The Predictive Role of Proteomics in Blood Pressure Response of Hypertensive Patients Undergoing Renal Denervation.

Renal sympathetic nervous activity plays a crucial role in the development and maintenance of hypertension (HTN). Renal denervation (RDN) is a minimally invasive catheter-based treatment using mainly radiofrequency or ultrasound energy to selectively disrupt the sympathetic renal nerves. RDN has experienced rises and falls during its development as a treatment option for HTN in humans. Latest well-designed sham-controlled randomised trials with improved methodology confirmed significant blood pressure (BP) reduction in both office and 24-hour ambulatory BP. Although the safety of RDN procedures seems favourable thus, the rate of BP response to the procedure is variable, with response rates reported in the range between 60% and 70%. It is of great importance to identify biomarkers able to reliably predict subjects who would benefit from this treatment, in order to achieve better therapeutic results.

Proteomics is the study of the full complement of proteins produced or modified by a biological system (cell, tissue, organ, biological fluid, or organism). Proteomic analysis is used in different research settings to understand pathogenicity mechanisms and emerge biomarkers with predictive role in diagnosis and treatment of different diseases.

The main purpose of this study is to investigate the potential predictive role of the urine proteomics in BP response of patients undergoing RDN. This hypothesis may lead to the emergence of biomarkers in urine of hypertensive patients, in order to optimally select those who will undergo RDN.

This is a prospective observational study enrolling hypertensive patients, aged 18-80 years who will proceed in RDN as participants of randomized control trials. During baseline evaluation HTN diagnosis will be confirmed by office blood pressure measurement (OBPM) and ambulatory blood pressure measurement (ABPM), while urine sample will be collected before RDN for proteomic analysis. The participants will have a follow-up visit in 3 months since baseline procedure for office blood pressure (OBP) and ambulatory blood pressure (ABP) measurements. A cut off value of 5mmHg reduction in ABP or/and 10mmHg reduction in office blood pressure (OBP) on 3 months visit will be set to categorize the patients to responders or non-responders after RDN. The qualitative and quantitative differences of proteins between the two groups of patients will be investigated, based on proteomic analysis results, in order to determine specific urine proteins with predictive role in blood pressure response.

The study results are expected to determine the predictive role of urine proteomics in optimal selection of hypertensive patients who will undergo renal denervation.

Hypertension (HTN) is the most prevalent modifiable risk factor associated with cardiovascular morbidity and mortality, while its prevalence is rising globally. Despite the availability of safe and effective antihypertensive medication, in a large proportion of patients, blood pressure (BP) control to guideline-recommended targets is not achieved. The current knowledge is that sympathetic nervous system hyperactivity contributes to initiation, maintenance and progression of HTN.

Renal denervation (RDN) is a minimally invasive catheter-based method dedicated to disrupt renal sympathetic enervation using different ablation techniques, while is the most extensively investigated device-based therapy for ΗΤΝ. Current endovascular catheter systems access renal arteries and deliver radiofrequency energy, ultrasound energy or use neurotoxic agents such as alcohol, resulting in destruction of the peri-arterial afferent and efferent renal nerves. RDN has experienced ups and downs as a potential treatment option for HTN over recent years of investigation. The initial proof-of-concept, open label clinical trials produced promising results about RDN effectiveness in BP lowering, although, the first randomized sham-controlled trial failed to confirm the previous encouraging results. However, the latest randomized, sham-controlled trials with improved design and methodology demonstrated significant reductions in office blood pressure (OBP) and ambulatory blood pressure (ABP) in patients undergoing RDN. Despite demonstrated efficacy and safety of RDN procedure, about 30% to 40% of patients do not present with a clinically significant BP reduction, even in latest optimally designed clinical trials. This might be due to the pathophysiological substrate in these patients, while identifying a biomarker of BP response could result in a better selection of patients with better response.

The dynamic role of proteins to support the life is documented since the initial stages of biological research. The proteome can be defined as the overall protein content of a biological system such as cell, tissue, biological fluid, etc. Proteomics is the large-scale study of the proteome, as well as the identification and quantification of these proteins. There are some basic steps of proteomic analysis: sample acquisition, protein extraction, protein separation, protein sequence determination and protein identification with bioinformatic techniques in referential databases. Chromatography based techniques are used for protein extraction and high-resolution two-dimensional electrophoresis techniques for proteins separation, while mass spectrometry (MS)-based analysis is widely applied for the protein sequence determination. Proteomic analysis is used in various research settings in detecting diagnostic biomarkers and highlighting pathogenicity mechanisms, alteration of expression patterns in response to different signals or interpretation of functional protein pathways in different diseases. The ultimate goal of proteomics is to better comprehend the molecular complexity while establishing diagnostic and therapeutic healthcare algorithms for the prevention and treatment of diseases.

