PPDPF preserves integrity of proximal tubule by modulating NMNAT activity in chronic kidney diseases

Abstract

Genome-wide association studies (GWAS) have identified loci associated with kidney diseases, but the causal variants, genes, and pathways involved remain elusive. Here, we identified a kidney disease gene called pancreatic progenitor cell differentiation and proliferation factor (PPDPF) through integrating GWAS on kidney function and multiomic analysis. PPDPF was predominantly expressed in healthy proximal tubules of human and mouse kidneys via single-cell analysis. Further investigations revealed that PPDPF functioned as a thiol-disulfide oxidoreductase to maintain cellular NAD⁺ levels. Deficiency in PPDPF disrupted NAD⁺ and mitochondrial homeostasis by impairing the activities of nicotinamide mononucleotide adenylyl transferases (NMNATs), thereby compromising the function of proximal tubules during injuries. Consequently, knockout of PPDPF notably accelerated the progression of chronic kidney disease (CKD) in mouse models induced by aging, chemical exposure, and obstruction. These findings strongly support targeting PPDPF as a potential therapy for kidney fibrosis, offering possibilities for future CKD interventions.

Fig. 1.. The kidney disease risk gene, PPDPF, was up-regulated in the early stages of both human and mouse kidney diseases.

(A) Schematic representation of bulk RNA-seq and scRNA-seq on kidney samples of 1d and 5d UUO. (B) DEGs that were significant in UUO 1d but not significant in UUO 5d of bulk RNA-seq. (C) The UMAP of 18 distinct cell types identified by unsupervised clustering. Endo, endothelial cell; podo, podocyte; LOH, loop of Henle; DTL, descending thin limb of Henle’s loop; DCT, distal convoluted tubule; PC, principal cell; IC, intercalated cell; urothelial, urothelial cell; macro, macrophage; B, B lymphocyte; NK, natural killer cell; T, T cell; neutro, neutrophile; DC, dendritic cell; stroma, stroma cell; Immune1, immune cell type 1; Immune2, immune cell type 2. (D) Cell type–specific expression of top DEGs identified from (B). (E) Venn diagram exhibits the overlaps among indicated sets (up arrow: up-regulated DEGs). (F) The relative mRNA expression of Ppdpf in the cisplatin (Cis)–, FA-, and UUO-administered kidney samples. (G) Western blots of PPDPF in Cis-, FA-, UUO-, and IRI-administered kidney samples. Eight-week-old male mice were used. NC, negative control. (H) Representative images of double staining of PPDPF and KIM1 in the UUO-administered kidneys. Scale bar, 20 μm. (I) The relative mRNA expression of PPDPF in human AKI kidneys. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (A to D) n = 3, control; n = 3, UUO 1d mice; n = 2, UUO 5d mice. (F) For Cis: n = 6, control; n = 5, Cis-treated mice. For FA: n = 6, control; n = 5, FA-treated mice. For UUO: n = 6, control; n = 6, UUO-treated mice. (G) Three control mice and three diseased mice were included for each model. FC, fold change; GAPDH, glyceraldehyde phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 2.. Ppdpf is mainly expressed in the healthy PT subclusters.

(A) Expression of Ppdpf in the PT of control, UUO 1d, and UUO 5d mouse kidneys by scRNA-seq. (B) Bubble plot of Ppdpf expression in PT clusters of UUO by scRNA-seq (GSE190887). (C) Expression of Ppdpf in isolated LTL⁺ cells from control and UUO 1d. (D) Top: Western blots of PPDPF in isolated LTL⁺ cells from control and UUO 1d. Bottom: Western blots quantification. (E) UMAP of all PT cells in subclustering analysis. (F) UMAP of cell densities of PT cells between different treatment groups. (G) Bubble plot of Ppdpf expression in each subcluster of (E). (H) Feature plots of E2f2 (proliferating PT) and Tfdp1 (proliferating PT). (I) Representative FISH images. Scale bars, 20 μm. (J) Expression of PPDPF in the scRNA-seq of human kidneys (GSE202109). Left: UMAP of human kidney with cell type annotations. PEC, pericyte; CD, collecting duct. Middle: Bubble plots of injured cell cluster marker genes. Right: Expression of PPDPF in PT subcluster. (K) Expression of PPDPF in the scRNA-seq of human kidneys (GSE183276). Left: Bubble plots of injured cell cluster marker genes; right: PPDPF expression in PT subcluster. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (A) n = 3, control; n = 3, UUO 1d mice; n = 2, UUO 5d mice. (C) n = 3, control; n = 3, UUO 1d mice. (D) n = 3, control; n = 3, UUO 1d mice.

