Proteomics reveals the key transcription-related factors mediating obstructive nephropathy in pediatric patients and mice
Introduction Obstructive nephropathy is one of the leading causes of kidney injury and fibrosis . In infants and children, ureteropelvic junction obstruction (UPJO) is the major contributor to this condition . UPJO-induced hydronephrosis elevates intrapelvic and intratubular hydrostatic pressure, leading to tubular cell damage, reduced blood supply, chronic inflammation, and ultimately renal fibrosis . However, the precise cellular and molecular mechanisms underlying obstructive nephropathy remain incompletely understood. Transcription-related factors (TRFs), including transcription factors and cofactors, are proteins that bind to specific DNA sequences and regulate gene expression by controlling the initiation, elongation, and termination of transcription . Mounting evidence implicates TRFs in kidney fibrosis . For instance, elevated levels of COUP-TFII (also known as NR2F2) have been linked to enhanced myofibroblast activation and renal fibrosis . Deletion of proximal tubule-specific ZEB2, a transcription factor regulating the transforming growth factor (TGF)-β pathway, significantly mitigates tubulointerstitial fibrosis progression in an ischemia-reperfusion model . Furthermore, the neural transcription factor POU4F1 can drive renal fibrosis by inducing macrophage-myofibroblast transition . Nevertheless, a comprehensive understanding of dysregulated TRFs in obstructive nephropathy, particularly in pediatric patients, remains lacking. We previously conducted a proteomic study of renal fibrosis induced by UPJO in pediatric patients and unilateral ureteral obstruction (UUO) mice . Here, we further examined the kidney proteomes to investigate potential TRFs involved in the pathogenesis of obstructive nephropathy in both patients and mice. We identified 16 key TRFs that were induced by ureteral obstruction across species, some of which have been known to be associated with inflammation and TGF-β pathway during renal fibrosis. Importantly, we also identified several previously unexplored TRFs that may play a role in the development of obstructive nephropathy. 

Materials and methods 2.1. Patient samples and ethics statements The study included seven obstructed kidneys from pediatric patients with UPJO who underwent nephrectomy, as well as three healthy kidneys from pediatric patients with nephroblastoma as control , which was described in our previous study . The control kidney tissues were sampled from regions five centimeters away from the tumor. Written informed consent was obtained from the legal patients. The study was approved by the Research Ethics Committee of the Seventh Medical Center of Chinese PLA General Hospital. 2.2. Mice and unilateral ureteral obstruction (UUO) model The C57BL/6 background mice were purchased from Vital River (Beijing). All animal experimental procedures were approved by the Institutional Animal Care and Utilization Committees of Chinese PLA General Hospital. The mice were maintained in a specific pathogen-free condition. For the UUO model, male mice aged 8 weeks were used. Generally, after anesthesia, a median abdominal incision was performed and the left ureter was double ligated. The sham group underwent the same procedure except for ureteral ligation. Mice were sacrificed at the indicated time points (3, 7, and 14 days post UUO) and kidneys were harvested for further analysis. As for the proteomic study, UUO mice were sacrificed 7 days after UUO. 2.3. Histological analysis Human and mouse kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. The kidney sections were stained with Masson’s trichrome. The following primary antibodies were used: Prohibitin Polyclonal antibody (10787-1-AP, Proteintech), regulatory factor X 1 (RFX1) Polyclonal antibody (26859-1-AP, Proteintech), activity-dependent neuroprotector homeobox protein (ADNP) Polyclonal antibody (17987-1-AP, Proteintech), and ZEB2 Polyclonal antibody (14026-1-AP, Proteintech). Images of the kidney slides were obtained using a Nano Zoomer Slide Scanner (Hamamatsu Photonics). 2.4. Proteomic analysis We previously performed a data-independent acquisition (DIA)-based proteomic study of control and obstructed kidneys from UPJO patients with obstructive nephropathy and UUO mice. The resulting data were deposited in the ProteomeXchange Consortium (PXD039314) . We mined our data for the TRF profile using web sources ( http://bioinfo.life.hust.edu.cn/AnimalTFDB4/?#/Download ), which provides a list of human and mouse transcription factors and transcription cofactors . Protein-protein interaction networks were analyzed using STRING. 2.5. Western blot Kidney protein extracts were prepared according to standard protocols. Generally, tissue lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The following primary antibodies were used (all from Proteintech): ZEB2 polyclonal antibody (14026-1-AP), ADNP polyclonal antibody (17987-1-AP), prohibitin (PHB) polyclonal antibody (10787-1-AP), RFX1 polyclonal antibody (26859-1-AP), β-actin monoclonal antibody (66009-1-lg). The images were quantified by ImageJ. 2.6. Statistical analysis Statistical analyses were performed with GraphPad Prism version 6.0c or R version 4.1. Proteins with p-value <0.05, q-value <0.05, and fold change >2 or <0.5 were considered as up- or down-regulation. Data are presented as mean ± SEM. Two-tailed Student’s t -test and Fisher’s exact test were used for comparisons between the two groups. One-way ANOVA was used to analyze three or more comparisons. A p-value less than 0.05 was considered significant. 

