*2.9. Immunofluorescence*

Kidneys fixed in paraformaldehyde (PFA)-fixed were cut in 5-μm-thick paraffin sections. Sections were dewaxed, demasked for 20 min with proteinase K, and, after washing, pretreated with 10% normal goat serum in PBST for 60 min at room temperature (RT). Without rinsing, sections were incubated with polyclonal rabbit anti-pNCC (Novus Biologicals, 1:200) antibody at 4 ◦C overnight. After washing, sections were incubated for 1 h with goat anti-rabbit Alexa 594 (1:500, Invitrogen, Waltham, MA, USA). Nuclear staining was performed with DAPI (1:1000) for 5 min. Controls were performed by omitting primary antibodies. The slides were analyzed on a Zeiss LSM 880 Airyscan confocal microscope equipped with a 63× oil immersion lens (NA 1.3).

#### *2.10. RNA Isolation and Quantitative RT-PCR*

Total RNA was isolated from snap-frozen hearts after homogenization in a 24 Fast Prep machine using TRI Reagent. The nucleic acid concentration and integrity were determined by electrophoresis (Agilent Tapestation). Only samples that had a RIN value above seven were used. Two micrograms of RNA were transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Quantitative RT-PCR was performed on a qTOWER3/G qPCR device (Analytic Jena, Jena, Germany). The qPCR performed in a volume of 15 μL was composed of 1× PCR buffer B2 (Tris-HCl, (NH4)2SO4, and Tween-20; Solis Biodyne, Tartu, Estonia), 3.5 mM MgCl2, 200 μM dNTP mix (Solis Biodyne), 250 nM of each primer, and either 200 nM hydrolysis probe or 0.4 × EvaGreen (Biotium, Fremont, CA, USA), 1 U HOT FIREPol® DNA polymerase (Solis Biodyne), and 2 μL template DNA. For mouse primer sequences, see Supplementary Table S1. Cycling conditions consisted of an initial 15-min incubation step at 95 ◦C for polymerase activation and template denaturation, followed by 45 cycles of 95 ◦C denaturation for 15 s and 60 ◦C annealing and elongation for 60 s. All samples were measured in triplicate and normalized to two housekeeping genes (*Dpm1* and *Txnl4a*). The qPCR results were gained and primarily evaluated with the software qPCRsoft 4.1 (V4.1.3.0 Analytic Jena) and then analyzed using the standard delta delta Cq method.

## *2.11. Statistical Analysis*

Statistical analysis was performed using Graph Pad Prism 9 or SPSS. The data were analyzed by two-sided t-test for two groups, one-way or two-way analysis of variance (ANOVA) to assess the influence of genotype and treatment, as well as their two-way interactions followed by the Student–Newman–Keuls multiple comparison test when comparing more than two groups; *p*-values of less than 0.05 were considered significant. Data are presented as scatter dot plots with bars depicting means ± SEM.
