Fig 2: Analyses of implanted cells.a Immunofluorescence imaging using LIVE/DEAD™ staining demonstrated high cell viability on cell inserts after 3- and 7-day implants. Quantification of LIVE/DEAD™ staining demonstrated 95.6 ± 1.7 and 90.3 ± 4.5% viability (mean ± standard deviation) of cell inserts after 3- and 7-day implants, respectively (for 3-day implants n = 6 biologically independent samples from 3 animals, for 7-day implants n = 3 independent samples from 2 animals). b Cell inserts after 3- and 7-day implants demonstrated confluency and integrity of the cell monolayer, as examined by immunofluorescence imaging of tight junction protein ZO-1 (green) and DAPI (blue) nuclear staining (for 3-day implants n = 9, for 7-day implants n = 6). c Gene expression of cell transporters AQP1 and NHE3 and vitamin D 1a Hydroxylase were measured using qPCR as a surrogate of renal cell function. Relative to in vitro controls, AQP1 expression was 1.05x ± 0.35 (mean ± standard deviation), while 1a Hydroxylase and NHE3 were 1.56 ± 0.18 and 2.43 ± 0.27 x more expressed (n = 4 biologically independent samples). d The activity of damage biomarkers NAG and GGT was measured after days 3 and 7, using ELISA and normalized to in vitro controls. HREC from implants demonstrated NAG expression levels similar to those of in vitro controls and were significantly lower than a standard positive control (0.93 ± 0.15 for day 3) (2.01 ± 0.77 for day 7) (mean values ± standard deviation). Day 7 GGT levels were comparable to in vitro control cells and significantly lower than a standard positive control (1.37 ± 0.04) (n = 3 biologically independent samples). * or # p < 0.05, ** or ## p < 0.001, where * is for day 3 comparisons and # is for day 7. Source data are provided as a Source Data file.

Fig 3: Effects of sh‐NSUN4 and sh‐CAMK1 on renal injury in STZ‐induced DN mice. (A) The flow chart of the model construction. (B) NSUN4, SMURF1, and CAMK1 protein expression was detected with WB in control, STZ, STZ+sh‐NSUN4, and STZ+sh‐NSUN4+sh‐CAMK1 groups. (C, D) Statistical chart of body weight and blood glucose changes for 12 consecutive weeks. (E–H) Related kits were used to test Serum BUN, serum Cre, U‐NAG, and ACR levels. (I–K) H&E and PAS staining were performed to evaluate renal tubular damage score and glomerular damage score for assessing renal injury. (L) Cell apoptosis was measured using TUNEL staining. (M) DHE staining was performed to check ROS production. (N) TEM was used to observe the representative image of fragmented mitochondria. (O) Mfn1, Mfn2, Fis1, p‐Drp1 Ser616, and Drp1 protein expression was assessed with WB. The data are presented as the mean ± SD. (B, E–H, J–O) One‐way ANOVA with Tukey's post hoc test; (C, D) two‐way ANOVA with Tukey's post hoc test. n = 8, *p < .05, **p < .01, ***p < .001.

Fig 4: Effect of NSUN4‐cKO on renal injury and mitochondrial fission in STZ‐induced DN mice. (A) The flow chart of the model construction. (B) NSUN4 expression was measured using IHC staining in the kidney tissues of NSUN4‐WT, NSUN4‐cKO, STZ+NSUN4‐WT and STZ+NSUN4‐cKO groups. (C, D) Statistical chart of body weight and blood glucose changes for 12 consecutive weeks. (E–H) Serum BUN, serum Cre, U‐NAG, and ACR levels were detected with related kits. (I–L) Renal tubular damage score and glomerular damage score were assessed to evaluate renal injury by H&E and PAS staining. (M) Cell apoptosis was tested by TUNEL staining. (N) DHE staining was used to assess ROS production. (O) Representative image of fragmented mitochondria observed by TEM. (P) Mfn1, Mfn2, Fis1, p‐Drp1 Ser616, and Drp1 protein levels were evaluated using WB. The data are presented as the mean ± SD. (B, E–H, K–P) One‐way ANOVA with Tukey's post hoc test; (C, D) two‐way ANOVA with Tukey's post hoc test. n = 8, *p < .05, **p < .01, ***p < .001.