OF UROPERITONEUM

DESCRIPTION

Field of the Invention

The present invention relates in general to the field of medical biotechnologies, and more precisely, it relates to a method for the early diagnosis of uroperitoneum by determination of a biomarker in biological samples.

State of the Art

By the term "uroperitoneum" or "uroabdomen" is meant the collection of urine in the peritoneal or retroperitoneal cavity of a patient, which may originate from kidneys, ureter, bladder and proximal portion of urethra. The causes can be of traumatic or iatrogenic origin [1 ,2]; there may be also spontaneous ruptures of the urinary tract as a result of neoplasms or of chronic inflammation. The uroabdomen has serious consequences that can even compromise the patient's life; in particular, the delay in diagnosing this condition can expose the patient to a further worsening of his clinical condition, thus increasing morbidity and mortality. In fact, the presence of urine in the abdominal cavity triggers an inflammatory cascade and a series of electrolytic disorders having a significant impact on heart and kidney functions.

Uroabdomen causes dehydration, azotaemia, hyperkalaemia and metabolic acidosis. The urine, in fact, compared to the extracellular fluid (ECF), is a hyperosmolar solution. Its accumulation in the peritoneal cavity creates therefore a gradient of concentration that determines the displacement of water from the extracellular space and causes dehydration, and the diffusion towards the extracellular compartment of lower molecular weight solutes present in the urine, such as urea and potassium, causing increased azotaemia and hyperkalaemia, i.e. an excess of non-protein nitrogen and potassium in the blood, respectively. When potassium is reabsorbed into the systemic circulation beyond certain concentrations, it can trigger conduction disorders at cardiac level, and generates arrhythmias until it leads to patient’s death. In addition, a reduction in glomerular filtration was observed in patients with uroabdomen, resulting in a decreased clearance of urea and creatinine. Finally, the irritation caused by the presence of urine in the abdominal cavity is responsible for the paralytic ileus, which contributes to the displacement of fluids and albumin from the extracellular space into the abdomen.

Uroperitoneum can be observed in hospitalized, traumatized patients, in major abdominal surgery, but it is more often found following urological surgery. The most frequent cause of uroperitoneum is anastomotic dehiscence following operations involving the bladder and the urinary excretory tracts. In the literature, the incidence of perforation, rupture and anastomotic dehiscence in urological surgery greatly varies depending on the type of surgery performed and on the population studied (paediatric- adult) [3]. Particularly relevant in this regard is the augmentation cystoplasty surgery, in which the patient's natural bladder is surgically augmented thanks to the use of a tract of the intestinal wall, or cystectomy in which the bladder is completely replaced by intestinal walls. Augmentation cystoplasty is a surgical technique widely used in benign pathological conditions of the bladder, such as neurological bladder, exstrophy, or inflammatory lesions; it is however burdened by an incidence of perforations ranging from 4 to 13% [4] In radical cystectomy, used in cases of bladder cancer, although significant improvements have been achieved in the surgical technique and in the volume / pressure ratio of the bladder reservoir, complications are not uncommon. The most commonly observed complications, besides intestinal obstruction, urinary tract infections, electrolytic disorders, incontinence, and fistulas, are perforations and rupture with formation of uroperitoneum. More specifically, this type of surgery is burdened by an incidence of perforations of about 4.3% [4]

For the reasons explained above, an early diagnosis of uroperitoneum is extremely important [5]. To date, the diagnosis of uroperitoneum starts from the patient's symptoms, ranging from pain, abdominal distension, fever, urinary retention and oliguria/anuria [6], and it is confirmed by laboratory findings on blood, urine and abdominal fluid samples. In such laboratory tests, in particular, high levels of creatinine in the abdominal drainage are strongly suggestive of uroperitoneum when the ratio between the creatinine concentration in the peritoneum and that in the serum is equal to or greater than 2. However, this dosage of creatinine has proved to be a low sensitivity marker, linked to the patient's renal function and to the amount of diuresis produced, and capable of providing a reliable result only when the loss of urine in the abdomen is very abundant [1 ,7-9].

The gold standard in the diagnosis of uroperitoneum is currently represented by Computed Tomography (CT) of the abdomen [10]. This is certainly an effective diagnostic tool, but unfortunately, it requires moving the patient, who is usually in a post-surgical phase, characterized by strong clinical instability, bedridden in an intensive care unit from where the movement is technically complicated. In addition, it requires the administration of an iodinated contrast medium and the patient is subjected to an increased radiological risk.

