Expression of parathyroid hormone/parathyroid hormone-related peptide receptor 1 in normal and diseased bladder detrusor muscles: a clinico-pathological study

This study was authorized by the Institutional Review Board of Kyoto University (G279) and Tokyo Women’s Medical University (2089), and written informed consents for participation in the study were obtained from participants or parents. Normal kidney tissue, as a positive control of PTH1R, was obtained from a nephrectomy specimen from a patient with renal cell carcinoma without paraneoplastic hypercalcemia, and was used as a positive control for the staining procedure. Bladder specimens were obtained from the bladder dome by sagittal section in following patients. In total, 3 normal control bladder tissues were obtained during cystotomy for ureteral reimplantation for vesicoureteral reflux in two patients, and partial cystectomy for urachal cyst in one patient, who all underwent surgery at the Department of Urology, Kyoto University Hospital. These three patients had no symptoms of abnormal bladder storage or emptying. The clinical backgrounds of the control patients are summarized in Table 1. Data are expressed as mean ± S.D.

In total, 13 diseased bladder tissues were obtained from patients who underwent bladder augmentation at the Department of Urology, Tokyo Women’s Medical University. The clinical backgrounds of the patients are summarized in Table 2. The median age of the patients was 15 years (range 8–31 years, mean 17.8 ± 8.8 years), and included 9 males and 4 females. Seven patients had spinal disorders, three had posterior urethral valves and three had Hinman syndrome. All the patients underwent preoperative urodynamic studies combined with cystography. Bladder augmentation was indicated for low-compliance bladder and/or incontinence, despite aggressive anti-cholinergic regimens. The bladder capacity was presented as the percentage of the reported age-standard capacity for Japanese children, calculated by the formula 25 × (age in years + 2) for patients younger than 12 years, and 350 ml was defined as 100% for patients older than 12 years [9].

The specimens were fixed in formalin and paraffin-embedded. Sections were mounted on glass slides and used for immunohistochemical detection of PTH1R. Incubation and washing procedures were carried out at room temperature, unless otherwise specified. Deparaffinization and antigen retrieval were carried out in a microwave, as described previously, [10] and endogenous peroxidase activity was then blocked with 0.3% H₂O₂ in methyl alcohol for 30 min. The glass slides were washed six times in phosphate-buffered saline (PBS) for 5 min each, and mounted with 1% horse normal serum in PBS for 30 min for pre-blocking. Primary antibody against PTH1R (3D1.1, Santa Cruz Biotechnology, Dallas, TX, USA; 1:100) was then applied overnight at 4°C, followed by incubation with biotinylated horse anti-mouse serum (second antibody) (ABC-Elite, Vector Laboratories, Burlingame, CA, USA) diluted 1:300 in PBS for 40 min, followed by six washes in PBS (5 min each). Avidin-biotin-peroxidase complex (ABC-Elite, Vector Laboratories, Burlingame, CA) was applied for 50 min at a dilution of 1:100 in bovine serum albumin. After washing six times in PBS (5 min each), a coloring reaction was carried out using diaminobenzidine, with a uniform reaction time for all specimens. Nuclei were counterstained with haematoxylin. PTH1R protein-expression intensity was classified as negative or positive by two independent examiners, with reference to renal tubules as a positive immunostaining control.

In the kidney specimen, the tubules stained positive for PTH1R, in contrast to the glomeruli, which stained faint, as reported previously (Figure 1A) [11]. In the normal control bladder specimens, both the detrusor tissue and blood vessel stained positive for PTH1R. Staining in the detrusor tissue was mainly observed in the cytosol (Figure 1B).

Preoperative urodynamic study revealed that the bladders in all patients undergoing bladder augmentation had deteriorated storage function. The median bladder capacity was 146 ml (range 5–303 ml, mean 153.6 ± 84.0 ml), median percent capacity of age-standard was 43.6% (range 1.5–86.6%, mean 46.9 ± 23.2%), median intravesical pressure at maximal capacity was 30 cmH₂O (range 10–107 cmH₂O, mean 38.8 ± 23.3cmH₂O), and the median compliance was 3.93 ml/cmH₂O (range 0.05–30.3 ml/cmH₂O, mean 6.6 ± 7.8 ml/cmH₂O). Detrusor overactivities were noted in five patients and vesicoureteral reflux in eight patients (Table 2).

All of the bladder-augmentation specimens showed negative PTH1R staining in detrusor smooth muscle cells in the bladder wall, in contrast to the positive staining seen in normal control bladder specimen. However, blood vessels stained positive for PTH1R in seven out of 13 augmentation cases (53.8%), indicating that the negative staining was not the result of a technical failure, but rather reflected downregulation of PTH1R expression in these bladders (Figure 2).