Patient samples and ethics statements The study included seven obstructed kidneys from pediatric patients with UPJO who underwent nephrectomy, as well as three healthy kidneys from pediatric patients with nephroblastoma as control , which was described in our previous study . The control kidney tissues were sampled from regions five centimeters away from the tumor. Written informed consent was obtained from the legal patients. The study was approved by the Research Ethics Committee of the Seventh Medical Center of Chinese PLA General Hospital. 

Mice and unilateral ureteral obstruction (UUO) model The C57BL/6 background mice were purchased from Vital River (Beijing). All animal experimental procedures were approved by the Institutional Animal Care and Utilization Committees of Chinese PLA General Hospital. The mice were maintained in a specific pathogen-free condition. For the UUO model, male mice aged 8 weeks were used. Generally, after anesthesia, a median abdominal incision was performed and the left ureter was double ligated. The sham group underwent the same procedure except for ureteral ligation. Mice were sacrificed at the indicated time points (3, 7, and 14 days post UUO) and kidneys were harvested for further analysis. As for the proteomic study, UUO mice were sacrificed 7 days after UUO. 

Histological analysis Human and mouse kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. The kidney sections were stained with Masson’s trichrome. The following primary antibodies were used: Prohibitin Polyclonal antibody (10787-1-AP, Proteintech), regulatory factor X 1 (RFX1) Polyclonal antibody (26859-1-AP, Proteintech), activity-dependent neuroprotector homeobox protein (ADNP) Polyclonal antibody (17987-1-AP, Proteintech), and ZEB2 Polyclonal antibody (14026-1-AP, Proteintech). Images of the kidney slides were obtained using a Nano Zoomer Slide Scanner (Hamamatsu Photonics). 

Proteomic analysis We previously performed a data-independent acquisition (DIA)-based proteomic study of control and obstructed kidneys from UPJO patients with obstructive nephropathy and UUO mice. The resulting data were deposited in the ProteomeXchange Consortium (PXD039314) . We mined our data for the TRF profile using web sources ( http://bioinfo.life.hust.edu.cn/AnimalTFDB4/?#/Download ), which provides a list of human and mouse transcription factors and transcription cofactors . Protein-protein interaction networks were analyzed using STRING. 

Western blot Kidney protein extracts were prepared according to standard protocols. Generally, tissue lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). The following primary antibodies were used (all from Proteintech): ZEB2 polyclonal antibody (14026-1-AP), ADNP polyclonal antibody (17987-1-AP), prohibitin (PHB) polyclonal antibody (10787-1-AP), RFX1 polyclonal antibody (26859-1-AP), β-actin monoclonal antibody (66009-1-lg). The images were quantified by ImageJ. 

Statistical analysis Statistical analyses were performed with GraphPad Prism version 6.0c or R version 4.1. Proteins with p-value <0.05, q-value <0.05, and fold change >2 or <0.5 were considered as up- or down-regulation. Data are presented as mean ± SEM. Two-tailed Student’s t -test and Fisher’s exact test were used for comparisons between the two groups. One-way ANOVA was used to analyze three or more comparisons. A p-value less than 0.05 was considered significant. 