For these reasons, a strong need is still felt to identify a sensitive and specific marker of uroperitoneum, which therefore allows for an early diagnosis, and can be easily measured in biological samples. Moreover, an optimal marker should not be expensive, nor should its measurement be based on the use of expensive technologies, or which require the patient to be moved. In fact, being able to intervene promptly without moving the patient is in these cases the best possible treatment, which can avoid further serious consequences for the patient.

Summary of the Invention

Now inventors have identified a biomarker having a high sensitivity and specificity in the diagnosis of uroperitoneum, also in early diagnosis, in the presence of non- abundant amounts of urine in the patient’s abdomen.

Besides assisting the physician in making a diagnosis of uroperitoneum, the method of the invention allows a rapid identification of urine in patient’s biological samples, with a very limited invasiveness and without the need to move the patient from the intensive care, as is instead necessary if the patient has to undergo Computed Tomography (CT). Furthermore, the present method does not require expensive reagents or equipment.

It is therefore subject of this invention a method for the diagnosis of uroperitoneum, whose essential features are defined in the first of the annexed claims.

Characteristics and advantages of the method according to the present invention will be more clearly illustrated in the following detailed description of a non-limitative, exemplary embodiment thereof.

Detailed Description of the Invention Inventors have found that the uromodulin protein is a biochemical marker detectable in biological samples coming from the abdominal drainage, and that its concentration can be correlated to the presence of urine in the abdomen in case of uroperitoneum, in particular in patients in post-urological surgery. Inventors have also found how this marker is extremely specific to the presence of uroperitoneum.

Uromodulin or Tamm-Horsfall Protein (THP) is a kidney-specific glycoprotein as it is synthesized exclusively by the epithelial tubular cells of the thick ascending limb of the loop of Henle in the nephron [1 1] Uromodulin is the most abundant protein excreted in urine under physiological conditions (50-100 mg/die) [12]. This protein is mainly localized on the apical plasmatic membrane of the cells of the thick ascending tubule of the loop of Henle, where it is then released in the urine. Small concentrations of this protein (about 1000 times lower than concentrations in urine) can also be found in the blood since, after its production within the tubular cells, it is transported to the basolateral side of the cells and released in blood [13]. The biological functions of this protein are not yet fully known. Its chemical-physical characteristics suggest its possible role in protecting against urinary infections, in preventing the formation of urinary stones and in regulating the sodium and water balance. Mutations in the gene encoding for uromodulin lead to the development of severe and diverse clinical entities identified under the term "uromodulin-associated kidney disease" (including familial juvenile hyperuricemic nephropathy, Type 2 medullary cystic disease, and glomerulocysts with hyperuricemia) [1 1 ,14]. Recently, genomic association studies have identified uromodulin as a risk factor for chronic kidney disease (CKD) and hypertension, and have suggested that uromodulin level in urine could be a useful biomarker for the diagnosis of CKD [12,15,16].

As far as inventors are aware of, literature has not highlighted so far any links between the presence of uromodulin in abdominal fluids and its correlation with a diagnosis of uroperitoneum. Unexpectedly, inventors have now found that the uromodulin concentrations can be used to determinate the presence of urine in abdominal fluids and therefore to diagnose uroperitoneum. In fact, uromodulin is present in infinitesimal concentrations (<50 ng) in samples from abdominal drainage of patients under physiological conditions, while it can be found in measurable concentrations in this kind of samples in case of rupture or perforation of the bladder with release of urine in the patient’s abdominal cavity (uroperitoneum). In addition, this observation also applies to the very early phases of uroperitoneum, thus allowing an early diagnosis of this severe pathological condition so that the physician can promptly intervene, avoiding the patient more severe consequences.

The presence of uroperitoneum, even at the onset of uroperitoneum, i.e. when the amount of urine in the patient's abdomen is still low, can therefore be detected by searching for this uromodulin biomarker, in samples of patients who need a diagnosis in this sense, particularly in urological patients, and even more particularly in patients in the post-operative phase after urological surgery, or in any case in all those patients suspected of contamination with urine. These patients, who undergone urological surgery, represent the population of patients at the highest risk of developing uroperitoneum as a complication in the post-operative phase.

The method of the invention is therefore a method for the diagnosis of uroperitoneum in a patient in need thereof, comprising the following steps:

a) preparation of a sample from abdominal drainage previously taken from said patient;

b) determination of the presence of uromodulin in said sample; and

c) association of the presence of uromodulin in said sample from abdominal drainage to a pathological condition of uroperitoneum in said patient.

In the present invention by“sample from abdominal drainage” is meant a sample of the biological fluid taken from the abdominal cavity of a patient by means of the medical-surgical device called“drainage”.

