differences between healthy and stenotic tissue samples in Ctgf gene expression levels were observed, with the samples from afflicted tissue showing lower expression. No statistical difference in expression levels of Tgfb1, Timp1, Vegfa, Mmp1, Pai1 was found. Discussion and conclusions: These findings suggest that tissue fibrosis similar to other tissues and organs is not the leading cause of stenosis, at least at the moment of surgery. Decreased CTGF ex-pression is indicative of developmental origin of obstruction.

Keywords:

Subject: Medicine and Pharmacology - Pediatrics, Perinatology and Child Health

1. Introduction

Congenital ureteral stenosis is the leading cause of impaired urinary drainage and subsequent dilatation of urinary collecting system, condition known as hydronephrosis or ureterohydronephrosis, when ureteric dilatation is present. Ureteral obstruction is a very serious clinical condition, leading to damage of renal parenchyma because of obstruction induced increased pressure, reduced glomerular filtration, activation of profibrotic factors, cell apoptosis and subsequent loss of kidney function which has been investigated in animal models in a series of studies. [1], [2] Early recognition of obstruction, followed by timely treatment usually results in recovery of a kidney, mainly because of the better reproduction potential of renal stem cells and recovery of parenchyma at an early age. [3]

Several theories of pathogenesis leading to the development of hydronephrosis were learnt from animal models. The reasons of hydronephrosis can be generally regarded as obstructive and non-obstructive.

In systematic analysis which reviewed data from 10 articles was revealed that 15 genes had changed expression in ureteral stricture tissue. Genes which were up regulated were (ET1, 87 ACTA2, MCP-1, TGFB1, NFKB1, IL-6, HIF1A, S100A1, SYP) and the expression of six genes was down regulated (ADM, NOS2, EGF, PDGFRA, UCHL1, NGFR). The products of these genes are components of HIF-1 signaling pathway and participate in the development of vasculature. Some of these factors act as inductor of signaling pathways and particularly affect Ras signaling system. There may also be a connection of current factors with others that are related with tissue hypoxia, fibrosis and the inflammation. [4]

Histological studies of obstructed human ureters identified excessive amount of collagen in extracellular matrix and impaired distribution of its isoforms, increased proportion of elastin and apoptosis of myocytes. Altogether it leads to the abnormal development of tissue. [5], [6] In the intracellular level, immunofluorescence and PCR analysis revealed that cytoskeleton structural proteins were impaired in PUJO smooth muscle cells probably explaining the increased apoptosis of smooth muscle cells in PUJO specimens. [7] Aforementioned pathological findings suggest that the pathogenesis of the development of ureteral obstruction involves the processes of fibrosis, apoptosis and cell differentiation arrest.

The aim of our study was to analyze several fibrosis related factors that are known to participate in general mechanism of fibrosis in multiple sites of human body and to discuss their involvement in previously described chains of pathogenesis.

We have chosen to investigate fibrosis-associated genes expression in congenitally obstructed ureteral tissue. As obstruction involves abnormal cell differentiation as well as changes in extracellular matrix, expression profiles of Tgfb1, Ctgf, Vegfa, Timp1, Pai1 and Mmp1 genes were chosen to be analyzed according to previous studies where they were analyzed as the fibrosis inducing factors. [8,9,10,11,12,13] We could not identify any studies describing the expression analysis of CTGF, MMP1, PAI-1 in obstructed ureteral tissue over the last 10 years. Nevertheless, these genes were chosen as closely related to Tgfb pathway and implicated in the development of tissue fibrosis in other organs, such as the heart. [14]

2. Materials and Methods

Collection of samples

It is a monocentric study in which patients were included prospectively from 2019 to 2021. Samples of pyeloureteric junctions and 2 ureterovesical junctions were collected during surgery and stored in -80°C until RNA extraction.

The decision of whether or not to operate on the patient was based on our local protocol: the operation was performed when there was an increase in the anterior-posterior diameter of renal pelvis by 20% on subsequent ultrasound scans, when the differential renal function (DRF) was <40% of the affected kidney or a decrease in DRF by >5% was observed on subsequent renal scans, or when symptoms could be attributed to hydronephrosis.

RNA extraction and qPCR

Tissue RNA was extracted after freezing the samples in liquid nitrogen, mechanically dissociating them and applying Trizol reagent (Thermo Fisher) according to the manufacturer’s protocol. 1 mL of Trizol reagent was added per 100 mg of tissue. Isolated RNA was dissolved in nuclease-free water, and its quantity and quality were evaluated using the NanoDrop device (Thermo Fisher).

cDNA synthesis was performed using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher). Each sample had reverse transcription negative (RT-) control. qPCR was performed using Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher) with the Eppendorf realplex4 quantitative PCR device. Expression levels of Gapdh and Gpi genes (geometric average) were used to calculate relative expression of the investigated genes. The Common base method, as described in [15] was used to calculate relative expression of the genes, factoring the qPCR reaction efficiency. Primer sequences used for qPCR were derived from Primer Bank (https://pga.mgh.harvard.edu/primerbank/)

Statistical analysis

Results were analyzed using RStudio Version 1.2.5033. Outliers were removed prior to analysis. Wilcoxon rank-sum test was carried out to determine the significance of differences between healthy and obstructed tissue (significance level α = 0.05), and assessment of confidence intervals was carried out to further understand the differences between the groups.