Results 3.1. Obstructive nephropathy led to renal fibrosis in UPJO patients and UUO mice A representative intravenous pyelography of a patient with bilateral duplex kidneys was shown, indicating the loss of function of the upper pole of the kidney due to right hydronephrosis . To reproduce the UPJO phenotype in mice, we utilized the UUO mouse model. Masson staining analysis confirmed significant fibrosis in both human and mouse obstructed kidneys . We then took advantage of our previously performed DIA-based proteomics data  to study the atlas of TRFs in obstructive nephropathy . 3.2. The alteration of TRFs in pediatric patients with obstructive nephropathy We analyzed human kidney proteomics to identify the dysregulated TRFs in pediatric patients. A total of 140 human TRFs were detected, with 28 upregulated and 1 downregulated . Gene ontology (GO) analysis was conducted on the dysregulated proteins to elucidate the key pathways in obstructive nephropathy. The analysis revealed significant enrichment of signaling pathways including janus kinase/signal transducer and activator of transcription (JAK-STAT), interferon, TGF-β, IL-6, PDGFR-β, and CD209 in obstructed kidneys compared to control kidneys . These pathways have been consistently implicated in the pathogenesis of kidney fibrosis. Notably, the pathway analysis also uncovered the involvement of sumoylation of transcription cofactors , indicating a potential role of sumoylation-mediated transcriptional regulation in obstructive nephropathy. 3.3. The alteration of TRFs in UUO mice Then, we performed a proteomic analysis of kidneys from sham and UUO mice. We revealed 160 TRFs, with 88 showing upregulation and 1 showing downregulation . GO analysis with the upregulated proteins showed significant enrichment of tumor necrosis factor (TNF), TGF-β, Th17 cell differentiation, Th1 and Th2 cell differentiation, JAK-STAT, NF-κB, interleukin-1 processing, and EGFR1 signaling pathways . Of note, some of the enriched pathways were consistent with the analysis of human proteomes, including JAK-STAT and TGF-β. These results suggest that ureteral obstruction leads to the upregulation of TRFs associated with the inflammatory responses and TGF-β pathways. Furthermore, qPCR analysis revealed a significant increase in the expression of Il1b , Il6 , Il4 , Il13 , Tgfb1 , Nfkb1 , Ifng , and Pdgfra in the kidneys of UUO mice compared to those in sham-operated mice , these genes are the target of the TFs Rfx1, Adnp, Creb1, Atf2, and Stat3. 3.4. Key dysregulated TRFs in both patients and UUO mice To uncover the key dysregulated TRFs involved in the pathogenesis of obstructive nephropathy in patients and mice, we performed a cross-species analysis and revealed 15 upregulated proteins implicated in the fibrotic process. These TRFs include Zinc finger E-box-binding homeobox 2 (ZEB2), Lactotransferrin (LTF), Sp110 nuclear body protein (SP110), High mobility group protein B2 (HMGB2), STAT1, Pre-B-cell leukemia transcription factor 1 (PBX1), High mobility group protein HMG-I/HMG-Y (HMGA1), COUP transcription factor 2 (NR2F2), Protein max (MAX), Transcriptional enhancer factor TEF-1 (TEAD1), High mobility group protein B3 (HMGB3), Activity-dependent neuroprotector homeobox protein (ADNP), Transcriptional repressor p66-beta (GATAD2B), MHC class II regulatory factor RFX1 (RFX1), and Dachshund homolog 1 (DACH1) . Additionally, our analysis identified Prohibitin (PHB) as the only TRF that was decreased in obstructed kidneys across species . Notably, several upregulated TRFs have been demonstrated to be critical in the organ fibrotic process, such as ZEB2 , NR2F2 , DACH1 , and STAT1 . However, the roles and mechanisms of the rest dysregulated TRFs in obstructive nephropathy remain unclear. To validate the proteomic findings, we conducted immunoblot analyses of four selected proteins, including PHB, RFX1, ADNP, and ZEB2. The expression levels of these proteins were examined at day 3, 7, and 14 post-UUO induction. Following UUO surgery, PHB exhibited a notable decrease in obstructed kidneys at day 3. RFX1 and ADNP showed a progressive increase from day 3 to day 7, while ADNP levels declined by day 14. ZEB2 was used as a positive control due to its established involvement in renal fibrosis, and it displayed a significant upregulation at day 7 post-UUO . Similarly, immunoblots and immunohistochemistry analysis showed that PHB was decreased, while RFX1, ADNP, and ZEB2 were upregulated in UPJO patients compared to healthy controls , indicating a shared mechanism of obstructive nephropathy between patients and murine models. 

Obstructive nephropathy led to renal fibrosis in UPJO patients and UUO mice A representative intravenous pyelography of a patient with bilateral duplex kidneys was shown, indicating the loss of function of the upper pole of the kidney due to right hydronephrosis . To reproduce the UPJO phenotype in mice, we utilized the UUO mouse model. Masson staining analysis confirmed significant fibrosis in both human and mouse obstructed kidneys . We then took advantage of our previously performed DIA-based proteomics data  to study the atlas of TRFs in obstructive nephropathy . 

The alteration of TRFs in pediatric patients with obstructive nephropathy We analyzed human kidney proteomics to identify the dysregulated TRFs in pediatric patients. A total of 140 human TRFs were detected, with 28 upregulated and 1 downregulated . Gene ontology (GO) analysis was conducted on the dysregulated proteins to elucidate the key pathways in obstructive nephropathy. The analysis revealed significant enrichment of signaling pathways including janus kinase/signal transducer and activator of transcription (JAK-STAT), interferon, TGF-β, IL-6, PDGFR-β, and CD209 in obstructed kidneys compared to control kidneys . These pathways have been consistently implicated in the pathogenesis of kidney fibrosis. Notably, the pathway analysis also uncovered the involvement of sumoylation of transcription cofactors , indicating a potential role of sumoylation-mediated transcriptional regulation in obstructive nephropathy. 

