Isolation of glomeruli

To isolate mouse glomeruli, anesthetised mice first were perfused with 1× HBSS to remove circulating blood, followed by perfusion of Dynabead (14013, Thermo Fisher). Minced kidneys were digested with collagenase IV (1 mg/ml, Thermo Fisher) plus DNase I (50 U/ml, Thermo Fisher) at 37 °C for 30 min. After passing through two 100-µm strainers, cell suspensions were pelleted by centrifugation (500 × g at 4 °C for 5 min). The glomerular pellet was re-suspended in HBSS and then the beads-containing glomeruli were gathered by the magnet holder and washed three times.

To isolate human glomeruli (mean 150 µm in diameter), we considered maximally to include Bowman’s capsule, which is often stripped off during classical serial sieving (425-, 250- and 150-µm sieves). To this end, we used only a single 300-µm sieve allowing easy passage of human glomeruli together with Bowman’s capsule. Briefly, minced kidney biopsies were first incubated with collagenase IV (1 mg/ml) plus DNase I (50 U/ml) at 37 °C for 15 min, followed by a single sieving using a 300-µm strainer (PluriStrainer). Then a 100-µm strainer (Pluristrainer) was used to separate human glomeruli from tubules. Glomeruli retained on 100-µm strainers were collected by turning the strainer upside down followed by multiple washes and pelleted by centrifugation (500 × g at 4 °C for 5 min). Purity of isolated glomeruli was validated under a dissection microscope. All kidney tissues were kept on ice except for during enzymatic digestion.

To exclude whether bead perfusion and the magnet separation process in glomerulus isolation could lead to subsequent transcriptome alterations compared with the sieving method, we isolated mouse glomeruli using a recently reported bead-free method without bead perfusion²⁸. In brief, minced mouse kidneys were incubated with collagenase IV (1 mg/ml) without DNase in 1× HBSS buffer at 37 °C for 15 min. After spinning down, the re-suspend tissue mixture passed through two sieves from 100- to 75-µm strainers. The filtrate containing glomeruli and tubular fragments was collected using a 40-µm strainer. Then enriched glomeruli were obtained after settling the mixture on a 10-cm culture dish for 1–2 min allowing adherence of tubules to the dish bottom. We unbiasedly sorted these cells.