Fig. 3.. Loss of Ppdpf impairs mitochondrial function.

(A) The stress score of control, UUO 1d, and UUO 5d kidney samples from scRNA-seq. (B) UMAP plot of the stress score. (C) KEGG enrichment analysis in UUO 1d-specific PT cells by scRNA-seq. (D) Representative transmission electron microscopy images of WT and Ppdpf KO kidney tubules (8 weeks old). (E) Quantifications of damaged mitochondria in (D). (F) Relative mtDNA levels in WT and Ppdpf KO kidneys analyzed by amplifying ND1 and 16S genes and normalized against the hexokinase 2 (HK2) gene. (G) Complex I enzyme activity of kidneys from WT and Ppdpf KO mice. (H) Left: Western blots of complex I (NDUFB9 and NDUFS3) and complex II (SDHA and SDHC) in primary cells of WT and Ppdpf KO kidneys. Right: Quantifications of NDUFB9 and NDUFS3. (I) Quantifications of fluorescent JC1 signaling (green/red ratio) and fluorescent mitoSOX signaling in primary cells from WT and Ppdpf KO mice. (J) Representative images and quantifications of MitoTraker of primary cells from WT and Ppdpf KO mice. Scale bars, 10 μm. (K) Analysis of oxygen consumption rate (OCR) in primary cells from WT and Ppdpf KO mice. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. **P < 0.01; ****P < 0.0001. (A) n = 3, control; n = 3, UUO 1d mice; n = 2, UUO 5d mice. (D and E) n = 6, WT; n = 6, KO. (F to H) n = 3, WT; n = 3, KO. (I) n = 4, WT; n = 4 KO. (J) n = 10, WT; n = 10, KO. (K) n = 3, WT; n = 3, KO. TNF, tumor necrosis factor; a.u., arbitrary units; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; mOD, measure optical density.

Fig. 4.. Ppdpf regulates the cellular NAD⁺ levels.

(A) Heatmap of top 20 up-regulated and down-regulated genes of WT and Ppdpf KO kidney from bulk RNA-seq analysis. (B) GSEA analysis of mitochondrial respiratory chain complex I assembly from Ppdpf KO over WT kidneys from bulk RNA-seq data. NES, normalized enrichment score; P value, nominal P value. (C) KEGG enrichment analysis of DEGs from Ppdpf KO versus WT kidney by bulk RNA-seq. (D) NAD⁺ content of whole-cell lysates and mitochondria isolated from WT and Ppdpf KO kidney. (E) Quantification of FiNad- and SoNar-positive primary cells isolated from WT and Ppdpf KO kidney. (F) Heatmap of energy metabolites of WT and Ppdpf KO kidneys measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). (G) Targeted metabolomics of Ppdpf KO and WT kidney measured by LC-MS/MS. Significantly changed metabolites were determined by setting a false discovery rate (FDR) of 5% and are represented in red in a volcano plot. (H) NAD⁺ concentration of Ppdpf KO and WT kidney in targeted metabolomics measured by LC-MS/MS. (I) Schematic process of NAD⁺ metabolism and quantifications of NAD⁺ synthesis–related metabolites measured by LC-MS/MS. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (A to C) n = 2, WT; n = 2, KO. (D) For total lysate: n = 4, WT; n = 4, KO. For mitochondria: n = 3, WT; n = 3, KO. (E) For SoNar: n = 42, WT; n = 34, KO. For FiNad: n = 67, WT; n = 69, KO. (F to H) n = 4, WT; n = 4, KO. (I) n = 6, WT; n = 6, KO. PPAR, peroxisome proliferator–activated receptor; ADP, adenosine 5′-diphosphate; AMP, adenosine 5′-monophosphate; GMP, guanosine 5′-monophosphate.

Fig. 5.. PPDPF deficiency inhibits NAD⁺ synthesis through NMNATs.

