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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2026 Mar 26;37(9):1895–1909. doi: 10.1681/ASN.0000001053

The Impact of Low-Protein Diet on the Molecular and Cellular Development of the Fetal Kidney

Kieran M Short 1,2, Giovane G Tortelote 3, Lynelle K Jones 1,2, Fabiola Diniz 3, Francesca Edgington-Giordano 3, Luise A Cullen-McEwen 1,2, Jan Schröder 4,5, Ashley Spencer 1,2, Andrew Keniry 6,7, Jose M Polo 2,4, John F Bertram 1,2, Marnie E Blewitt 6,7, Ian M Smyth 1,2,✉, Samir S El-Dahr 3,✉
PMCID: PMC13340419  NIHMSID: NIHMS2173090  PMID: 41885952

Visual Abstract

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Keywords: kidney development, renal development, nephron development

Abstract

Key Points

  • Maternal low protein diet reduced kidney size and altered branching without changing ureteric tip number, with fewer nephrons at birth and adulthood.

  • Nephron progenitor proliferation and commitment were impaired, pretubular aggregate formation decreased, consistent with a reduced nephron number.

Background

Low nephron number has a direct effect on the development of hypertension and CKD later in life. Although intrauterine growth restriction caused by maternal low-protein diet (LPD) is believed to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes that drive nephron formation are poorly understood.

Methods

We conducted a comprehensive characterization of the effect of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphologic features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during development of the kidneys to affect nephron number.

Results

Single-cell analysis revealed differential expression across metabolic, cell cycle, epigenetic, and reciprocal inductive signaling pathways in most cell types, shifting cellular energy production and developmental trajectories. In nephron progenitor cells, LPD impeded commitment and differentiation toward pretubular aggregates and renal vesicles, accompanied by downregulated Wnt signaling. Confocal microscopy showed fewer pretubular aggregates and reduced progenitor proliferation, consistent with impaired commitment. Critically, nephron progenitor cell proliferation remained reduced through P0, whereas ureteric tip proliferation, although reduced earlier, had normalized by P0. Branching morphology also changed, with optical projection tomography showing shorter tip and tip-parent lengths, consistent with subtle patterning defects.

Conclusions

This study demonstrates that gestational LPD reduced nephron endowment by impairing nephron progenitor cell commitment, with concurrent alterations in branching morphogenesis.

Introduction

Nephron endowment has a direct effect on kidney function and is inversely related to the risk of hypertension and adult kidney disease.1–3 Endowment can vary 13-fold in normal human populations,4 but the intrinsic drivers of these differences are not fully known. Although genetic factors no doubt account for some of this variation,5 maternal disease and diet during pregnancy are also likely to significantly influence this measure because all nephrons in humans (and most in mice) are formed in utero. One such dietary variable is protein deficiency, a common global nutritional deficit6–8 exacerbated by the recent coronavirus disease 2019 pandemic.9 However, the precise molecular mechanism by which maternal low-protein diet (LPD) affects embryo and kidney development remains unclear.

Studies in rodent models have shown that an isocaloric LPD impairs kidney development and reduces nephron endowment,10,11 which predisposes them to the development of hypertension and kidney disease later in life.12–17 Therefore, a mechanistic understanding of how genetic and epigenetic changes influence nephron endowment in this context is of clinical importance. Recent studies on LPD in rats18 and calorie-restricted diets18 in mice report significant reductions in nephron number and nephron progenitor cell proliferation at embryonic day (E) 14.5. Although providing important insights, neither study directly addressed how diet affects nephrogenesis, ureteric bud branching, or cellular transcriptional changes linked to nephron deficiency.

In this study, we sought to define how gestational LPD leads to reduced nephron endowment. We hypothesized that nephron deficit arises from disruption of key developmental processes required for nephron formation. To delineate these processes, we integrated quantitative imaging of branching morphogenesis and nephrogenic niche architecture with single-cell transcriptomic profiling across developmental time points.

Methods

Institutional Animal Care and Use Committee Statement

All animal experiments follow the NIH Guide for the Care and Use of Laboratory Animals where applicable and were approved by the Monash University Animal Ethics Committee (MARP/18204) and Tulane University Institutional Animal Care and Use Committee (1558).

Animals and Diets

First time CD1 and C57BL6/J female mice were fed either a normal-protein diet (NPD) (20%–18%) or low-protein diet (LPD) (6%–8%) for 2 weeks. Differences in diet composition because of international transport restrictions are detailed in Supplemental Methods. Despite differences, the kidney phenotype in LPD-exposed offspring was consistent. Pregnant females were euthanized at E14.5 and E19.5, and pups at postnatal day (P) 0, P2, P9, P14, and P21 according to ethical guidelines. Embryos were staged according to limb staging,19 and kidneys were microdissected and processed according to experimental needs.

Where possible, paired median-weight male and female offspring were used for analyses, with n reflecting litters, such that multiple samples were typically obtained per litter. The litter and sample numbers for experimental analyses are available in Supplemental Methods.

Niche Assessment

Kidneys at E14.5, P0, and P2 were stained with antibodies (SIX2, TROP2, and PHH3—Supplemental Methods), cleared, and confocal microscopy imaged to a typical depth of 70 µm.20 Assessments included cell counts, proliferation, cap mesenchyme, and tip volumes in 3D for 78 niches (a niche includes one tip and associated cap) at E14.5 (from eight kidneys derived from eight litters), 564 at P0 (from 20 kidneys derived from ten litters), and 289 at P2 (from 14 kidneys derived from seven litters). Nephric structure assessment was performed by identifying nuclei patterns with or without Six2 in Z slices around ureteric tips to locate nephron progenitor cells, pretubular aggregates, renal vesicles, comma/S-shaped bodies, and nephrons on the basis of morphology.

Histologic Staining

Kidneys at E14.5 and P0 were histologically sectioned at 4 µm thickness and stained with primary antibodies for Wnt4, Wnt9b, and Six2 and appropriate secondary antibodies (HRP, Alexa Fluor 555, DBA, and Hoechst-33342). See Supplemental Methods for antibody details. RNAscope was applied to formalin-fixed, paraffin-embedded tissue sections of E14.5 and P0 kidneys using the Multiplex Fluorescent Assay (Cat. No. 323100; Advanced Cell Diagnostics) following the manufacturer's recommendations. See Supplemental Methods for probe details.

