The study protocol was approved by the Local Clinical Research Ethics Committee (CDD25918). The study conforms to the principles outlined in the Declaration of Helsinki and we obtained written consent from each enrolled patient.

We performed a prospective longitudinal study on 32 ADPKD patients consecutively recruited at the University Hospital “Policlinico Umberto I” of Rome, Sapienza University of Rome, Italy. Patients were enrolled between May 2019 and January 2022. Clinical, laboratory and instrumental parameters were evaluated at baseline (T0) and at 24 months (T1).

Patients aged > 18 years with a diagnosis of ADPKD, defined according to Pei's criteria [18], were eligible. We enrolled both normotensive and hypertensive patients. Arterial hypertension was defined as the use of anti-hypertensive drugs (angiotensin converting enzyme (ACEIs), Angiotensin II receptor blockers (ARBs), beta-blockers, calcium antagonists, alpha1-receptor antagonists and/or diuretics) or by the presence of blood pressure above 140/90 mm Hg in three consecutive measurements.

We excluded ADPKD patients with: eGFR ≤ 30 ml/min/1.73 m2, history of kidney surgery or cyst drainage procedures, other diseases that could potentially affect kidney function, metal clip, pregnancy or nursing, on therapy with tolvaptan, or history of cancer in the previous 5 years. Moreover, patients that refused to give consent or with missing data were also excluded.

Patients were divided into two groups: Rapid disease progression (RP, n 15) and Non-rapid disease progression (NRP, n 17), according to the Mayo Clinic classification criteria based on radiologic haTKV classification [12]. Classes 1A and 1B include patients with NRP, while patients with RP are classified as 1C, 1D, 1E to 1E.

Blood samples were obtained in the morning after 12 h fasting. Fasting plasma glucose (mg/dL), insulin (µU/mL), total serum cholesterol (mg/dL), triglycerides (mg/dL), high-density lipoprotein (HDL, mg/dL), serum creatinine (SCr, mg/dL), serum nitrogen (mg/dL), serum uric acid (SUA, mg/dL), calcium (mg/dL), phosphorus (mg/dL), serum sodium (mEq/L), serum potassium (mEq/L), and C-reactive protein (CRP, μg/L) levels were measured using standard automated techniques. Low-density lipoprotein-cholesterol (LDL, mg/dl) was calculated using the Friedewald equation: TCho − HDL − (Tg/5).

T0 plasma samples were taken for quantitative evaluation of h-miR17-5p, h-miR21-5p and h-miR199a-5p by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a single miRNA-specific assay (miRNeasy Serum/Plasma Kit (Qiagen)). H-miR16-5p was used as a normalizer. Starting from aliquots of the frozen plasma, total RNA (including the fraction of microRNA) was extracted using Qiagen miRNeasy serum/plasma kit (Qiagen) according to the manufacturer's instructions (supplementary protocol: Purification of total RNA, including small RNAs, from serum or plasma using the miRNeasy Mini Kit) and with the use of synthetic oligonucleotides cel-miR-39, such as spike-in. Ten ng of RNA was reverse transcribed using the TaqMan ™ MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions, followed by the pre-amplification reaction (same kit) which allows for uniform amplification of all miRNAs prior to quantification by RT-PCR. For qPCR, pre-amplified 1/5 diluted samples are prepared using TaqMan ADV MIRNA ASSY (miRNA probes) and TaqMan Fast Advance MMIX (both Applied Biosystems) as per the manufacturer's instructions. Relative quantification was performed by the 2-ddCT method normalizing the data with respect to miR16-5p as an endogenous control and using the NT3 (non-fast progressing) sample as a calibrator.

All patients underwent an MRI protocol with 3 T magnet (Discovery MR 750, 3T, GE Healthcare) after positioning of the 32-channel surface coil. The acquisition protocol included morphological sequences, single shot T2-weighted (SSFS)(TR 850 ms, TE 105 ms; Flip Angle 90°; FoV 320 × 320; Matrix 320 × 224) acquired on axial, sagittal and coronal planes and sequences Gradient Echo (GRE) T1-weighted (TR 5 ms; TE 1 ms; Flip Angle 15°; FoV 420 × 420; 288 × 192 matrix). To evaluate parenchymal perfusion, we used ultrafast GRE T1-weighted sequences, acquired in the coronal plane (TR 2 mS; TE 1 mS; Flip Angle 13°; Thickness 200 mm; FoV 300 × 300 mm, matrix 192 × 138) during administration of i.v. contrast (Gadobutrol 1 mmol/ml, Gadovist, Bayern) using a perfusion technique, with high temporal resolution of 4 s, for a total duration of about 8 min. The start of dynamic sequences coincides with the administration of i.v. contrast medium [18, 19]. Evaluation of Total Perfusion Volume (TPV) and TFV results from perfusion MRI after a qualitative and quantitative approach. Each parameter was the result of post-processed slice-by-slice renal segmentation respectively in the early arterial phase (1st minute of perfusion) and the late perfusion phase (8th minute of perfusion). Segmentation was guided using colorimetric maps. After segmentation, software Workstation vers. 4.6 was used for 3D volume, resulting in a semiquantitative estimation of vascularized and fibrotic parenchyma. These parameters can estimate the residual functional parenchyma. In the HASTE T2 and T1-weighted 3D GRE morphological images, the following parameters were evaluated: kidney size on 3 planes of acquisition (axial sagittal and coronal), cortical thickness, cortico-medullary differentiation, cyst volume [13]. In the 3D T1 weighted GRE sequences, the morphological characteristics and the signal intensity relating to the presence or absence of high protein content were evaluated, allowing the identification of hemorrhagic cyst content, infected cysts and heteroplastic cysts. MRI evaluation of coronal, axial and sagittal renal diameters allowed us to calculate haTKV and to classify each patient according to class of risk of disease progression according to the Mayo classification [12](http://www.mayo.edu/research/documents/pkd-center-adpkd-classification/doc).

Data management and analysis were performed using IBM® SPSS® Statistics 22.0 for Windows® software (IBM Corporation, New Orchard Road Armonk, New York, United States). The normality of variables was tested using the Shapiro–Wilk method for normal distributions. All continuous variables were expressed as mean ± standard deviation, categorical variables were expressed as number (percentage). Student's t-test was used to determine the between-group difference, and the binomial or Chi-square test was used for the comparison of categorical data. Bivariate correlations and the degree of associations between variables were obtained by Pearson's test. A value of p < 0.05 was considered statistically significant.