Dear colleague,

On the occasion of the 63º Congress of the Italian Society of Nephrology, there will be the first meeting of the Centers participating in the CLIgAN study in which the organization of the project and the ancillary clinical studies requested so far will be illustrated. Therefore, your participation would be appreciated or, if busy at the same time in another meeting, to want to designate one of your collaborators who will participate to the study. The meeting will be held on Thursday 6 October from 14.30 to 15.30 in Sala Borgo 2 (first floor) of the congress venue in Rimini. Looking forward to meeting you, I send cordial greetings

F.P. Schena

1. Presentation of the CLIgAN pilot project presented to the participating Centers.
2. Eligible IgAN patients: age ≥ 18 years; eGFR ≥ 30 ml/min/1.73 mq; Proteinuria ≥ 0.5 g/ 24 hours
3. Local Ethics Committees.
4. Dapagliflozine prescription
5. Person of the Center who will follow the project and will talk with the CRO.
6. Randomization performed by eResult in collaboration with the Schena Foundation.
7. Submission of the questionnaire on the practice of kidney biopsy. Reading venue. Collaboration during the study.
8. Local reading of the kidney biopsy and subsequent sending of the slides (4 colours: HE, PAS, AM, Masson) to Anatomia Patol. Bicocca for their scanning with the Aperio system for the Pathology Review (Rossini, Bari; Pagni-L'Imperio-Ferrario, Milano-Bicocca; M. Haas, Cedar Sinai-Los Angeles). To read the kidney biopsy, fill in the Oxford classification form (MEST-C).
9. Enrollment and start of therapy within 2 weeks (Active) and 4 weeks (Chronic or Moderate Lesions) from the kidney biopsy.
10. Collection of kidney biopsies for the BIO-D study.
11. Collection of biological samples (blood, urine). Methods for collecting samples.
12. Practical notes on therapy.
13. Non-responder patients (may receive other non-randomized therapies but followed for 36 months in total).
14. ACIgAN patients non-responders to therapy after 8 months may receive a second kidney biopsy.
15. Patient Responders (prot ≤ 0.15g/24 hour); partially responders (50% reduction); confirm data 30 days after last value, non-responders.
16. Publication of the works: the director of the Center and their collaborators will be included among the authors of the publication. The order of the author's position will be based on the number of enrolled patients who have completed the study.

Grade 1. Patient enrollment with visits and laboratory tests.
Grade 2. Samples of blood, urine, storage at -20ºC and shipment.
Grade 3. Kidney biopsy study with shipment of kidney tissue sections.
Grade 4. Participation in ancillary projects.

1. RENAL TISSUE PRESERVATION

A. The renal tissue included in FFPE, after it has been used for the histological study, must be kept in the refrigerator at +4ºC. For the CLIgAN study a slide, containing 3 - 4 sections for each staining (2 micron thickness) must be sent to the Pathological Anatomy of Milano-Bicocca, Monza. In total, 4 slides will be prepared for each biopsy, one for each staining.

B. For the center participating in the BIO-D study, also send two slides containing 4 sections of FFPE tissue (thickness 5 microns). Sections from the first slide will be used for spatial transcriptomics, the other slide for histological (H-E) staining. One section will be used to evaluate the RNA quality technique (DV 200 > 50%). Tissue included in OCT, after it has been used for immunofluorescence, should be stored at -80ºC. Sections of 5 microns measuring 75x25 must be cut within 4 weeks. After cutting the tissue, the remaining part of the kidney biopsy must be covered with OCT and frozen at -80ºC. One of the 8 sections will be used to evaluate the RNA quality (RIN>7). The kidney sections will be obtained with a cryostat at a temperature of -10 -20ºC. The cryostat blade must be at -10ºC. The sections must remain in the cryostat for 30 min.

C. A part of the kidney biopsy core (0.5 cm long), dedicated to immunofluorescence, will be immersed in HypoThermososl FRS, cold (+4ºC) preservation solution and subsequently transferred into cold CryoStor CS10. The part of the biopsy core will be subsequently transferred to -20ºC and finally it will be stored at -80ºC in order to be subsequently transferred to the laboratory for sequencing.


2. PATHOLOGY REVIEW OF KIDNEY BIOPSY
After reading the locally performed kidney biopsy, the slides containing 4 sections of tissue, 2µm thickness (4 colours: HE, PAS, A, Masson) will be sent, after having accumulated a certain number of biopsies, to the Institute of Pathological Anatomy of the Milano-Bicocca University to scan them with the Aperio system. This procedure will be used for the Pathology Review process (Rossini, Bari; Pagni-L'Imperio-Ferrario, Milano-Bicocca; M. Haas, Cedar Sinai-Los Angeles). The biopsies will be anonymous and will only contain the date and the patient's code number. The kidney images will be stored in a 2 terabyte portal, accessible to each participant center. The entire file will be available to the Peer Review Committee, responsible for the project. However, the file will be available to anyone wishing to carry out a study, after having received the consent of the Organizing Committee of the study.