The alteration of TRFs in UUO mice Then, we performed a proteomic analysis of kidneys from sham and UUO mice. We revealed 160 TRFs, with 88 showing upregulation and 1 showing downregulation . GO analysis with the upregulated proteins showed significant enrichment of tumor necrosis factor (TNF), TGF-β, Th17 cell differentiation, Th1 and Th2 cell differentiation, JAK-STAT, NF-κB, interleukin-1 processing, and EGFR1 signaling pathways . Of note, some of the enriched pathways were consistent with the analysis of human proteomes, including JAK-STAT and TGF-β. These results suggest that ureteral obstruction leads to the upregulation of TRFs associated with the inflammatory responses and TGF-β pathways. Furthermore, qPCR analysis revealed a significant increase in the expression of Il1b , Il6 , Il4 , Il13 , Tgfb1 , Nfkb1 , Ifng , and Pdgfra in the kidneys of UUO mice compared to those in sham-operated mice , these genes are the target of the TFs Rfx1, Adnp, Creb1, Atf2, and Stat3. 

Key dysregulated TRFs in both patients and UUO mice To uncover the key dysregulated TRFs involved in the pathogenesis of obstructive nephropathy in patients and mice, we performed a cross-species analysis and revealed 15 upregulated proteins implicated in the fibrotic process. These TRFs include Zinc finger E-box-binding homeobox 2 (ZEB2), Lactotransferrin (LTF), Sp110 nuclear body protein (SP110), High mobility group protein B2 (HMGB2), STAT1, Pre-B-cell leukemia transcription factor 1 (PBX1), High mobility group protein HMG-I/HMG-Y (HMGA1), COUP transcription factor 2 (NR2F2), Protein max (MAX), Transcriptional enhancer factor TEF-1 (TEAD1), High mobility group protein B3 (HMGB3), Activity-dependent neuroprotector homeobox protein (ADNP), Transcriptional repressor p66-beta (GATAD2B), MHC class II regulatory factor RFX1 (RFX1), and Dachshund homolog 1 (DACH1) . Additionally, our analysis identified Prohibitin (PHB) as the only TRF that was decreased in obstructed kidneys across species . Notably, several upregulated TRFs have been demonstrated to be critical in the organ fibrotic process, such as ZEB2 , NR2F2 , DACH1 , and STAT1 . However, the roles and mechanisms of the rest dysregulated TRFs in obstructive nephropathy remain unclear. To validate the proteomic findings, we conducted immunoblot analyses of four selected proteins, including PHB, RFX1, ADNP, and ZEB2. The expression levels of these proteins were examined at day 3, 7, and 14 post-UUO induction. Following UUO surgery, PHB exhibited a notable decrease in obstructed kidneys at day 3. RFX1 and ADNP showed a progressive increase from day 3 to day 7, while ADNP levels declined by day 14. ZEB2 was used as a positive control due to its established involvement in renal fibrosis, and it displayed a significant upregulation at day 7 post-UUO . Similarly, immunoblots and immunohistochemistry analysis showed that PHB was decreased, while RFX1, ADNP, and ZEB2 were upregulated in UPJO patients compared to healthy controls , indicating a shared mechanism of obstructive nephropathy between patients and murine models. 