(A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD⁺ from NMN. (D) NAD⁺ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD⁺ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.

Fig. 6.. PPDPF functions as a thiol-disulfide oxidoreductase.

(A) Schematic representation of the domain organization of TGR, TR1, TR3, GR, LDH, and PPDPF. These six proteins have active disulfide center (the CxxxxC motif; two cysteines separated by four other residues) of the pyridine nucleotide disulfide oxidoreductase family. TGR, thioredoxin (Trx) and GSSG reductase; TrxR1, cytosolic Trx reductase; TrxR2, mitochondrial Trx reductase; GR, GSSG reductase; LDH, lactate dehydrogenase. (B) Determination of the redox potential of recombinant human PPDPF by subjecting it to increasing dithiothreitol (DTT) concentrations. The band intensity increases as the redox environment becomes more reducing. This indicates that the disulfide bond is gradually being reduced and resulting in an increase in free cysteine thiols for MPB labeling. (C) Optical density readout of an insulin turbidity assay. (D) Readout of a di-eosin-GSSG assay using purified proteins. RFU, relative fluorescence unit. (E) Readout of an PPDPF RNase disulfide isomerase assay. (F) Left: Representation workflow of MPB labeling. PPDPF reduces disulfide bonds, exposing free thiol groups within NMNATs. The free thiols are labeled with MPB, and the proteins are separated by SDS–polyacrylamide gel electrophoresis. Right: Western blot of MPB-labeled NMNATs in the presence of PPDPF. (G) Schematic representation of the transfection of PPDPF WT, C30S, and C35S constructs into HEK293T followed by quantifications of NAD⁺ levels and NMNAT activity. (H) Western blots of PPDPF-WT, PPDPF-C30S, and PPDPF-C35S overexpression in HEK293T cells. (I) Quantifications of NMNAT activity (top) and NAD⁺ content (bottom) in cells transfected with indicated constructs. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (C to E) n = 2 for each group. (H) n = 2 for each group. (I) For NMNAT activity, n = 4 for each group. For NAD⁺ measurement, n = 4 for each group. OD₆₅₀, optical density at 650 nm.

Fig. 7.. Ppdpf deficiency exacerbates renal injury in aged mice.

(A) Scheme of the experimental process. Kidneys, serum, and urine were harvested at the age of 12 months. (B) Serum creatinine levels in WT and Ppdpf KO mice at 12 months old. (C) Serum BUN levels in WT and Ppdpf KO mice at 12 months old. (D) Serum cystatin C levels in WT and Ppdpf KO mice at 12 months old. (E) Urine albumin levels in WT and Ppdpf KO mice at 12 months old. (F) Representative images of H&E- and Masson-stained kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bar, 20 μm. (G) Quantification of tubular injury score and Masson staining area from (F). (H) Relative mRNA levels of fibrosis markers Col3a1, Col1a1, and Vimentin in kidneys of WT and Ppdpf KO mice at 12 months old. (I) Western blots and quantifications of vimentin, ACTA2, and KIM1 in kidneys of WT and Ppdpf KO mice at 12 months old. (J) Representative images of vimentin and KIM1 staining in kidney sections from WT and Ppdpf KO mice at 12 months old. Scale bars, 20 μm. (K) NAD⁺ content of whole kidney lysates of WT and Ppdpf KO mice at 12 months old. P value was calculated by two-tailed t test. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B and C) n = 4, WT; n = 9, KO. (D) n = 3, WT; n = 5, KO. (E) n = 8, WT; n = 8, KO. (F) n = 6, WT; n = 11, KO. (H) n = 7, WT; n = 13, KO. (H) n = 3, WT; n = 3, KO. (K) n = 4, WT; n = 4, KO.

Fig. 8.. Ppdpf deficiency exacerbates renal injury in the long-term cisplatin model.