Optical Projection Tomography, Branching, and Niche Number Analysis

E14.5 kidneys stained with TROP2 were imaged in 3D by optical projection tomography using a Bioptonics OPT3001 instrument, and ureteric trees mapped and quantified using Tree Surveyor.20,21 Branch patterning, tip number, and niche number at P0 were quantified, with SIX2+ niche clusters manually counted in Imaris.22 Each niche represents a single ureteric/collecting duct tip.

Statistics

Linear mixed-effects models and two-factor ANOVA were applied for repeated measurements using R, with two-sample Kolmogorov–Smirnov (KS) tests and unpaired two-tailed T tests used for pseudotime association and other comparisons.

Nephron Counting

Pups were weighed and sexed, and kidneys were dissected in PBS and fixed in methacarn (60% methanol, 30% chloroform, and 10% glacial acetic acid) and processed into paraffin. Kidneys were sectioned at 4 µm (P0) or 5 µm (P21), and ten evenly spaced section pairs were sampled. Sections were stained with lectin peanut agglutinin (PNA, see Supplemental Methods) to identify podocyte membranes, counterstained with hematoxylin, and projected using a light microscope. All PNA+structures, representing glomeruli from S-shaped body to capillary loop stages, were counted using the physical dissector/fractionator method.23

Single-Cell and Nuclear RNA-Sequencing and Data Analysis

E14.5 kidneys (n=10) from two females per diet were dissociated into single-cell suspensions using 10× Genomics protocols.24 At P0, paired kidneys were snap-frozen and underwent cell lysis, centrifugation, and flow cytometry to extract nuclei.25 10× Genomics protocols, kits, and tools were used for the workflow. About 10,000 E14.5 cells or P0 nuclei per sample were emulsified, and approximately 50,000 reads per bead were generated. Cell Ranger mapped extracted sequences to mm10 (v3). Analyses and cell quality control were conducted in R using Seurat (v3.2).26 Canonical kidney genes identified cell types after supervised clustering.27 Differential gene expression used limma28 and Goanna, STRING, and DAVID29 aided Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes analyses. Pseudotime analysis used Slingshot30 and TradeSeq.31 See Supplemental Methods for details. Data are available at Gene Expression Omnibus, ID GSE240044.

Results

Maternal LPD Reduces Postnatal Body Mass and Nephron Number

Mice were fed isocaloric LPD-modified chow for 2 weeks before conception, throughout pregnancy, and until weaning. At E14.5, body mass was similar between diets and placental mass was reduced by approximately 7% (Supplemental Dataset 1 and Supplemental Figure 1). At birth and P21, body mass in LPD offspring was also lower than that of controls (9.5% and 16%, respectively, Figure 1, A–C). Both newborn and 3-week-old LPD mice (Figure 1, D and E) exhibited 19% and 23% less nephrons, respectively (Supplemental Dataset 1). The deficit in nephron formation after P0 suggests a continued postnatal effect of prenatal programming compounded by deficient lactation limiting nephrogenesis. No sex differences in body mass (Supplemental Figure 1, B and C) or nephron number were observed.

Figure 1.

Figure 1

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Maternal LPD affects the mouse embryo and kidney. At embryonic day E14.5, body mass is no different depending on diet (A, P = 0.78), but this changes by birth (B, P = 0.002). Postnatally, a LPD continues to affect postnatal body mass through to P21 (C, P < 0.001). At birth, total nephron numbers are 19% lower in LPD than NPD (D, P = 0.006) and by postnatal day 21, nephron number is 23% lower in LPD (E, P < 0.001). No sex differences in these parameters were found. Orders for sample numbers are NPD/LPD. Sample numbers with litter and sex breakdowns available in Supplemental Methods. E, embryonic day; F, female; LPD, low-protein diet; M, male; NPD, normal-protein diet; P, postnatal day.

Maternal LPD Alters Kidney Branching

Given the importance of signaling between the ureteric bud and nephron progenitor cells during kidney development, we investigated whether LPD affected ureteric bud branching. We assessed branching at E14.5 using optical projection tomography32 and at P0 by counting peripheral SIX2+ nephrogenic niches.21 Although P0 niche numbers showed a slight 9% reduction (Figure 2A), E14.5 ureteric tip numbers were unchanged (Figure 2B and Supplemental Dataset 1). However, the E14.5 LPD kidneys had 25% less volume and an 18.4% reduction in surface area, resulting in a 16% increase in ureteric tip density (Figure 2, C–E). Terminal branch generations in LPD offspring were also shorter, with tips reduced by 22.2% and parent branches by 15% (Figure 2F). To determine whether these changes altered ureteric tree morphology, we analyzed kidney branch patterning33 and observed an increase in imbalanced Fibonacci patterns, similar to kidneys with increased tip packing (Figure 2F). Nearest-neighbor distances between tips were reduced by approximately 25% (Figure 2G), analogous to changes observed in Spry1-mutant mice.33 These defects in branching, growth, and tip packing may influence or be influenced by nephron formation.

Figure 2.

Figure 2

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The effect of LPD on branching morphogenesis and kidney growth. At the day of birth, the number of Six2-positive nephrogenic niches is lower in LPD pups, and there are no differences between the sexes (A, n=16/16, P = 0.02 for diet). This represents a reduction in tip number of the ureteric tree. Earlier at embryonic day E14.5, the tip number is unchanged (B, P = 0.52), but those tips are packed into a smaller kidney (C, P = 0.008) with a smaller surface area (D, P = 0.005), leading to a higher tip density (E, P = 0.01). The outer branches of the ureteric tree starting at the tips toward the ureter are consistently shorter in length in the LPD kidneys (F, tip P = 0.01, tip parent P = 0.02, tip great grandparent P = 0.03). The shorter branches, which are packed more densely, have shorter tip-to-tip distances (G, P < 0.001) with less space to grow. This tighter packing partially affects the LPD branching pattern. Kidney branching shows mild asymmetry (half delay pattern), whereas more asymmetric (Fibonacci) or perfectly symmetric (perfect) patterns are less common.33 In LPD kidneys, we observed an increase in imbalanced Fibonacci patterns, similar to kidneys with increased tip packing at a greater proportion than the NPD kidneys (H, P = 0.004). n=6/6 NPD/LPD litters for whole kidney measurements and branches analyzed per kidney are n>1000. UB, ureteric bud.