Discussion Although increasing evidence suggests that TRFs are associated with the development of kidney diseases, a global landscape of their alterations in pediatric patients with obstructive nephropathy was lacking. Here, our proteomic analysis in patients and UUO mice identified key dysregulated TRFs and uncovered several previously unexplored TRFs that may play a role in this condition. Obstruction-induced tubular injury results in the release of damage-associated molecular patterns (DAMPs), initiating the inflammatory response . Prolonged inflammation stimulates the production of pro-fibrotic proteins, such as TGF-β, FGF, and PDGF, to support the activation of myofibroblasts, which are the key contributors to extracellular matrix deposition during renal fibrosis . We identified several dysregulated TRFs that are enriched in pathways involved in kidney inflammation and fibrosis, including the JAK-STAT pathway. It has been established that the JAK-STAT pathway is associated with renal fibrosis in diabetic kidney disease . Activation of the JAK-STAT pathway contributed to the proliferation of renal fibroblasts, and JAK inhibitors can ameliorate albuminuria in diabetic kidney disease . Interestingly, interferon signaling was notably enriched in the upregulated TRFs (including PIAS1, STAT1/2, and IRF9) in patients with hydronephrosis. Studies have indicated that mitochondrial damage leads to the release of mitochondrial DNA and the activation of the cGAS-stimulator of interferon genes (STING) pathway, thus inducing interferon expression and immune cell recruitment . Therefore, the activation of interferon signaling could be related to obstruction-induced mitochondrial dysfunction. We revealed a significant activation of the TGF-β pathway in patients and mice, which is the master regulator of organ fibrosis . Previous studies have consistently reported an increased expression of TGF‑β in renal fibrosis. TGF-β signals through the canonical and non-canonical pathways . Canonical signaling via Smad proteins has a central role in myofibroblast activation by inducing the transcription of profibrotic molecules, including α-smooth muscle actin (α-SMA), collagen I, and tissue inhibitor of matrix metalloproteinases (TIMP) . Hence, TGF‑β is a potential target for renal fibrosis. Our study identified several novel TRFs in obstructive nephropathy, including TEAD1, PHB, RFX1, and ADNP. TEAD1 is required for the activation of YAP, the key component of the Hippo pathway . Increasing evidence underscores the critical involvement of the Hippo pathway in renal fibrosis . Upon activation, YAP translocases into the nucleus and interacts with TEAD and Smad1/Smad4 to faciliate epithelial-mesenchymal transition (EMT), a key process in renal fibrosis development . Our findings indicate a significant upregulation of TEAD1 in the obstructed kidneys. However, further functional investigations are required to elucidate the roles and mechanisms of TEAD1 in obstructive nephropathy. There are two isoforms of PHB, PHB1 and PHB2, which belong to the prohibitin domain family . PHB1 is located in the inner mitochondrial membrane and interacts with PHB2 to stabilize mitochondrial respiratory enzymes and maintain mitochondrial integrity . Moreover, PHB is required for mitophagy, a cellular process required for removing damaged mitochondria . Therefore, expression changes in PHB are always associated with detrimental consequences caused by mitochondrial dysfunction. Of note, mitochondrial dysfunction is one of the hallmarks of kidney disease . Our study revealed that PHB was significantly downregulated in both patients with obstructive nephropathy and UUO mice. This is consistent with a previous study showing that PHB expression is significantly reduced in UUO rats . Furthermore, PHB can act as a transcription regulator by suppressing the expression of E2F1, a gene associated with liver and lung fibrosis . These findings suggest that decreased PHB levels may contribute to obstructive nephropathy by damaging mitochondrial function and activating profibrotic genes. RFX1 is an evolutionarily conserved TF that governs diverse cellular processes such as cell cycle, cell proliferation, and apoptosis . Deficiency in RFX1 leads to enhanced differentiation of naïve CD4 + T cells into Th17 cells, thereby exacerbating autoimmune diseases . Recent research suggests that RFX1 promotes the polarization of M1 macrophages through DNA demethylation in the context of autoimmune inflammation . However, the impact of RFX1 on obstructive nephropathy and renal fibrosis remains to be fully elucidated. ADNP was first described as a neural protective protein and has been implicated in various neural developmental disorders and cancers . Mutations in ADNP underlie the complex neurological and developmental disorder known as ADNP syndrome . Deletion of ADNP in mice leads to compromised brain formation and embryonic lethality  and it promotes neural differentiation by modulating Wnt/β-catenin signaling . Moreover, it has been reported that ADNP is indispensable for immune reactions to allergen in Th2 response. ADNP contributes to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1, resulting in type 2 cytokine expression . However, there is little characterization of ADNP in the context of kidney diseases, which needs further investigation. The EMT of tubular cells is orchestrated by TFs including ZEB2 . Knockout of ZEB2 ameliorates progression of renal fibrosis in a mouse model of ischemia-reperfusion injury. Moreover, ZEB2 is responsible for maintaining the cell fate of stromal progenitors during kidney development. Loss of ZEB2 leads to differentiation of stromal progenitors into myofibroblasts and kidney fibrosis. One of the limitations of the study is the small size of the patient samples, partially because it is relatively difficult to recruit a large number of pediatric patients with obstructive nephropathy. This may affect the validity of our findings. Future studies should focus on expanding the sample size to strengthen the conclusions. Moreover, it is of great significance to investigate the clinical relevance of the expression of TRFs in patients, which may provide evidence as to whether the selected TRFs can be considered as biomarkers or therapeutic targets in obstructive nephropathy. 

Conclusion Collectively, our study uncovered key TRFs and provided additional molecular insights into obstructive nephropathy. 

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