In a preferred aspect of this invention, the present method for the diagnosis of uroperitoneum comprising a step of detection of the uromodulin concentration in a sample from abdominal drainage, and of comparison of the concentration obtained with a reference value, provides as the reference value the value of uromodulin concentration detected in a urine sample taken from the same patient at the same time when the abdominal drainage sample is taken. The accordance between the value of uromodulin concentration in the urine sample and the value of uromodulin concentration in the abdominal drainage sample is in fact a confirmation of the presence of uroperitoneum in the patient.

In the present invention by “accordance” between the values for uromodulin concentration a coincidence is meant, or a similarity with a 10% margin of difference.

For example, the determination of the presence of uromodulin and the detection of the related values of concentration in a sample can be carried out according to this invention by an immunological assay using monoclonal, polyclonal or recombinant anti- uromodulin antibodies, preferably monoclonal antibodies that recognise epitopes of the uromodulin sequence (SEQ ID NO: 1 ).

The immunological assay used in the method of this invention is preferably a direct ELISA assay of the type called“sandwich”. By ELISA assay is meant the well-known assay that takes its name from the English expression“Enzyme-Linked ImmunoSorbent Assay”, and that is used to detect the presence of an antigen by using one or more antibodies, one of which is linked to an enzyme.

In the direct sandwich ELISA assay, usually carried out in microtiter plates with wells, the bottom of these wells is coated with an antibody specific for the antigen to be detected (primary antibody). Then, after at least one washing, the sample where the presence of the antigen is to be detected and optionally the antigen’s concentration is to be determined, is added and incubated for the time necessary for the antigen to bind to the antibody. Then at least one further washing is carried out to eliminate the antigen in excess. At this point, a second specific antibody (secondary antibody) able to bind the antibody-antigen complex that may be present is added into the well, thus obtaining a sort of sandwich, from which the assay just takes its name. An enzyme that allows the chromogenic detection of the antigen is linked to the second specific antibody added. After further washings, by adding into the well the substrate of the enzyme linked to the second antibody, a coloured product is formed. The development of the colour is a signal of the presence of the antigen and, by spectrophotometric measurement of the colour, gives the possibility to obtain the concentration of the antigen present in the sample.

The colour development can be also obtained by directly binding the antibody to a chromophore substance.

The method according to the invention therefore allows a diagnosis to be made on patients suffering from uroperitoneum, even at the onset of this disease. The method of this invention can also be used to confirm diagnoses already proposed based on an objective examination of the patient and/or results of an analysis on urine samples from the same patient. In addition, it is a non-invasive method, which does not require medical treatments other than those already used for a patient in the post- operative phase after urological surgery; the use of expensive reagents or equipment is not required as well. Furthermore, there is no need to move the patient.

The diagnosis by the method of this invention allows detecting the presence of urine inside the abdominal cavity (uroperitoneum) in case of detection of the uromodulin protein in the sample under examination. The method of this invention is extremely sensitive and allows detecting even minimal amounts of uromodulin in a biological sample.

Experimental Part

In the validation experiments conducted by the inventors, portions of blood, urinary and abdominal drainage samples were used, taken from selected patients in the population indicated below, after having received their written, free and informed consent, to the sampling and study carried out on the material collected, and described below, which was also approved by the local Ethics Committee of the University of Pisa. Samples were not stored, but destroyed after analysis.

For the uromodulin dosage, portions of blood, urinary and abdominal drainage samples are used, already taken daily from patients for the normal standard therapy and monitoring practices. In particular, blood samples are taken from the arterial line and urine samples from the urine collection bag, while the abdominal fluid is taken from the drainage system. The selected patients undergo clinical monitoring, like all other hospitalized patients, in addition to the dosage of creatinine and to the urinary volume. They also undergo the evaluation of the patient’s severity indexes SAPS II (Simplified Acute Physiology Score) and SOFA (Sequential Organ Failure Assessment) according to the standard practice for the monitoring of patients admitted to the Intensive Care Unit (ICU). No additional stress is therefore created in the patient, due to the control and dosage of uromodulin.

The population, where the subjects meeting the inclusion criteria listed below are selected, consists of patients admitted to the Intensive Care Unit, in the University Anaesthesia and Resuscitation Department IVa of the University of Pisa. The selected patients are enrolled upon admission to the department when the investigator explains the characteristics of the study, and has the patient read the information text, the consent form and the form on personal data protection. The inclusion criteria are as follows:

1. Age > 18 years;

2. Patients able to provide their consent;

3. Patients subjected to major abdominal or urological surgery, particularly urological patients, urological patients with uroperitoneum and non-urological patients;

4. Patients requiring a post-surgery hospitalization in ICU;

5. Patients who provided their consent to take part to the study and to the treatment of personal data.

Depending on the type of surgery they underwent and the possible development of uroperitoneum in the post-operative phase, the patients enrolled were divided into the following three groups:

- GROUP OF UROLOGICAL PATIENTS: patients subjected to major urological surgery, that may be burdened by perforation of the bladder or of the excretory tract, such as: radical cystectomy with bladder reconstruction (orthopic ileal neobladder, uretero-ileus cutaneostomy), radical cystectomy with ureterocutaneostomy and radical prostatectomy. This study group is characterized by patients with a regular post-operative clinical course, without clinical complications.

