The Expression of Ptch1 and Ptch2 in the Stenotic Tissue of Congenital Ureteropelvic Junction Obstruction in Children

Objective Ptch1 and Ptch2 are expressed in tubular epithelium and stromal cells adjacent to the UPJ. They mediated inhibition of smoothened, a transmembrane protein expressed on the cell surface. If the pathway was disturbed, UPJO might be onset. The aim of this study was to determine the expression of Ptch1 (P1) and Ptch2 (P2) in stenotic segments in children with congenital ureteropelvic junction obstruction (UPJO) versus in normal control subjects. Materials and methods P2 that in the cytoplasm of in two groups there statistical differences the two groups in P1 and P2 with Meanwhile there were no statistical differences with Western and and respectively). But we found that in the immunofluorescence P1 were diffused in controls and mainly surrounding the nucleuses of smooth muscle cells in UPJO.

patients with Wilm's tumor, and the tissues were confirmed histologically to be unaffected.
Immunofluorescence, Western blot and real-time PCR were used to investigate the expression of P1 and P2. Statistical methods were used to find the differences between the two groups Results P1 and P2 were identified that located in the cytoplasm of smooth muscle in two groups through Immunohistochemistry. However, there were no statistical differences between the two groups in P1 and P2 with immunohistochemistry (P=0.31 and P=0.3, respectively). Meanwhile there were no statistical differences with Western blot (P=0.75 and P=0.9, respectively) and real-time PCR (P=0.52 and P=0.45, respectively). But we found that in the immunofluorescence P1 were diffused in controls and mainly surrounding the nucleuses of smooth muscle cells in UPJO.

Conclusions
The expression of P1 and P2 between the two group had no statistically significant. P1 mainly surrounding the nucleuses of smooth muscle cells in UPJO. The P1 pathway might be disturbed by the abnormal distribution of P1 rather than the quantity.

Background
Ureteropelvic junction obstruction (UPJO) is the most common form of congenital urinary tract obstruction (1). Most cases of UPJO are caused by intrinsic narrowing of the ureter at the pelvic-kidney 3 junction, while the underlying pathogenic mechanisms are undefined exactly (2). Researchers found that Sonic hedgehog (Shh) signaling pathway was involved in kidney and ureter development during embryonic period (3,4). Several researchers had found that the pathway was disturbed by experimental models and UPJO children (5).
Shh pathway was important in renal development. There were Shh and its receptor Patched in the tubular epithelium and stromal cells of the UPJ, (6). Shh bound to Ptch and relieved Smoothened, which was a transmembrane Ptch-mediated inhibition protein expressed on the cell surface (5). Fulllength Gli3 proteins translocated to the nucleus after bounding, acting as transcriptional activators (7). If the pathway was disturbed, UPJO might be onset.
There are two Ptch homologs in mammals, Ptch1 (P1) and Ptch2 (P2), both of which bind to Shh ligands with similar affinity (8). Despite rigorous investigations, the Ptch in UPJO children remains unclear, and whether there was variation yet to be proved (9). The expression of P1 and P2 were compared in hydronephrotic model or contiguous segments in same ureters of UPJO patients, but the actual human ureteropelvic junction segments were not (5,10).
Here, we investigated the expression of P1 and P2 in stenotic segments in children with PUJO versus in normal control subjects using immunohistochemistry, Western blot and real-time PCR methods, aiming to find the possible pathogenic mechanisms in congenital hydronephrosis due to UPJO. The control specimens were obtained from ureteropelvic junction segments of Wilm's tumor patients.

Patients and control samples
This study was approved by the Ethics Committee (2019-k-368). Stenotic segments of ureter tissues were obtained from 20 UPJO patients. The diagnosis was based on the accessory examination.
Surgical indications followed EUA guideline. Intrinsic stenosis was confirmed during operation.
Extrinsic stenosis, such as vessel and ureteral polyp, were excluded. The control ureter specimens were obtained from 10 patients with Wilm's tumor, and the tissues were confirmed histologically to be unaffected. The samples of ureteropelvic junction were immediately stored at -80℃ immediately after operation.
Immunofluorescence Of P1 And P2 4 Immunofluorescence was performed on 4 µm paraffin sections; Dako serum-free protein block was used for blocking, primary and secondary incubation buffers. Whole-mount immunofluorescence was performed as usual. Antibodies were used: P1 (Primary antibodies: goat, lot: ab39266; Fluorescent secondary antibodies: CY3 Labeled rabbit anti-goat IgG) and P2 (Primary antibodies: rabbit, lot: ab238338; Fluorescent secondary antibodies: 488 Labeled goat anti-rabbit IgG) (Abcam company, 1:100). Every patient's tissue was divided into three parts, every part was photographed under fluorescent microscopy in five different areas and calculated the average value.

Western Blot Of Ptch1 And Ptch2
Pre-cooled RIPA protein extraction reagent (50 mMTris-HCl(pH 7.4), 150 mM NaCl, 1% np-40, 0.1% SDS) were added to Protease inhibitor cocktail (Roche) (phosphorylated protein needs to be added to phosphatase inhibitor at the same time) (according to the volume ratio of 50:1).The tissue was cut into pieces, and 50 mg of tissue was added into 500 ul lysate. The tissue was centrifuged at 13000 rpm for 20 min. Remove the supernatant and store at -80 degrees for Wb. The separation gel All data were presented as mean ± SD. Significance of differences was evaluated by using two-sample t test, Linear regression analysis was used to test the correlation among the parameters, P value < 0.05 was considered to be statistically significant.

