An RNA transmethylation pathway governs kidney nephrogenic potential

Abstract

The adult kidney lacks the ability to generate new nephrons, placing individuals born with low nephron counts at greater risk for chronic kidney disease as they age. Limited nutrient availability hinders nephron formation; however, the key metabolic dependencies remain unclear. Here we show that S-adenosylmethionine (SAM) and cellular transmethylation status are crucial determinants of the kidney's nephrogenic capacity. The RNA methyltransferase METTL3 serves as a SAM sensor and is essential for the fate determination of nephron progenitor cells (NPCs). Reducing transmethylation or inhibiting METTL3 blocks NPC differentiation and nephrogenesis, whereas enhancing transmethylation or increasing METTL3 activity facilitates an induced NPC population and increases nephron production. Additionally, we identify Lrpprc mRNA, encoding a mitochondrially enriched protein, as a key direct target of METTL3-mediated transmethylation. Accordingly, inhibiting LRPPRC negates the nephrogenic effects of SAM and METTL3. Our findings reveal a modifiable methionine-SAM-RNA transmethylation pathway that can be targeted to enhance nephron formation.

Fig. 1. Methionine–METTL3–m⁶A pathway is activated during NPC differentiation.

A Principal component analysis of the metabolomics data, showing segregation of renewing (rNPCs) and differentiated NPCs (dNPCs) samples (N = 4). B LC/MS-measured methionine and C ELISA-assessed SAM levels in dNPCs compared to rNPCs. D Immunoblot analysis shows the expression of indicated proteins in rNPCs compared to dNPCs. Actin serves as the loading control. Each lane represents pooled lysate from 3 to 4 biological replicate primary NPC cultures. E ELISA (N = 5; biological replicates) and F RNA dot blot analysis showing m6A enrichment in dNPCs compared to rNPCs (N = 3). Methylene blue (MB) serves as a loading control. G–I Images showing MAT2A, METTL3, and m⁶A immunostaining and DBA labeling in kidney sections from E16.5 embryos (N = 4). Anti-MAT2A, anti-METTL3, and anti-m⁶A (red); DBA (green); DAPI (blue). Scale bars 25 μm (G–I). Error bars represent the standard error of the mean (SEM). Statistical analysis: two-tailed, unpaired t-test (B, C, and E). Source data are provided as a Source Data file. N indicates biological replicates.

Fig. 2. SAM and METTL3 mediate NPC fate transition towards differentiation.

A Immunofluorescence and B immunoblots showing SIX2 and PAX8 expression in NPCs cultured for 48 h in 1.25 μM CHIR NPEM media supplemented with vehicle, 100 μM SAM, 100 μM SAH, or 100 μM SAM and 40 μM METTL3 inhibitor. Actin serves as the loading control. Each lane represents pooled lysate from three biological replicate NPC cultures. C Images showing GFP and PAX8 immunostaining in NPCs treated with different doses of SAM or M3A (METTL3 activator). The average percentages (mean ± SD) of NPCs that are double-positive for GFP and PAX8 are presented in relevant images (N = 3). Source data are provided as a Source Data file.

Fig. 3. Inhibiting the methionine–METTL3 axis blocks NPC differentiation and nephrogenesis.

A Immunostaining for PAX8, SIX2, and E-cadherin in E12.5 kidneys cultured in regular or methionine-depleted media supplemented with vehicle, 250 μM SAM, or 100 μM SAH. The number (average ± SD) of PAX8-positive tubules and SIX2-positive NPC niches is depicted in the relevant images. B H&E-stained, SIX2, Pan-CK, and PAX8 immunostained, and DBA-labeled kidney sections from P1 mice of the indicated genotypes. White dotted circles indicate PAX8+ tubules. The graphs depict the number of glomeruli and pre-tubular structures. C PAX8, SIX2, and E-cadherin immunostaining in E12.5 Six2:eGFP^TGC/+ (control) and Six2:eGFP^TGC/+; Mett3^F/F (Six2-Mettl3-KO) kidneys cultured ex vivo for 48 h. Six2-Mettl3-KO kidneys were grown in regular media (vehicle) or media supplemented with 250 μM SAM. Control (wildtype) kidneys were grown in regular media (vehicle) or supplemented with M3i or M3i plus SAM. The number (average ± SD) of PAX8 tubules and SIX2 NPC niches is depicted in relevant images. ** Indicates P < 0.05. Statistical analysis: one-way ANOVA with Tukey’s multiple-comparisons test (A, C); two-tailed, unpaired t-test (B). Error bars represent SEM. Source data are provided as a Source Data file.