(A) Scheme of the experimental approach. Eight-week-old male mice were intraperitoneally injected with cisplatin (Cis; 7 mg/kg) and euthanized after 28 days. (B) Serum creatinine levels of WT and Ppdpf KO mice with or without cisplatin treatment. (C) Urine albumin levels in WT and Ppdpf KO mice with cisplatin treatment. (D) Representative images of H&E- and Masson-stained kidney sections from WT and Ppdpf KO mice following saline or cisplatin injection. Scale bars, 20 μm. (E) Quantification of tubular injury score and Masson staining area from (D). (F) The relative expression of fibrosis markers Col3a1, Fibronectin, and Vimentin in control or cisplatin-treated WT and Ppdpf KO mice. (G) Western blots of fibrosis markers ACTA2, vimentin, fibronectin, and mitochondrial marker DRP1 in WT and Ppdpf KO mice with or without cisplatin treatment. (H) NAD⁺ content of whole kidney lysates of WT and Ppdpf KO mice with or without cisplatin treatment. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 4, WT CTR; n = 3, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (C) n = 5, WT Cis; n = 5, KO Cis. (E) n = 3, WT CTR; n = 3, KO CTR; n = 6, WT Cis; n = 6, KO Cis. (F) n = 4, WT CTR; n = 4, KO CTR; n = 5, WT Cis; n = 5, KO Cis. (G) n = 3, WT CTR; n = 3, KO CTR; n = 3, WT Cis; n = 3, KO Cis. (H) n = 4, WT CTR; n = 4, KO CTR; n = 4, WT Cis; n = 4, KO Cis.

Fig. 9.. NAD⁺ but not NMN alleviates the renal injuries in Ppdpf KO mice with FA treatment.

(A) Scheme of the experimental approach. Eight-week-old male mice were intraperitoneally injected with NAD⁺ (3 mg/g) or NMN (500 mg/kg). FA (250 mg/kg) was injected 3 days later, and mice were then euthanized after 7 days. (B) Serum creatinine levels of WT and Ppdpf KO mice with or without FA, NAD⁺ or NMN treatment. (C) Representative images of H&E- and Sirus red–stained kidney sections from WT and Ppdpf KO mice following FA and NAD injection. Scale bar, 20 μm. (D) Quantifications of tubular injury score and Sirus red staining area in the kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD⁺ administration. (E) Western blots of vimentin in kidneys of WT and Ppdpf KO mice with or without FA and NAD⁺ treatment. (F) The relative expression of fibrosis markers Fibronectin and Col3a1 in kidneys of WT and Ppdpf KO mice with or without FA, NMN, or NAD⁺ administration. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA. (D) n = 10 for each group. (E) n = 2 for each group. (F) n = 4, WT; n = 4, KO; n = 3, WT + FA; n = 3, KO + FA; n = 6, WT + NMN + FA; n = 6, KO + NMN + FA; n = 5, WT + NAD + FA; n = 3, KO + NAD + FA.

Fig. 10.. Overexpression of Ppdpf alleviates kidney fibrosis induced by FA administration.

(A) Schematic diagram of Ppdpf overexpression in mice. Eight-week-old male mice were in situ injected with AAV9 encoding Ppdpf or scramble. Post–4-week injection, the mice were subjected to FA treatment. (B) The relative expression levels of Ppdpf in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (C) Western blots of Flag and PPDPF in kidneys with AAV9-Ctrl or AAV9-PPDPF injections. (D) Western blots of PPDPF in LTL-positive cells from AAV9-Ctrl or AAV9-PPDPF kidneys. (E) Representative images of H&E- and Sirus red–stained kidney sections from AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. Scale bars, 20 μm. (F) Quantifications of tubular injury score and Sirus red staining area from (E). (G) The relative expression levels of fibrosis markers Col3a1, Vimentin, and Fibronectin, as well as injury markers Kim1 and Lcn2 in kidneys of AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. *P < 0.05. (H) Representative images of vimentin, fibronectin, and Col3a1 staining on kidney sections from AAV9-Ctrl– or AAV9-PPDPF–injected mice following FA administration. Scale bars, 50 or 20 μm. (I) NAD⁺ content of whole kidney lysates from AAV9-Ctrl– or AAV9-PPDPF–injected mice. (J) NMNAT activity of primary cells isolated from AAV9-Ctrl– or AAV9-PPDPF–injected kidneys. P value was calculated by two-tailed t test (B, F, I, and J) and two-way ANOVA (G). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (B) n = 3, AAV9-Ctrl; n = 4, AAV9-PPDPF. (C and D) n = 3 for each group. (F) n = 10 for each group. (G) n = 3, FA-AAV9-Ctrl; n = 4, FA-AAV9-PPDPF. (I) n = 4 for each group.