Maternal LPD Reduces Nephron Progenitor Cell Proliferation and Commitment

The LPD-induced differences in branching and kidney size prompted us to investigate whether proliferation of ureteric bud cells and nephron progenitor cells was affected. We imaged and counted markers for nephron progenitor cells (SIX2), epithelial ureteric bud cells (TROP2), and proliferative cells (PHH3) in whole-mount stained kidneys. After LPD exposure, nephrogenic niches at E14.5 exhibited a reduction in normalized proliferation in both the ureteric bud cell (26%) and nephron progenitor cell (27%) niches (Figure 3, A–C). At P0, proliferation in nephron progenitor cells was further reduced (37%), but ureteric bud cell proliferation had normalized (Figure 3, A and B, and Supplemental Dataset 1). By P2, no differences in either niche were noted (Figure 3, A and B). These changes occurred without any detectable differences in apoptosis at P0 (Supplemental Figure 2). To determine the effect of LPD on nephrogenesis, we quantified the number of developing nascent nephrons scoring for pretubular aggregates, renal vesicles, comma-shaped bodies, S-shaped bodies, and stage 4 (nearly mature) nephrons. Despite the preservation of nephrogenic tip niches at E14.5, LPD caused a consistent reduction in pretubular aggregate number (60%, 37%, and 41%, respectively) and the total number of nephrons at any stage of differentiation (32%, 19%, and 27%, respectively) (Figure 3, C and D). The number of connected nephric segments was unchanged in utero, but by P2, there was a 17% reduction in these structures (Figure 3E), suggesting that LPD affects the ability of nephron progenitor cells to commit to nephron formation.

Figure 3.

Figure 3

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Analysis of proliferation and early nascent nephrons at E14.5, P0, and P2. Proliferation of the ureteric bud tip in LPD offspring is reduced at E14.5 but is indifferent later in development (A), although nephron progenitor cell proliferation is affected at E14.5 and P0 but not P2 toward the end of nephrogenesis (B). The number of pretubular aggregates (C) and total nephrogenic segments per niche are reduced throughout development in LPD offspring (D). Although the number of distal-linked segments per niche seems to be indifferent at E14.5 and P0, they are reduced by P2 (E). Sample numbers, n=38/40 niches (n=4/4 kidneys; NPD/LPD) at E14.5, n=282/282 niches (9/11 kidneys; NPD/LPD) at P0, n=145/144 niches (n=7/7 kidneys; NPD/LPD) at P2.

Single-Cell Sequencing Profiles the Effects of Maternal LPD

To understand the molecular basis of these phenotypes, we performed single-cell and single-nuclear RNA sequencing at E14.5 and P0. Quality controlled and filtered datasets were clustered (Figure 4, A and B) and assigned to cell types using a priori known markers (Figure 4, C and D), indicating that diet did not alter clustering or Uniform Manifold Approximation and Projection structure (Supplemental Figure 3). P0 kidneys had more differentiated cell types, with the most variably expressed genes being common and related to age (Figure 4D, 2968 and 2546 genes at E14.5 and P0). Gene ontology analysis of gene expression, performed for each cell type at both ages, revealed that the top three upregulated and downregulated pathways are mostly broadly associated with developmental and metabolic processes (Figure 4E and Supplemental Dataset 2).

Figure 4.

Figure 4

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Single-cell and nuclear analysis reveals overarching developmental and metabolic changes in gene expression. Viewed in UMAP-reduced dimension space, clusters identified at E14.5 from single-cell sequencing of 30,845 cells (A), partitioned into known kidney cell types typified by canonical cell type markers, and similarly from P0 single nuclear sequencing of 17,954 cells (B), the clusters represent known developmental cell types by canonical marker expression (C and D). (E) Alluvial plot with central cluster blocks; flows to the left are downregulated GO terms, right are upregulated. Ribbon width is proportional to the number of enriched terms per cluster; colors indicate GO supercategories defined as development, metabolism, and other. The flows show a pervasive metabolic signal and a pronounced enrichment of developmental terms on the downregulated side. Cort.Str, cortical stroma; DT, distal tubule; DT.LoH, distal tubule/loop of Henle; GO, Gene Ontology; IC-B, type B intercalated cell; immune, macrophage; Med.Str, medial stroma; NPC, nephron progenitor cell; NPC.com, committing NPC; NPC.Str, NPC-associated stroma; Pap.Str, papillary stroma; Pelv.Str, pelvic stroma; Pod, podocyte; PT, proximal tubule; RV.SSB, renal vesicle/S-shaped body; UMAP, Uniform Manifold Approximation and Projection; Vasc, endothelial cell; VSMC, vascular smooth muscle cell.

LPD Causes Uniform Disruption to Metabolism and Epigenetic Gene Expression

Reflecting the primary effect of dietary intervention on cellular metabolism, we observed consistent cellular and temporal disruption in several metabolic pathways, including ATP production, RNA metabolism, and metabolic stress (Supplemental Dataset 2). Key ATP metabolism genes such as Atp1a1, Atp5c1, Cpt1a, Gapdh, and Trp53 were affected. Notably, Trp53, downregulated in all 18 clusters at E14.5, plays a critical role in nephron progenitor cell renewal and commitment.34 Its deletion significantly affects Pck1, a gluconeogenesis gene,35 also downregulated in nephrogenic lineages at both E14.5 and P0 (Supplemental Dataset 2). LPD also affected RNA metabolism, including genes involved in amino acid regulation, ribosomal protein transcription, and translation. Notable downregulated genes included Hnrnpa0 and Ybx3, which regulate RNA and amino acid levels (Table 1 and Supplemental Dataset 2). Metabolic stress genes were altered by LPD at E14.5 and P0, as shown in Table 1 and Supplemental Dataset 2. We observed upregulation of Glo1, Ldhb, Dera, Rdh10, AP-1 factors (Fosb and Jun), and Atf4. Atf4 regulates genes involved in oxidative stress, amino acid synthesis, and cell differentiation,36 potentially contributing to the proliferation and differentiation deficits in LPD kidneys.