Fig. 4. Activating SAM–METTL3 promotes NPC differentiation and ex vivo nephrogenesis.

A Kidney sections of E12.5 control or Six2:eGFP^TGC/+; Mettl3^OE (Mettl3-OE) mice immunostained with SIX2 and pan-cytokeratin, or subjected to in situ hybridization using probes against Cited1 and C1qdc2. B H&E staining, SIX2 (red), and pan-cytokeratin (green) immunostaining of kidney sections from P1 control or Mettl3-OE mice. White arrowheads point to precocious PAX8 expression. C E12.5 kidneys from control, Mettl3-OE, or wildtype embryos were cultured in regular media for 48 h and co-stained for PAX8 and E-cadherin or SIX2 and E-cadherin. The media of wild-type kidneys was supplemented with a vehicle, M3A (100 or 400 μM), or SAM (250 or 1000 μM). The number (average ± SD) of PAX8 tubules and SIX2 NPC niches is depicted in relevant images. ** Indicates P < 0.05. Statistical analysis: One-way ANOVA, Tukey’s multiple comparisons (C). Source data are provided as a Source Data file.

Fig. 5. Activating SAM–METTL3 promotes in vivo nephrogenesis.

A Principal component analysis of the metabolites in E16.5 and P3 nephrogenic zone cells (NZCs). B Volcano plot showing differential metabolite abundance between P3 and E16.5, filtered by P value (horizontal dashed line). Methionine and SAM are highlighted as black circles. C LC/MS-measured methionine and SAM levels at P3 and E16.5. D Schematic of the SAM or M3A treatment regimen. Created in BioRender. Patel, V. (2025) https://BioRender.com/6239npyE Kidney-weight-to-body-weight (KW/BW) ratio and F glomerular counts of P10 mice, and G eGFR values of P40 mice treated with vehicle, SAM, or M3A. H Low- and high-magnification images of P4.5 kidneys expressing Six2:eGFP. The number (average ± SD) of GFP clusters per 1 mm² kidney section area is depicted in the relevant images. I Distribution of the number of Six2:eGFP-positive cells within NPC clusters. At least 30 clusters from three biologically independent samples were counted for each condition. J Images of P4.5 kidneys immunostained for PAX8 and GFP are shown. The percentage of PAX8- and GFP-double-positive NPC clusters is indicated. Statistical analysis: Two-tailed, unpaired t-test (C, E–G, I). Error bars indicate SEM; ** indicates P < 0.05. Source data are provided as a Source Data file.

Fig. 6. LRPPRC is a downstream target of SAM and METTL3.

A Pathway analysis of RNA-Seq datasets showing downregulation of mitochondrial metabolism genes in E16.5 Six2^Cre-Mettl3-KO (M3^KO) compared to control kidneys. B IGV tracks display the input-normalized m⁶A signal on Lrpprc mRNA. C Q-PCR analysis showing Lrpprc (mouse cells) and LRPPRC (human cells) transcripts are enriched in samples recovered after RNA immunoprecipitation using an anti-METTL3 antibody compared to an anti-IgG antibody. N = 3. D Immunoblots showing LRPRRC and oxidative phosphorylation complex expression in Six2^Cre-Mettl3-KO (M3^KO) and Mettl3-OE (M3^OE) kidneys compared to their respective controls. Actin serves as a loading control. E LRPPRC, SIX2, or PAX8 immunostaining, or live-cell MitoTracker labeling of NPCs cultured for 48 h in 1.25 μM CHIR NPEM media and co-treated with 100 μM M3A or 100 μM SAM in the presence or absence of 20 μM GAA. F–H Newborn pups were injected from P1 to P4 with a vehicle, 20 mg/kg GAA, 25 mg/kg SAM, or both 20 mg/kg GAA and 25 mg/kg SAM. Kidney weight-to-body weight (KW/BW) ratio (F) and glomerular count (G) of P10 mice, and eGFR (H) of P40 mice, are shown. I–K Newborn pups were injected daily from P1 to P4 with a vehicle, 20 mg/kg GAA, 2.17 mg/kg M3A, or both 20 mg/kg GAA and 2.17 mg/kg M3A. KW/BW ratio (I) and glomerular count (J) of P10 mice, and eGFR (K) of P40 mice are shown. Statistical analysis: Fisher’s Exact Test (A), two-tailed unpaired t-test (C), and one-way ANOVA with Tukey’s multiple comparisons (F–K); error bars indicate SEM. Source data are provided as a Source Data file.