Senescent Kidney Cells in Hypertensive Patients Release Urinary Extracellular Vesicles.

Background Hypertension may be associated with renal cellular injury. Cells in distress release extracellular vesicles (EVs), and their numbers in urine may reflect renal injury. Cellular senescence, an irreversible growth arrest in response to a noxious milieu, is characterized by release of proinflammatory cytokines. We hypothesized that EVs released by senescent nephron cells can be identified in urine of patients with hypertension. Methods and Results We recruited patients with essential hypertension (EH) or renovascular hypertension and healthy volunteers (n=14 each). Renal oxygenation was assessed using magnetic resonance imaging and blood samples collected from both renal veins for cytokine-level measurements. EVs isolated from urine samples were characterized by imaging flow cytometry based on specific markers, including p16 (senescence marker), calyxin (podocytes), urate transporter 1 (proximal tubules), uromodulin (ascending limb of Henle's loop), and prominin-2 (distal tubules). Overall percentage of urinary p16+ EVs was elevated in EH and renovascular hypertension patients compared with healthy volunteers and correlated inversely with renal function and directly with renal vein cytokine levels. Urinary levels of p16+/urate transporter 1+ were elevated in all hypertensive subjects compared with healthy volunteers, whereas p16+/prominin-2+ levels were elevated only in EH versus healthy volunteers and p16+/uromodulin+ in renovascular hypertension versus EH. Conclusions Levels of p16+ EVs are elevated in urine of hypertensive patients and may reflect increased proximal tubular cellular senescence. In EH, EVs originate also from distal tubules and in renovascular hypertension from Henle's loop. Hence, urinary EVs levels may be useful to identify intrarenal sites of cellular senescence.

Companied P16 genetic and protein status together providing useful information on the clinical outcome of urinary bladder cancer.

SPEC P16/CEN3/7/17 Probe fluorescence-in-situ-hybridization (FISH) has become the most sensitive method in indentifying the urothelial tumors and loss of P16 has often been identified in low-grade urothelial lesions; however, little is known about the significations of other P16 genetic status (normal and amplification) in bladder cancer.We detected P16 gene status by FISH in 259 urine samples and divided these samples into 3 groups: 1, normal P16; 2, loss of P16; and 3, amplified P16. Meanwhile, p16 protein expression was measured by immunocytochemistry and we characterized the clinicopathologic features of cases with P16 gene status.Loss of P16 occurred in 26.2%, P16 amplification occurred in 41.3% and P16 gene normal occurred in 32.4% of all cases. P16 genetic status was significantly associated with tumor grade and primary tumor status (P = .008 and .017), but not with pathological tumor stage, overall survival, and p16 protein expression. However, P16 gene amplification accompanied protein high-expression has shorter overall survival compared with the overall patients (P = .023), and P16 gene loss accompanied loss of protein also had the tendency to predict bad prognosis (P = .067).Studies show that the genetic status of P16 has a close relation with the stages of bladder cancer. Loss of P16 is associated with low-grade urothelial malignancy while amplified P16 donotes high-grade. Neither P16 gene status nor p16 protein expression alone is an independent predictor of urothelial bladder carcinoma, but combine gene and protein status together providing useful information on the clinical outcome of these patients.

p16(INK4a) /Ki-67 dual labelling as a marker for the presence of high-grade cancer cells or disease progression in urinary cytopathology.

OBJECTIVE: Overexpression of p16(INK4a) independent of the presence of E6-E7 oncoproteins of high-risk papillomaviruses has been identified in bladder carcinoma in situ lesions with or without concurrent papillary or invasive high-grade (HG) urothelial carcinoma. As p16(INK4a) and Ki-67 co-expression clearly indicates deregulation of the cell cycle, the aim of this study was to investigate the frequency of p16(INK4a) /Ki-67 dual labelling in urinary cytology samples. METHODS: Immunolabelling was performed in demounted, destained Papanicolaou slides after ThinPrep(®) processing. A total of 84 urinary cytology samples (18 negative, 10 low grade, 19 atypical urothelial cells and 37 high grade) were analysed for p16(INK4a) /Ki-67 co-expression. We assessed underlying urothelial malignancy with cystoscopy, histopathology and follow-up data in every case. RESULTS: Compared with raw histopathological results, p16 (INK4a) /Ki-67 dual labelling was observed in 48 out of 55 (87.3%) HG lesions and in 11 out of 29 (37.9%) negative, papillary urothelial neoplasia of low malignant potential or low-grade carcinomas (P = 0.05). All cases with high-grade/malignant cytology were dual labelled. Sixteen out of 17 (94.1%) carcinoma in situ cases and eight out of 14 (57.1%) cases with atypical urothelial cells matching with HG lesions were dual labelled. Extended follow-up allowed three cases of progression to be diagnosed in dual-labelled cases with negative/low-grade cytology results after a 9- to 11-months delay. CONCLUSIONS: The data show that p16(INK4a) /Ki-67 co-expression allows most HG cancer cells to be detected initially and in the follow-up period. Additional studies are needed in order to determine whether dual labelling can be used as a triage tool for atypical urothelial cells in the urine.

[Expression of the tumormarker p16INK4a in cytology specimens of the urinary bladder. A new means for early recognition and surveillance of urothelial cancer]. / Vorkommen einer p16INK4a-Expression in Zytologien der Harnblase. Ein neuer Weg zur Früherkennung und Verlaufskontrolle beim Urothelkarzinom.

BACKGROUND: This study was carried out to learn whether cytological specimens from urinary bladder lavages express the tumor suppressor gene p16INK4a, whether an abnormally increased expression indicates a cancerous state and whether cytological measurements are comparable regarding sensitivity and specificity with measurements made in histological sections of biopsies. PATIENTS AND METHODS: A total of 82 urine specimens of patients suspected of having a bladder tumor were examined for the presence of p16INK4a. RESULTS: Out of 46 patients with urothelial carcinoma 29 expressed p16INK4a in the cells in the urine specimens. Out of 36 patients free of cancer 30 expressed no p16INK4a in cytological specimens. The sensitivity of the expression proved to be 63% and the specificity 83%. Well-differentiated carcinomas seldomly expressed an increased p16INK4a (sensitivity 27%), whereas moderately differentiated carcinomas showed a sensitivity of 69% and poorly differentiated carcinomas a sensitivity of 77%. CONCLUSION: Compared to other minimally invasive tumor markers, such as NMP22, the expression of p16INK4a in cytology specimens of urine appears to be a sensitive marker for urothelial carcinoma, especially for the detection of poorly differentiated carcinomas. Its high specificity makes it ideal for use in tumor screening.