Table 1.

Differential gene expression between normal-protein diet and low-protein diet nephron progenitor cells at E14.5 and P0

Gene LogFC LogCPM P Value Age Direction
Hnrnpa0 −1.318 9.277 1.49E-84 E14.5 Down
Ybx3 −1.090 9.351 8.79E-68 E14.5 Down
Ptn −0.884 9.201 6.82E-35 E14.5 Down
Rcc2 −0.546 9.109 2.50E-15 E14.5 Down
Ubb −0.540 10.748 1.06E-61 E14.5 Down
Gpc3 −0.525 9.417 7.92E-19 E14.5 Down
Snhg12 0.552 9.060 5.38E-14 E14.5 Up
Tgfbi 0.580 9.102 1.23E-15 E14.5 Up
Erh 0.589 9.130 1.67E-17 E14.5 Up
Crym 0.990 9.655 3.99E-65 E14.5 Up
Glo1 1.178 9.389 1.12E-76 E14.5 Up
Aldh1a2 1.284 9.000 7.46E-53 E14.5 Up
Rbfox1 −0.950 12.154 2.14E-55 P0 Down
Lhfp −0.840 11.150 3.04E-23 P0 Down
Cdh4 −0.821 11.245 3.39E-22 P0 Down
Dach1 −0.769 11.188 1.39E-21 P0 Down
Pdgfc −0.744 11.482 7.81E-25 P0 Down
Mdga2 −0.670 11.177 5.76E-14 P0 Down
Strbp 0.422 11.213 2.80E-08 P0 Up
App 0.427 11.269 9.03E-09 P0 Up
Malat1 0.435 16.608 1.72E-47 P0 Up
Fgfr2 0.444 11.221 4.68E-09 P0 Up
Hdac8 0.523 11.345 5.44E-12 P0 Up
H3f3b 0.568 11.212 1.42E-13 P0 Up
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The table presents the differentially expressed genes between nephron progenitor cells from normal-protein diet and low-protein diet cohorts (with P value < 0.05 and an absolute log fold change (|logFC|) >0.5). The differentially expressed genes were determined using EdgeR as described in the Methods. LogCPM, log of counts per million reads mapped during single-cell sequencing; LogFC, log of the fold change difference.

To address whether LPD affects differentiation toward multiple nephron epithelial lineages, we conducted pseudotime analysis of nephron progenitor transitions to podocytes, proximal tubules, and distal tubules. Slingshot30 and tradeSeq31 based differential gene expression analysis identified a significant temporal reduction in Ezh2 expression across all three lineages (trajectories) at P0, including nephron progenitor-to-podocyte, nephron progenitor-to-distal tubule, and nephron progenitor-to-proximal tubule transitions, as well as in nephron progenitor-to-renal vesicle/S-shaped body trajectories (Supplemental Figure 4). Reduced Ezh2 expression was also observed along the broader nephron progenitor cell pseudotime, committing nephron progenitor cell trajectories and S-shaped body–derived lineages (S-shaped body to proximal tubule, and S-shaped body to distal tubule; Supplemental Dataset 1 and Supplemental Figure 4). When assessing collective gene expression in entire cellular compartments by RNAscope fluorescence, no changes in Ezh2 were identified (P > 0.05, Supplemental Dataset 1 and Supplemental Figures 5 and 6). However, this approach collectively analyzes cells at distinct stages of the differentiation pathway identified by single-cell analysis. The common reduction of Ezh2 across these diverse differentiation paths suggests that LPD impairs epigenetic mechanisms necessary for nephron progenitor maintenance and their transition to multiple nephron epithelial fates.

Maternal LPD Impairs the Differentiation of Nephron Progenitor Cells

Using traditional cluster gene expression analysis, we identified downregulation of several cell cycle and proliferation-related genes (Ybx3, Ptn, Gpc3, and Rcc2) in LPD nephron progenitor cells at E14.5, aligning with decreased nephron progenitor cell proliferation (Figure 3B). GO analysis linked these changes to cell proliferation in metanephric development (GO:0072203), indicating LPD's effect on progenitor maintenance (see Supplemental Dataset 2). Within the P0 nephron progenitor cell cluster, LPD induced downregulation of several genes, including Dach1, which interacts with Eya1 in nascent nephrons,37,38 and Pdgfc, which is expressed in nephron progenitor cells at the ureteric tip at E11.539,40 (Table 1). Both Pdgfc and Eya1 are critical for the establishment, maintenance, and differentiation of nephron progenitor cells,41,42 suggesting that LPD disrupts reciprocal signaling during branching morphogenesis (Figure 2F). In committing nephron progenitors, genes related to RNA metabolism, nuclear division, and mitosis (Rcc2, Cdk2ap1, Ccnd1, and Hnrnpa) were differentially expressed at E14.5, with misexpression of genes such as Crym (Table 2 and Supplemental Dataset 2) indicating compromised transitions to renal vesicles. At P0, downregulation of nephron progenitor identity genes such as Pax2, Bmper, Wt1, and Magi2 (Table 2) supported the observed reduction in pretubular aggregates (Figure 3C).

Table 2.

Differential gene expression between normal-protein diet and low-protein diet in the committing nephron progenitor cell cluster at E14.5 and P0