3. Results

17 patients underwent Hynes-Anderson pyeloplasty due to the PUJO, 2 patients underwent ureteroneocystostomy due to ureterovesical junction obstruction (UVJO). Six patients were chosen for control group: 5 underwent nephrectomies due to the kidney tumor and 1- upper pole heminephrectomy due to the duplex kidney with normal pyeloureteric junctions in all. Median age was 15.2 months [9.07; 66.2] at the time of the surgery and median age of control group (4 boys and 2 girls) was 60 months [35; 80.3].

The expression of fibrosis-related genes in every specimen are depicted in the heatmap. (Figure 1). Variable expression of all studied genes was noted both in obstructed and in normal junctions.

Figure 2. Relative expression of fibrosis-related genes in control and strictured ureter tissue samples. Medians (dot/triangle) and 95% confidence intervals (whiskers) shown. Star (*) indicates statistical significance between the groups (p < 0.05) as determined by Wilcoxon rank-sum test. Sample sizes are 6 for control and 19 for strictured group.

Wilcoxon rank-sum test showed significant differences between groups in Ctgf gene expression level. We further decided to compare the confidence intervals between groups, and the intervals of Ctgf gene showed little overlap, indicating differences between obstructed and control tissue samples. (Figure 2) No statistically significant differences between the groups were found in the expression of other studied genes.

Figure 1. Heatmap of relative expression of fibrosis-related genes in control and strictured ureter tissue samples. Results standardized to highest gene expression levels for each gene. PUJ – pyeloureteric junction obstruction; Tumor – pyeloureteric junction from kidney tumor specimen; Heminephr. – pyeloureteric junction from heminephrectomy specimen; VUJ – vesicoureteral junction obstruction.

4. Discussion

Out of our selected markers the best described group of fibrosis-related factors in the literature belong to transforming growth factor (TGF) family, particularly TGFs 1 to 3, sharing similar functions with minor structural differences. [16] Out of the three, the most well studied and ubiquitous is TGFβ1, a polypeptide that regulates cell differentiation, proliferation, adhesion and immune behavior. It is found in sites of injury, inflammation, cancer and elsewhere. The protein positively and negatively regulates other growth factors. The mature peptide may form heterodimers with other TGF beta family members. [17] It is stored in the extracellular matrix (ECM) as a complex with its pro-domain. Activation of TGFβ-1 requires the binding of alpha-V integrin, present on the membrane of residing cells, to an RGD sequence in the TGF prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGFβ binding proteins (LTBP). As the synthesis and release of TGFβ involves many steps of proteolytic processing, binding and release, the expression and activity of TGFβ is both time and situation-dependent.

Among the disease-relevant ROS-dependent genomic targets (reactive oxygen species), TGF-beta1 stimulates expression of the potent profibrotic matricellular PAI-1 and CTGF genes as well as those that code ECM structural elements (fibronectin, collagen I). PAI-1 is one of the most highly regulated part of the TGFβ1/SMAD3-induced group, a prominent ROS responsive gene and involved in tissue fibrosis process. As the main inhibitor of plasmin generation, PAI-1 limits ECM degradation alleviating accumulation of matrix structural elements at the injury site. PAI-1 deficiency is renal-protective whereas transgenic PAI-1 overexpression promotes an increased fibrotic response with associated recruitment of inflammatory cells, macrophages and myofibroblasts. As proof-of-concept, unilateral ureter-obstructed PAI-1^−/− mice develop a significantly attenuated inflammatory response suggesting that PAI-1 directly promotes infiltration of macrophages and T-cells. [17]

However, it is worth mentioning that out of these studies we could identify only one when polymerase chain reaction (PCR) analysis was applied in human ureters and showed that the amount of TGFβ1 mRNA in stenotic tissue was higher than in controls. [18] Another study found that immunohistochemical staining of TGFβ3 in obstructed tissue was higher than in the control tissue. [19] Even though the results of our study of TGFβ in patients with PUJO could not replicate these findings, this could be due to the observations that TGFβ1 is often expressed in the early stages of diseases, especially when macrophage activity is high, as well as when inflammation is present. Experimental studies have also advocated the increase of mRNA TGF-β in postoperative UPJO. Under these circumstances such finding can be explained by an acute onset of obstruction activating muscle fiber and collagen elaboration. [20]