- GROUP OF UROLOGICAL PATIENTS WITH UROPERITONEUM: patients subjected to major urological surgery that have developed uroperitoneum during or after hospitalization in intensive care unit. Included in this group are the patients having signs and/or symptoms of a bladder perforation (abdominal pain with signs of peritoneal irritation, presence of suspicious material in abdominal drainages, onset of fever, increase in flogosis indexes), with uroperitoneum confirmed by abdominal TC. - GROUP OF NON UROLOGICAL PATIENTS: patients subjected to major abdominal non urological surgery, then without intra-operatory involvement of the kidneys, excretory tract, or prostate.

Scheme of the validation study

This is a monocentric study with the creation of an ad hoc database, accessible only to the investigator and conducted according to the international standards of Good Clinical Practice, in accordance with the latest revision of the Declaration of Helsinki (2008). For each patient selected according to the aforementioned criteria, the study provides for the execution of the following investigations at the time of admission to the ICU (TO) within 6 hours from the admission to the ICU (time T1 ), after 12 hours from admission to the ICU (T2), after 18 hours from admission to the ICU (T3), after 24 hours from admission to the ICU (T4), and after 48 hours from admission to the ICU (T5):

taking a urine sample from the bladder catheter for measuring uromodulin and creatinine; about 5 ml of urine are taken and placed in a standard container suitable for collecting urine;

taking of blood from the arterial line for measuring uromodulin and creatinine by means of a Vacutainer® system. 6 ml of blood are taken from an arterial line already present on the patient for the normal standard procedures of therapy and monitoring; blood is collected in a Falcon tube;

taking of a sample from abdominal drainage for measuring uromodulin and creatinine; 6 ml of fluid are taken from an abdominal drainage already present on the patient subjected to abdominal and urological surgery; fluid drained from blood is collected in a separate tube.

For all patients enrolled the main clinical data are collected, such as age, gender, reason of hospitalization, 24-hours diuresis, urinary volume, body mass index (BMI), type of surgery, creatinemia, urea, volume of the fluid in the abdominal drainage, without any additional taking). For all patients SAPS II (after 24 hours from hospitalization) and SOFA (daily) are calculated. It is moreover noted also any possible development of uroperitoneum confirmed by abdominal TC. The concentrations of uromodulin in the abdominal fluid, in urine and in blood are compared, among each other for a same patient and for patients in the same group, as well as among the three study groups, urological, urological with uroperitoneum and non-urological.

This study has showed the presence of a significant difference between the uromodulin concentrations in patients with uroperitoneum after urological surgery. In fact, the uromodulin concentration in the fluid from abdominal drainage in the absence of uroperitoneum has infinitesimal values when compared with the values in the presence of uroperitoneum.

Analysis and interpretation of the results

In order to calculate the number of patients to be included in this study (or “sample size”) the indications of Charan and Biswas [17] were followed. The sample size was calculated assuming an expected proportion (p) of perforations of about 4.3% [18] and an absolute error (d) of 5%. The precise estimate of the sample size is 32; therefore, a total number of 40 patients was recruited to have sufficient statistical power. Data are presented as mean ± standard deviation (SD) or as a percentage (%). All statistical analyses are performed by using SPSS (IBM SPSS software version 21 ). Dosages of uromodulin are compared between groups of patients using t-test, or Mann-Whitney U-test where appropriate, and then Receiver Operating Characteristic (ROC) curves are constructed to evaluate the diagnostic value of the above parameters in predicting uroperitoneum. The area under the ROC curve for uromodulin is thus estimated as well as its sensitivity and specificity.

Dosage of uromodulin in the samples

The determination of the presence of uromodulin and the detection of the related values of concentration in the samples was carried out by using the ELISA technique and the kit for dosage Biovendor Human Uromodulin. The samples taken from patients are incubated in microtiter wells pre-coated with a polyclonal antibody anti-human uromodulin.