Gene LogFC LogCPM P Value Age Direction
Hnrnpa0 −1.400 9.276 1.73E-125 E14.5 Down
Ybx3 −0.940 9.309 2.62E-67 E14.5 Down
Cdk2ap1 −0.813 9.016 2.97E-41 E14.5 Down
Rcc2 −0.730 9.136 4.59E-39 E14.5 Down
Wfdc2 −0.612 9.043 9.58E-17 E14.5 Down
Ccnd1 −0.602 9.278 3.34E-15 E14.5 Down
Aldh1a2 0.725 9.111 3.33E-27 E14.5 Up
Hist1h2ae 0.870 9.032 1.74E-31 E14.5 Up
Crym 1.020 9.241 1.52E-56 E14.5 Up
Glo1 1.074 9.249 5.10E-79 E14.5 Up
Hist1h2ap 1.609 9.404 1.53E-99 E14.5 Up
Tenm4 −1.269 11.449 8.27E-62 E14.5 Up
Dach1 −1.125 11.602 5.83E-57 P0 Down
Pax2 −0.984 11.896 3.97E-52 P0 Down
Dab1 −0.905 11.480 1.90E-32 P0 Down
Bmper −0.890 11.703 5.07E-33 P0 Down
Pdgfc −0.870 11.491 3.63E-32 P0 Down
Ptprg −0.807 11.532 3.00E-30 P0 Down
Rbm39 0.357 11.815 1.41E-07 P0 Up
Rev3l 0.358 11.463 5.12E-06 P0 Up
Malat1 0.369 16.936 1.57E-40 P0 Up
Hmcn1 0.386 11.577 4.38E-07 P0 Up
Klhdc10 0.399 11.435 1.84E-06 P0 Up
Epo 0.802 11.390 6.50E-22 P0 Up
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The table presents the differentially expressed genes between committing nephron progenitor cells from normal-protein diet and low-protein diet cohorts (P value < 0.05 and an absolute log fold change (|logFC|) >0.5). The differentially expressed gene list was generated with EdgeR as described the Methods. LogCPM, log of counts per million reads mapped during single-cell sequencing; LogFC, log of the fold change difference.

To assess LPD's effect on nephron progenitor cell differentiation, we conducted pseudotime analysis30 (Figure 5, A and B). tradeSeq analysis (Figure 5C) further highlighted downregulated metabolic and cell cycle genes across pseudotime (Figure 5C, E14.5 clusters 8 and 4; P0 cluster 6, see also Supplemental Dataset 2), with key nephron progenitor cell commitment genes (Pax2, Fgf8, and Wnt4) showing reduced or shifted expression (Figure 5D, Supplemental Dataset 3, and Supplemental Figures 10 and 11). Notably, Fgf8 was downregulated at E14.5, whereas Pax2 and Wnt4 were reduced at both E14.5 and P0 in nephron progenitor cell lineages. In addition, Jag1, Notch2, and Notch3 were downregulated at P0 (Figure 5D and Supplemental Figure 9), indicating a decrease in NOTCH and WNT signaling. GO analysis confirmed enrichment of Wnt signaling genes (GO:0016055 and GO:0030177) across nephron progenitor and S-shaped body–derived lineages, including transitions to podocyte, distal tubule, proximal tubule, and renal vesicle/S-shaped body lineages, indicating a potential Wnt deficiency caused by LPD. Supporting this, confocal microscopy indicated that Wnt4 levels were significantly decreased in E14.5 and P0 LPD kidneys (P < 0.001 at E14.5 and P = 0.02 at P0, Figure 5E and Supplemental Figure 7).

Figure 5.

Figure 5

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Analysis of the nephrogenic lineage indicates changes in cell development and nephrogenesis-promoting gene expression. Nephron progenitor cell, committing nephron progenitor cell (NPC.com), RV/S-shaped body, and podocytes (Pod) were subset from the whole kidney dataset and reclustered (A). Slingshot was used to infer pseudotime and the resulting timeline recapitulated known nephrogenic progression from nephron progenitor cells to podocytes (B). Clustering of the dynamic changes in expression of the differentially expressed genes across pseudotime (C) indicates half of the clusters represent genes involved in cellular metabolic and nephrogenesis pathways, including Wnt pathways. Wnt, Pax2, Lhx1, and Jag1 genes important for nephrogenesis are downregulated over pseudotime (D). (E) Expression of Wnt4 (cyan) in the developing nephrons is reduced in the E14.5 and P0, with nephron progenitor cells marked by SIX2 (red), ureteric tree by cytokeratin (green), and DNA (blue). Cell density plotted over pseudotime reveals an enrichment of LPD cells at the transition between nephron progenitor cell and committing nephron progenitor cell identities at E14.5 and P0 (F). These enriched cells are also apparent in UMAP plots where LPD-specific nephron progenitor cells are apparent at E14.5 but not as strongly at P0 (G). Increased expression of Crym at the end of pseudotime indicates the epithelialization of the committing nephron progenitor cells is reduced (H). Changes in gene expression across the nephron progenitor cell trajectory indicate multiple pathways related to cell differentiation, specification, cell cycle, and mitochondrial function are affected (I). DEG, differentially expressed genes.

Uniform Manifold Approximation and Projections of subclustered nephron progenitor cells revealed diet-specific cell enrichment at P0 and a unique LPD-only pool at E14.5 (Figure 5F). Pseudotime analysis identified a greater proportion of nephron progenitor cells toward the end of nephron progenitor cell pseudotime (D=0.44, P < 2.2e-16 at E14.5; D=0.39, P < 2.2e-16 at P0, KS test) and a greater proportion of committing nephron progenitor cells at the beginning of committing nephron progenitor cell pseudotime (D=0.14, P = 1.502e-11 at E14; D = 0.27, P < 2.2e-16 at P0, two-sample KS test; Figure 5G). These results indicate that within the nephron progenitor cell cluster, a subset of LPD nephron progenitors occupies a more advanced differentiation state (Supplemental Figure 8A). A comparison of gene expression between advanced and regular nephron progenitor cells revealed 158 differentially expressed genes that were (|log2FC | ≥ 0.4), including upregulation of Crym (Log2FC=0.48; Figure 5H, Tables 1 and 2, Supplemental Figure 9, C and D) and downregulation of Cited1 (−2.20 Log2FC) and Meox1 (−0.48 Log2FC). The changes in Crym over pseudotime were not reflected in RNAscope fluorescence in cap mesenchyme (P > 0.05, Supplemental Dataset 1 and Supplemental Figures 5 and 6) likely because RNAscope cannot resolve colocalized cells with subtle expression differences across commitment states. Gene ontology analysis revealed that advanced nephron progenitor cells in LPD kidneys exhibit reduced expression of genes involved in metabolic processes, alongside increased expression of genes related to cell differentiation and commitment (Supplemental Dataset 2 and Supplemental Figure 8B). Despite this transcriptional shift, these cells continued to express naive markers and showed only limited activation of canonical commitment genes (e.g., Wnt4, Itga8; Supplemental Figure 9C). This mixed identity was also evident across nephron progenitor cell pseudotime. At E14.5, genes associated with nephron progenitor cell fate determination pathways (WNT, AKT, and HOX) were significantly downregulated in specific pseudotime clusters (clusters 3–5), whereas increased expression of known inhibitors was observed in other gene clusters (clusters 1–2; Figure 5I, Supplemental Dataset 3). By P0, LPD kidneys showed reduced expression of genes crucial for epithelialization and renal vesicle formation, including Magi2, Pax2, Itga8, and Shroom3 (Figure 5I, P0 clusters 5–7, Supplemental Dataset 3). These gene expression changes correlated with decreased cell proliferation and reduced differentiation (Figure 3) in the progenitor niche indicating that LPD disrupts the gene regulation necessary for commitment of nephron progenitor cells to differentiation and the accumulation of cells at these critical checkpoints.