Α. Aim of the study:

The main purpose of this study is to investigate the potential predictive role of the urine proteomics in BP response of patients undergoing RDN. This hypothesis may lead to the emergence of biomarkers in urine of hypertensive patients in order to optimally select those who will undergo RDN procedure.

B. Study design:

This is a prospective, non-interventional, observational study enrolling a total of 100 patients, with uncontrolled hypertension, treated or untreated, aged 18-80 years, undergoing RDN in the context of randomised clinical trials. During baseline evaluation HTN diagnosis will be confirmed by OBP and ABP measurements, while all patients have to be untreated or treated with maximum of 3 antihypertensive drugs (at least on 50% of maximum dose). Patients with moderate to severe chronic kidney disease (eGFR \<45 ml/min), renal artery abnormalities, type I diabetes mellitus and secondary HTN will be excluded. The enrolled cases will have a follow-up visit 3 months after RDN. Data will be collected using a specific form (CRF). These include demographic and somatometric data, past medical history, OBP measurements, ABP measurements, laboratory tests (estimated renal function, serum potassium, serum sodium and serum glucose levels). All patients have to sign an informed consent to participate in the study. Ethics approval has been obtained from the hospital ethical committee board.

C. Methods:

Screen visit: During baseline visit BP will be evaluated with OBP and ABP measurements. Patients with systolic OBP ≥140 mmHg and systolic ABP ≥130 mmHg will be included in the study. The patient profile will include serum tests (glucose, urea, creatinine, sodium, potassium), as well as renal ultrasound in order to exclude anatomic or structural abnormalities of kidneys. Secondary HTN will be excluded, according to HTN guidelines. Urine samples will be collected from all patients and stored in freezer till sending them to Proteomics Research Unit for analyses.

Renal denervation: RDN procedure will be performed 1 to 2 weeks after screen visit by an experienced interventional cardiologist followed by a 1-day hospital stay.

Follow up visit: A follow up visit will be scheduled 3 months after RDN procedure. New OBP and ABP measurements and serum tests (glucose, urea, creatinine, sodium, potassium) will be collected. Data gathering will be completed by statistical analysis. Patients will be categorized as responders or non-responders according to OBP or/and ABP differences between baseline visit and 3 months follow up visit. A 5mmHg reduction in systolic ABP and a 10mmHg reduction in systolic OBP will be set as cut-off values to categorize the patients. At that time point, results from proteomic analyses of baseline urine samples will contribute to the identification of significant qualitative and quantitative differences in proteins between the two groups of patients (responders and non-responders). Information about specific proteins' identity, structure, functions and metabolic pathways in which they are involved, will be collected through biological databases.

Statistical analysis: Normal distribution of all data will be checked with Kolmogorov-Smirnov test. Continuous variables will be expressed as mean and standard deviation (SD), while categorical variables will be presented as percentages (%). Comparisons between categorical variables will be done with χ2 (Pearson's chi-squared test). Comparisons between continuous variables with normal distribution will be done via Student's t-test for unpaired samples, while comparisons between continuous variables which follow non-normal distributions will be done via Mann-Whitney U test. Correlation analysis will be done via Pearson Phi coefficient or Spearman's rho (ρ) based on the distribution of the data set. Statistically significant will be differences with p-value \<0.05. Analysis of all data will be performed by SPSS 26 (SPSS Inc, Chicago, Illinois, USA) software.

D. Estimated research outcome:

The study results are expected to determine the predictive role of urine proteomics in blood pressure response of hypertensive patients undergoing renal denervation.

Eligibility Criteria:
Inclusion Criteria:

* All treated patients have to be on standard antihypertensive regimens for at least 8 weeks
* Age 18-80 years
* Office systolic blood pressure ≥140 mmHg and
* Ambulatory systolic blood pressure ≥130 mmHg

Exclusion Criteria:

* eGFR \<45mL/min/1.73m2
* Renal artery abnormalities
* Type I diabetes mellitus
* Secondary hypertension
* Pregnant or breastfeeding women
* Psychiatric or neurological disease which does not allow adequate co-operation
* Active cancer on treatment