We found only one study analyzing the role of PAI-1 in an obstructed kidney model and showing that TIMP-1 and PAI-1 in rat model are involved in the progression of tubulointerstitial scarring of renal tissue. [21]

With regard to one of the theories of UB branching, AngII is named as one of the enhancers for this process. PAI-1 has also been described as a downstream target of AngII in endothelial cells.[22] Is there a relation between potentially decreased PAI-1 and AngII, as its potential effector in congenitally obstructed ureter, remains to be answered. PAI-1 has been shown to play a role in ECM maintenance and remodeling, counteracting the effects of matrix metalloproteinases. [23]

We were able to identify only one study where VEGF was investigated in UPJO and a control group in human specimens: no color staining of VEGF was found in both obstructed and control groups and subsequently showed no difference in staining potential regardless of obstruction pattern. [24] Our results in VEGF expression have corresponded to the results of an aforementioned study, finding no difference in this gene expression in stenotic versus healthy tissue.

We could not identify any previous publication regarding Ctgf expression profile in congenital ureteral obstruction.

Our results have demonstrated that relative expression of Ctgf in stenotic ureteral tissue was significantly lower than in the control tissue supporting a potential role of this gene in the processes of stricture development. Ctgf has important roles in many biological processes, including cell adhesion, migration, proliferation, angiogenesis, skeletal development, and tissue wound repair, and is critically involved in fibrotic disease and several forms of cancers [9], [25]. It is thought that CTGF can cooperate with TGFβ to induce fibrosis and extracellular matrix production in association with other fibrosis-related conditions. [26] Overexpression of Ctgf in fibroblasts promotes fibrosis at the beginning of the development of stricture in the skin, kidney, and lung. We may only speculate that the reduction in our patients may be indicative on the stage of fibrosis – for example, in a nephropathy model in mice, it was shown that the expression level of Ctgf gradually increases over a 2-week period and is static afterwards; our results indicate there may be a significant reduction in the expression of this gene following the primary stage of disease development or gene can be only expressed for a certain period of time. [27]

Evident similarity in expression of other studied genes may suggest that fibrosis process is not the most important in the development of congenital PUJO and we should rather concentrate on other aspects of development.

Considering other aspects of cell distribution, the most prominent alteration comprises interstitial cells of Cajal and telocytes which are significantly reduced in comparison to normal ureteral tissue in both UPJO and UVJO. Differentiation of Cajal cells may be stimulated by overexpression of CTGF gene. [28] Also, SOX9 was found regulating CTGF/CCN2 transcription in chondrocytes. [29] Hypomotility of the ureter was induced by Sox9, a homeobox gene, the loss of which led to smooth muscle cell differentiation arrest. [30] Another study showed an essential role of Sox9 in regulation of differentiation of ureteric mesenchyme. Primary dilatation of renal collecting system without ureteral obstruction was induced by Keap1 knockout and expression of aquaporins with spontaneous mutations which caused urinary hyperproduction and secondary dilatation of renal collecting system. [31]

The drawback of our study is the relatively low and heterogeneous by age sample size. Although statistical analysis was possible, more specimens would be needed to elucidate the potential differences in the expression of our studied genes. Another drawback is a small and non-homogenous control group when 5 pyeloureteric junctions were obtained from renal tumor specimens and 1 pyeloureteric junctions from duplex kidney system. None of them had macroscopic signs of stricture, however the results can be influenced by preoperative chemotherapy and maldevelopment of the ureteral wall when compared to normal ureters. [32] Observed tendencies comparing PUJ, UVJ, dilated UVJ and normal PUJ after chemotherapy may be further investigated.

In summary, our findings suggest that decreased Ctgf expression in strictured ureteral specimen may be a sign of a progressive failure determined by a genetic trigger occurring at the embryonic period. Further studies have to be conducted to verify hypotheses.

5. Conclusions

Author Contributions

All authors have accepted responsibility for the entire content of this manuscript and approved its submission. All authors prepared the manuscript.

Funding

This project has received funding from European Regional Development Fund No 01.2.2-LMT-K-718-03-0087 under grant agreement with the Research Council of Lithuania (LMTLT).

Institutional Review Board Statement

This study was approved by the Vilnius Regional Bioethics Committee (158200-18/6-1044-544, the date of approval 05/06/2018).

Conflicts of Interest

None.

Abbreviations

UB	ureteric bud;
PUJO	obstructed pyeloureteric junction;
EGFR	epidermal growth factor receptor;
AT1R	angiotensin type 1 receptor;
AT2R	angiotensin type 2 receptor;
UVJO	ureterovesical junction obstruction;
ChKD	chronic kidney disease;
ESRD	end stage renal disease;
LTBP	latent TGFβ binding proteins

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