The Ureteric Epithelium Has Underlying Defects in Proliferative and Developmental Pathways

Given the perturbations to both branching and nascent nephron formation, we focused on the known determinative role of the ureteric epithelium in the nephron progenitor cell commitment process by examining gene expression changes in the ureteric bud (Figure 6, A and B). The observed reduction in ureteric tip lengths and cellular proliferation at E14.5 was reflected in the reduction in proliferation and mitosis gene expression at E14.5 (Hnmpa0, Ybx3, Rcc2, Gpc3, and Cdk2ap1, see Table 3). The decreased growth is also associated with metabolic stress with upregulation of Glo1 and Aldh1a3.43,44 Pseudotime analysis correctly ordered cells from ureteric bud tip through to ureteric bud stalk or trunk/collecting (Figure 6C) and revealed differences in cell distribution (Figure 6, C and D), as well as a reduction in Wnt9b expression with a mostly trunk/collecting duct identity at P0 (Figure 6E). Wnt9b triggers Wnt4 expression45 to initiate nephron progenitor cell commitment and is normally expressed in the base of the ureteric bud and trunk. Although RNAscope staining did not reveal any major changes in Wnt9b levels in ureteric bud cells (P = 0.69, Supplemental Figure 12), Wnt9b heterozygote mice exhibit a weaker induction of Wnt4.45 This aligns with our observations of decreased Wnt4 expression and delayed nephron progenitor cell commitment. Broader assessment of gene expression in the ureteric clusters and across pseudotime trajectories revealed effects on several pathways (Figure 6, E and F, and Supplemental Dataset 2) including differences in cell proliferation (Hdac9, Erbb4, and Fgf12; Table 3) and cytoskeleton remodeling and cell adhesion (Shroom3, Tenm2, Ctnnd2, Robo1, and Nrg3; Supplemental Dataset 2). Together, these changes indicate an effect of LPD on ureteric tip growth and inductive signal expression via disruption in metabolic and cell cycle gene expression leading to reduced cellular proliferation and expression of Wnt factors necessary for nephron progenitor commitment.

Figure 6.

Figure 6

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Single-cell analysis of ureteric development at E14.5 and P0. The ureteric lineage from tip to stalk in early development (E14.5) and from tip to trunk/collecting duct at P0 was subset from the entire single-cell dataset and reclustered (A). Pseudotime trajectory analysis followed the established developmental progression of ureteric development (B and C). The distribution of cells indicates a greater proportion of LPD cells in the middle of pseudotime at the approximate point of trunk differentiation (D=0.14, P value < 2.2e-16 at E14.5, and D=0.09, P value < 0.001 at P0, KS test; D). Wnt9b, a nephron progenitor commitment and renal vesicle specification gene was downregulated near the middle of pseudotime (E). Clustering of gene expression levels across pseudotime (F) revealed many genes with similar expression profiles having altered expression in metabolic, cell cycle, and other developmental genes. The top GO ontology terms of these clusters are mostly associated with metabolism pathways (G, presented in a manner similar to Figure 4F). KS, Kolmogorov–Smirnov.

Table 3.

Differentially expressed genes in the ureteric bud clusters between normal-protein diet and low-protein diet at E14.5 and P0

Gene LogFC LogCPM P Value Age Direction
Hnrnpa0 −1.193 9.183 1.09E-41 E14.5 Down
Ybx3 −0.992 9.002 7.21E-27 E14.5 Down
H19 −0.703 9.025 1.80E-11 E14.5 Down
Rcc2 −0.686 9.180 7.43E-20 E14.5 Down
Gpc3 −0.685 9.192 1.55E-16 E14.5 Down
Cdk2ap1 −0.676 8.940 3.96E-14 E14.5 Down
Hoxb9 0.530 8.884 7.31E-08 E14.5 Up
Hist1h2ae 0.645 9.130 5.76E-13 E14.5 Up
Aldh1a3 0.957 9.075 2.89E-25 E14.5 Up
Btg2 1.071 8.882 5.89E-27 E14.5 Up
Glo1 1.187 9.341 2.00E-60 E14.5 Up
Hist1h2ap 1.407 9.651 2.17E-68 E14.5 Up
Hdac9 −0.972 11.559 7.01E-17 P0 Down
Frmpd4 −0.906 11.514 5.37E-12 P0 Down
Erbb4 −0.857 11.973 1.47E-16 P0 Down
Nos1ap −0.843 11.362 1.72E-12 P0 Down
Fgf12 −0.821 11.286 1.32E-11 P0 Down
Ctnnd2 −0.803 11.800 9.07E-14 P0 Down
Rbm25 0.410 11.387 1.04E-03 P0 Up
Mbnl2 0.455 11.290 3.97E-04 P0 Up
Rbm39 0.464 11.678 8.10E-06 P0 Up
Strbp 0.501 11.418 3.21E-05 P0 Up
Esrrg 0.506 11.417 1.11E-04 P0 Up
H3f3b 0.517 11.353 2.27E-05 P0 Up
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The table lists differentially expressed genes identified between ureteric bud cells from normal-protein diet and low-protein diet cohorts using edgeR, as described in the Methods. Genes were considered significant at P < 0.05 with an absolute log fold change (|logFC|) >0.5. LogCPM, log counts per million mapped reads during single-cell sequencing; logFC, log fold change.