After 60 minutes of incubation followed by a washing, the biotinylated polyclonal anti-uromodulin antibody is added and incubated with the captured uromodulin for 60 minutes. In the well, it is then added a second specific antibody (secondary antibody) able to bind the antibody-antigen complex, the streptavidin-HRP conjugate. After 30 minutes of incubation and a last washing phase, the remaining conjugate can react with the solution of substrate. To the second specific antibody added is bound an enzyme that allows the chromogenic detection of the antigen: after further washings, following addition in the well of the substrate of the enzyme bound to the second antibody, a yellow coloured product is formed. At this point the absorbance of the resulting yellow product is measured, which is proportional to the concentration of uromodulin. A standard curve is obtained by plotting absorbance values against the uromodulin concentrations of the standards and the concentrations of unknown samples are determined using this standard curve.

The present invention was described so far with reference to a preferred embodiment thereof. It is to be understood that other embodiments may exist which belong to the same inventive core, as defined by the scope of the claims set forth in the following.

Bibliographic References

[1 ] Delacroix SE, Jr., Winters JC. Urinary tract injures: recognition and management. Clinics in colon and rectal surgery 2010; 23: 104-12.

[2] Nikolaos A, Nikolaos K, Dimitrios V, Georgios V. Uroperitoneum after Caesarean Section. Case Reports in Obstetrics and Gynecology 2013; 2013.

[3] Farnham SB, Cookson MS. Surgical complications of urinary diversion. World journal of urology 2004; 22: 157-67.

[4] Singh S, Choong S. Rupture and perforation of urinary reservoirs made from bowel. World journal of urology 2004; 22: 222-6.

[5] Rosenberg S, Gofrit ON, Hidas G, Landau EH, Pode D. Diagnosing spontaneous ileal neobladder perforation: Too often delayed. Canadian Urological Association journal 2013; 7: E817-9.

[6] Mansson W, Bakke A, Bergman B, Brekkan E, Jonsson O, Kihl B, Nurmi M, Pedersen J, Schultz A, Sorensen B, Urnes T, Wolf H. Perforation of continent urinary reservoirs. Scandinavian experience. Scandinavian journal of urology and nephrology 1997; 31 : 529-32. [7] Arun KG, Salahauddin, Leela V, Noel J, Venkatesh K, Ramakrishnan S, Dilip R. Intraperitoneal bladder rupture mimicking acute renal failure. Indian Journal of Nephrology 2008; 18: 26-7.

[8] Braun U, Nuss K. Uroperitoneum in cattle: Ultrasonographic findings, diagnosis and treatment. Acta Veterinaria Scandinavica 2015; 57.

[9] Wilson DG, MacWilliams PS. An evaluation of the clinical pathologic findings in experimentally induced urinary bladder rupture in pre-ruminant calves. Canadian journal of veterinary research = Revue canadienne de recherche veterinaire 1998; 62: 140-3.

[10] Leyland JW, Masters JG. Conservative management of an intraperitoneal rupture of an augmentation cystoplasty and continent urinary diversion in an adult. The Journal of urology 2003; 170: 524.

[1 1] Brunati M, Rampoldi L. [Kidney diseases associated with uromodulin (Tamm- Horsfall protein)]. Giornale italiano di nefrologia : organo ufficiale della Societa italiana di nefrologia 2015; 32 Suppl 64.

[12] Rampoldi L, Scolari F, Amoroso A, Ghiggeri G, Devuyst O. The rediscovery of uromodulin (Tamm-Horsfall protein): from tubulointerstitial nephropathy to chronic kidney disease. Kidney international 201 1 ; 80: 338-47.

[13] Jennings P, Aydin S, Kotanko P, Lechner J, Lhotta K, Williams S, Thakker RV, Pfaller W. Membrane targeting and secretion of mutant uromodulin in familial juvenile hyperuricemic nephropathy. Journal of the American Society of Nephrology : JASN 2007; 18: 264-73.

[14] Scolari F, Izzi C, Ghiggeri GM. Uromodulin: from monogenic to multifactorial diseases. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2015; 30: 1250-6.

[15] Fugiel A, Kuzniewski M, Fedak D. [Uromodulin - can it be a new marker of kidney damage?]. Przeglad lekarski 2013; 70: 976-82.

[16] Prajczer S, Heidenreich U, Pfaller W, Kotanko P, Lhotta K, Jennings P. Evidence for a role of uromodulin in chronic kidney disease progression. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2010; 25: 1896-903. [17] Charan J, Biswas T. How to calculate sample size for different study designs in medical research? Indian journal of psychological medicine 2013; 35: 121 -6.

[18] Nippgen JB, Hakenberg OW, Manseck A, Wirth MP. Spontaneous late rupture of orthotopic detubularized ileal neobladders: report of five cases. Urology 2001 ; 58: 43-6.