Results
The UPJO patients ranged from 6 month to 12.5 years old, with a mean age of 3.3 years. The control group ranged from 1.2 month to 7 years old, with a mean age of 3.2 years. No statistically differences were found between the two groups with regard the age of patients (P = 0.28).
Immunofluorescence P1 was specifically stained red and P2 was green, the nucleuses of smooth muscle cells were stained blue. In UPJO group, expression of P1 and P2 were found in stenotic segments ( Fig. 1A and 1C). In control group, expression of P1 and P2 were detected in ureteropelvic junction segments ( Fig. 1B and   1D), linear regression analysis between P1 and P2 with age were P = 0.63 and P = 0.33, respectively.
There were no statistical differences between the two groups in P1 and P2. Results of the immunofluorescence and P values between the two groups were summarized in Table 1.

Western Blot
Western blot method was used to examine the expression level of P1 and P2 in UPJO and control groups (Fig. 2). Quantity was calculated the level of protein expression, followed by the calculation of the relative intensity through standardization using the expression of β-actin as the internal control.
P1 and was 0.58 ± 0.08 in UPJO and 0.57 ± 0.09 in controls; P2 was 0.44 ± 0.15 in UPJO and 0.44 ± 0.11 in controls. The expression of P1 and P2 between the two group had no statistically significant (P 0.05). Results of the Western blot and P values between the two groups were summarized in Table 1.

Real-time PCR
Amplification and melting curves were drawn according to the mRNA fluorescence values and cycle 6 numbers of P1 and P2, using Beta actin as the internal control (Fig. 3). Each sample was repeated five times in real-time PCR test and averaged as the Ct value. The 2 −△△ct relative quantitative method was used for mRNA expression analysis by ABI 7500 fluorescence quantitative PCR instrument. The differences in mRNA expression of P1 and P2 in the two groups had no statistically significant (P < 0.05) ( Table 1).

Discussion
There was a lot of research on molecules mechanisms underlying congenital intrinsic UPJO, but it is still poorly understood (11). Researches demonstrated that a Shh-Ptch-Gli3 dependent mechanism caused murine intrinsic UPJO and implicated dysregulated this signaling in the pathogenesis of human was increased in a single patient out of eight (5), but the control tissue was contiguous UPJO segment.
So, these results could not describe a real pathogenic mechanism underlying congenital UPJO.
Here we obtained 20 intrinsic UPJO tissues and 10 ureters from patients with Wilms tumor, Specimen was strictly collecting and obtaining UPJ segments in two groups. Even so, the results were 7 depressing, our results demonstrated that P1 and P2 were not changed in the quantity of UPJO patients through immunofluorescence, western blot and PCR. We also found that age was not related with quantity of both. All the results were negative and we reached an impasse in our research as well. Previous studies found that P1 was removed from cilia after binding of the Shh ligand to P1 and allowing Smoothened (A G-coupled transmembrane protein) to enter cilia and become activated. If P1 was not removed, it would prevent Smoothened's localization to cilia and disrupt the Gli transcription activator/Gli3 repressor (Gli3R) balance (7). According to this molecular biology, the P1's distribution may be abnormal in the UPJO patients if this pathway had been disturbed. We turned back to examine the immunofluorescence of P1 again, found that red-stained P1 were diffused in control group ( Fig. 1B), but were mainly surrounding the nucleuses of smooth muscle cells in UPJO. Based on our previous findings, we inferred that P1's quantity was unchanged, but the movement was restricted in UPJO. Thus, the whole Shh pathway was disturbed. It was only a hypothesis that we inferred from our founds, and it was needed further study to confirm this.
P2 in the shh signaling pathway remains ambiguous. Researches had shown that the two homologs had overlapping functions. But it cannot compensate for the loss of P1 activity (14). Studies found that P2 does not play a functional role in regulating Shh signaling activity during kidney development, but serves as a specific marker of the onset of UPJO (5). In some studies, P2 was increased in UPJO (5), but in our research, P2 was not changed and we could not find any regularities of distribution. We strictly followed the rules of specimen collection of uretero-pelvic-junction in UPJO and control groups, but in other researches, they were not quite exactly the human uretero-pelvic-junction in control group.

Limitations
But we only assayed two factors of P1 and P2. The upstream signal Shh, downstream target factors such as Smoothened and Gli3 were not tested in our research, because they were verified in many studies (3,4). So, our objective was to prove the variation of Ptch. Although we had only seen the abnormal distribution and unchanged quantity of Ptch. Besides, we found nothing. Next, we will carry further study in interaction between P1 and Smoothened to explore the pathogenesis of UPJO. Availability of data and materials: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate: The experimental protocol was approved by the Ethics Committee of Beijing Children's Hospital, Capital Medical University. We obtained all the written consents from the participants or their parents in the study.

Figure 1
Ptch immunohistochemistry stain (×400). A: P1 was specifically stained red in the stroma in UPJO group. P1's distribution was mainly surrounding the nucleuses of smooth muscle cells (stained blue). B: P1 was stained red and diffused in the stroma in normal control group.
C~D: P2 was specifically stained green in the stroma in two groups.
12 Figure 2 Western blot analysis of P1 and P2 protein expression in UPJO patients and in controls. X indicates UPJO group and N control group. The protein expression of P1 and P2 in UPJO tissue had no statistics significance comparing with control group.
13 Figure 3 Real-time PCR analysis of 1 and P2 mRNA expression in UPJO and control group. A~B: Amplification and melting curves for Beta actin; C~D: Amplification and melting curves for P1 mRNA expression; E~F: Amplification and melting curves for P2 mRNA expression.