Discussion

Nephron deficit is a key feature of intrauterine growth restriction caused by maternal malnutrition. We used a dietary model with reduced protein intake to induce an approximately 19% reduction in nephron number at birth and 23% loss by age 3 weeks. LPD was fed throughout pregnancy and lactation and likely affected both prenatal and postnatal development (via breastfeeding/lactation) resulting in low nephron endowment and growth restriction. We focused primarily on the morphologic, cellular, and molecular basis for reduced nephron endowment in this study. The LPD diet induced gene expression signatures associated with downregulated metabolic stress, RNA metabolism, and ATP production genes throughout ureteric and nephrogenic cell lineages. This downregulation is crucial as energy production genes play direct roles in nephron progenitor cell commitment46 and nephron number.47

Branching morphogenesis and nephron progenitor cell maintenance and commitment are interdependent processes critical for nephron endowment,22,45,48 and experimental reduction of the fetal nephron progenitor pool can constrain ureteric branching and adult nephron endowment.49 Although LPD reduces kidney size, it does not impair branching capacity of the ureteric epithelium (with ureteric tip number unchanged at E14.5), but instead limits branch growth leading to patterning defects and alterations in cell cycle, likely as a consequence of failure to meet metabolic demands.50 Although most RET pathway genes controlling ureteric bud branching were unaffected, Gdnf, Gfra1, and branching induction genes (Dach137,38; Ptn51) were downregulated at E14.5. Reduced Wnt9b expression associated with collecting duct maturation may affect ureteric trunk cell polarity,52 affecting commitment of adjacent nephron progenitors.45,53,54 LPD kidneys showed widespread changes characteristic of altered WNT pathway signaling, including reduced Wnt4 in advanced pretubular aggregates/early renal vesicles and pretubular aggregate cell accumulation.

In the nephrogenic niche, LPD significantly reduces nephron progenitor cell commitment to differentiate into pretubular aggregate cells with flow on effects on mature nephrogenic structures. Notably, the number of mature connected nephrons remains unchanged during embryonic development, suggesting the first round of nephron formation is unaffected by LPD. However, an approximately 50% reduction in pretubular aggregates during later development correlates with impaired nephron number at birth, indicating a persistent defect in nephron progenitor cell differentiation. Together, these findings indicate that the nephron deficit in LPD arises primarily from impaired or delayed nephron progenitor cell commitment and is therefore distinct from mechanisms in which a reduced fetal nephron progenitor cell pool limits ureteric branching.49

Pseudotime analysis revealed significant effects on WNT and NOTCH pathways, including reduced expression of metanephric development and renal vesicle formation genes, mirroring reductions in Wnt9b expression in the developing ureteric bud-tip/trunk. Collectively, our analysis suggests the formation of a unique nephron progenitor cell subpopulation with mixed identity after LPD exposure, with hindered commitment to nephron differentiation. These cells exhibit increased expression of hallmarks of more differentiated nephron progenitor cells while also coexpressing uncommitted cell markers. This unique cell population clustered with other nephron progenitor cells, whereas committed LPD and normal-protein diet nephron progenitor cells clustered separately, suggesting they reach the brink of differentiation but fail to commit and transition to pretubular aggregate cell identities. Although no equivalent subset of LPD cells was evident in P0 nephron progenitor cells, pseudotime analysis still profiled less-differentiated cells at the end of nephron progenitor cell pseudotime before commitment, despite reduced proliferation and similar cell numbers.

Ezh2 is the sole lysine 27 methyltransferase in mouse nephron progenitor cells in which it maintains self-renewal by closing chromatin in poised differentiation genes and cell cycle inhibitors.55 Our study found a significant reduction in Ezh2 gene expression along the nephron progenitor cell lineage trajectory. Loss of Ezh2 function leads to premature progenitor commitment and Wnt4 expression,55 yet differentiation into pretubular aggregate and renal vesicles fails. In the LPD nephron lineage, Ezh2 expression is reduced but not ablated, resulting in incomplete differentiation and reduced Wnt4 expression, suggesting LPD disrupts commitment earlier, whereas Ezh2 loss affects a later stage.

A recent LPD study reported a 28% reduction in nephron numbers in Wistar rats.18 Our single-cell sequencing revealed stable expression of mTor, Tgfb1 (increased in their study), Myc, and Ki67 (both decreased in their analysis) and limited changes in β-catenin expression. Interestingly, a similar trend in Zeb1 expression in nephron progenitor cells was present, with our study showing a 0.22 Log2FC increase, aligning with their reported 30% increase in Zeb1. However, their study did not investigate ureteric development, spatially quantify progenitor numbers, or identify the normalization of nephron progenitor cell and ureteric bud cell proliferation by P2 or transcriptional changes. It is unclear whether these differences reflect the experimental models used or the analysis undertaken.

Welham et al. reported a transient increase in apoptosis within renal mesenchymal compartments in midgestation kidneys in a rat maternal low-protein model.56 By contrast, we did not detect increased apoptosis at P0 in nephron progenitor regions or nascent nephron structures in our mouse LPD model (Supplemental Figure 2), nor did nephron progenitor cell transcriptomes show enrichment of apoptosis-related programs, supporting impaired proliferation and delayed nephron progenitor cell commitment as the dominant contributors to reduced nephron endowment in this study.

A mouse calorie-restricted diet study found that maternal methionine supplementation and enhanced mTorc1 signaling are able to ameliorate nephron deficits.57 Although both calorie-restricted diet and LPD reduced body mass, postnatal nephron number, and early reductions in nephron progenitor cell proliferation, we observed no changes in mammalian target of rapamycin signaling in nephron progenitor cells indicating a distinct metabolic response. Notably, nephron progenitor cell proliferation normalized by birth in calorie-restricted diet–treated kidneys but was delayed until P2 in LPD-treated kidneys, suggesting more severe developmental challenges are elicited by protein deficiency, which may be reflected in the differences in normalization after birth.

Our findings suggest that impaired nephron progenitor cell commitment is central to the nephron deficit observed under maternal LPD conditions. The consistent reduction of Wnt4 supports this, given its well-established role in driving committed nephron progenitor cells toward renal vesicle formation. Although Wnt9b reduction was observed in the P0 pseudotime analysis, its role cannot be entirely excluded. The unchanged ureteric tip number, alongside increased tip density, introduces the possibility that altered spatial arrangement may influence WNT signaling dynamics, particularly influencing Wnt4 expression. Furthermore, pseudotime analysis indicates that nephron progenitor cells under LPD conditions may experience delayed or stalled commitment, potentially because of insufficient WNT signaling or metabolic constraints related to nutrient availability. This complexity suggests that both signaling and metabolic factors may contribute to the observed phenotype, warranting further investigation.

Our findings demonstrate that LPD reduces nephron numbers through disruptions in cell proliferation, branching-related developmental programs, and nephrogenic differentiation—particularly during nephron progenitor cell commitment to differentiation. These alterations correspond to decreasing expression of key developmental regulatory genes and pathways, notably in WNT signaling, which are crucial for nephron progenitor commitment and differentiation. By identifying specific gene expression changes and pathways affected by LPD, this study highlights how reduced protein intake during critical developmental windows leads to deficiencies in nephron endowment. These insights contribute to a more comprehensive understanding of the developmental origins of health and disease, informing clinical approaches and public health strategies to mitigate the effect of prenatal nutritional deficits on long-term health outcomes.

Supplementary Material

jasn-37-1895-s001.pdf (2.7MB, pdf)
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jasn-37-1895-s002.pdf (5.6MB, pdf)
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jasn-37-1895-s003.xlsx (10MB, xlsx)
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jasn-37-1895-s004.xlsx (3.8MB, xlsx)
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jasn-37-1895-s005.xlsx (9.6MB, xlsx)
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Acknowledgments

The authors acknowledge Grace Jackel and Guizhi Sun for technical assistance and James LeFevre (University of Queensland) for bioinformatics discussions. We thank the staff of the Monash MARP, Monash Micro Imaging, Monash FlowCore, Monash Histology Platform, Monash Genomics and Bioinformatics Platform, and the Tulane Center for Translational Research in Infection & Inflammation NextGen Sequencing Core.

Footnotes

K.M.S. and G.G.T. are co-first authors.

See related editorial, “Mapping Alterations in Nephrogenesis Induced by Metabolic Stress,” on pages 1849–1851.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F771.

Author Contributions

Conceptualization: John F. Bertram, Marnie E. Blewitt, Luise A. Cullen-McEwen, Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Data curation: Luise A. Cullen-McEwen, Francesca Edgington-Giordano, Lynelle K. Jones, Kieran M. Short, Giovane G. Tortelote.

Formal analysis: Luise A. Cullen-McEwen, Fabiola Diniz, Francesca Edgington-Giordano, Samir S. El-Dahr, Lynelle K. Jones, Jan Schröder, Kieran M. Short, Ashley Spencer, Giovane G. Tortelote.

Funding acquisition: John F. Bertram, Marnie E. Blewitt, Samir S. El-Dahr, Jose M. Polo, Ian M. Smyth.

Investigation: Luise A. Cullen-McEwen, Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Methodology: Luise A. Cullen-McEwen, Andrew Keniry, Jan Schröder, Kieran M. Short, Giovane G. Tortelote.

Project administration: Samir S. El-Dahr, Ian M. Smyth.

Resources: Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Software: Kieran M. Short.

Supervision: Samir S. El-Dahr, Ian M. Smyth.

Validation: Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Visualization: Kieran M. Short, Giovane G. Tortelote.

Writing – original draft: Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Writing – review & editing: John F. Bertram, Samir S. El-Dahr, Kieran M. Short, Ian M. Smyth, Giovane G. Tortelote.

Funding

I.M. Smyth: National Health and Medical Research Council (APP1080746). S.S. El-Dahr: National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK118231 and 1RO1DK143918). J.F. Bertram and M.E. Blewitt: National Health and Medical Research Council (APP1080746). G.G. Tortelote: Tulane School of Medicine Startup Grant.

Declarative Statements

All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals or an equivalent standard that meets or exceeds the ethical and welfare requirements outlined in the NIH Guide. All protocols were approved by the appropriate institutional animal care and use committee as listed in the Methods.

Data Availability Statements

Original data generated for the study are available in a public access repository. Data Type: Raw Data/Source Data. Repository Name: Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE240044. All sequencing data are available at GSE240044, and raw data are available in the Supplemental Material.

Supplemental Material

This article contains supplemental material online, published as provided by the authors, at http://links.lww.com/JSN/F772, http://links.lww.com/JSN/F773, http://links.lww.com/JSN/F774, http://links.lww.com/JSN/F775.

Supplemental Methods.

Supplemental Figure 1. LPD reduces placental mass and offspring body mass.

Supplemental Figure 2. Apoptosis in the nephrogenic zone of P0 kidneys from NPD-exposed and LPD-exposed offspring.

Supplemental Figure 3. Partitioning E14.5 and P0 single-cell datasets by diet at E14.5.

Supplemental Figure 4. Temporal downregulation of Ezh2 during nephrogenesis and ureteric lineage differentiation.

Supplemental Figure 5. RNAscope does not reproduce single-cell pseudotime-based differential gene expression.

Supplemental Figure 6. RNAscope of Crym, Ezh2, Aldh1A2, and Six2.

Supplemental Figure 7. A comparison of Wnt4 protein levels in E14.5 and P0 NPD and LPD kidneys by fluorescence microscopy.

Supplemental Figure 8. Identification and characterization of an LPD-specific advanced nephron progenitor cell subpopulation by pseudotime analysis.

Supplemental Figure 9. LPD promoted polarization of nephron progenitor cell subpopulations at E14.5.

Supplemental Figure 10. Smoothed expression profiles of Fgf8, Jag1, Notch2, and Notch3 along nephron progenitor cell lineage pseudotime trajectories.

Supplemental Figure 11. Smoothed expression profiles of Pax2 and Wnt4 along nephron progenitor cell pseudotime trajectories.

Supplemental Figure 12. Wnt9b expression in LPD and NPD kidneys.

Supplemental Dataset 1 (Excel).

Supplemental Dataset 2 (Excel).

Supplemental Dataset 3 (Excel).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Original data generated for the study are available in a public access repository. Data Type: Raw Data/Source Data. Repository Name: Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE240044. All sequencing data are available at GSE240044, and raw data are available in the Supplemental Material.

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