SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in kidney epithelial cells

Acute kidney injury (AKI) is a common clinical condition associated with diverse etiologies and abrupt loss of renal function. In patients with sepsis, rhabdomyolysis, cancer, as well as cardiovascular disorders, the underlying disease or associated therapeutic interventions can cause hypoxic, cytotoxic, and inflammatory insults to renal tubular epithelial cells (RTECs) resulting in the onset of AKI. To uncover stress-responsive disease-modifying genes, here we have carried out renal transcriptome profiling in three distinct murine models of AKI. We find that Vgf nerve growth factor inducible gene upregulation is a common transcriptional stress response in RTECs to ischemia, cisplatin, and rhabdomyolysis-associated renal injury. The Vgf gene encodes a secretory peptide precursor protein that has critical neuro-endocrine functions; however, its role in the kidneys remains unknown. Our functional studies show that RTEC-specific Vgf gene ablation exacerbates ischemia, cisplatin, and rhabdomyolysis-associated AKI in vivo and cisplatin-induced RTEC cell death in vitro. Importantly, addback experiments showed that aggravation of cisplatin-induced renal injury caused by Vgf gene ablation is partly reversed by TLQP-21, a Vgf-derived peptide. Finally, in vitro and in vivo mechanistic studies showed that injury-induced Vgf upregulation in RTECs is driven by the transcriptional regulator Sox9. These findings reveal a crucial downstream target of the Sox9-directed transcriptional program and identify Vgf as a stress-responsive protective gene in kidney epithelial cells.


Introduction
Acute kidney injury (AKI) is a heterogeneous clinical syndrome that is associated with adverse short and long-term sequelae (1). AKI usually occurs in the setting of other diseases, such as sepsis (2), rhabdomyolysis (3), cardiovascular (4) and oncological diseases (5), where the underlying disease and or associated therapy cause abrupt loss of renal function. As a result, the pathophysiology of AKI is generally complex due to the existence of multiple etiologies such as the presence of sepsis, ischemia, and therapy-induced nephrotoxicity (6). AKI-associated mortality depends on the severity and can be significantly high in critically ill patients (7). Importantly, patients who survive an episode of AKI are at increased risk for major adverse cardiovascular events, as well as for progression to chronic kidney disease (8).
Disorders such as sepsis, cancer, rhabdomyolysis as well as therapeutic interventions such as cardiac surgery and chemotherapy are associated with inflammatory, toxic, and hypoxic insults to renal tubular epithelial cells (RTECs). The resulting RTEC dysfunction and cell death are the hallmarks of AKI (9). RTEC dysfunction and renal impairment clinically manifest as systemic electrolyte and fluid imbalances along with accumulation of metabolic waste, which can trigger multi-organ failure (7). The pathogenesis of AKI is multifaceted due to the involvement of various intracellular pathways (6,(10)(11)(12) in RTEC dysfunction and cell death (9) as well as the contribution of vascular (13)(14)(15) and immune cells (16,17) in renal impairment.
Both the etiology and pathophysiology of AKI is complex. To identify common stress responsive genes, we have carried out genome-wide transcriptome analysis in three mouse models of AKI. Our studies identify the nerve growth factor-inducible gene, Vgf (nonacronymic; unrelated to VEGF) as a stress-responsive gene that is upregulated during ischemic, nephrotoxic, and rhabdomyolysis-associated kidney injury. Vgf was originally identified as a nerve growth factor (Ngf) -inducible gene in a neuroendocrine cell line and is expressed in specific neurons and endocrine cells in the brain and periphery (18). Vgf gene encodes a precursor polypeptide, which is proteolytically cleaved to generate several bioactive peptides, the best studied of which are TLQP-2, TLQP-62, and AQEE-30 (19). In the central nervous system, the secreted Vgf-derived peptides regulate neuronal activity, survival and progenitor proliferation (20)(21)(22). Furthermore, germline Vgf knockout mice have significantly reduced body weight, increased energy expenditure, and are resistant to diet-induced obesity, indicating that Vgf-derived peptides are critical regulators of energy homeostasis (20,23).
Interestingly, the role of Vgf in kidney physiology and pathophysiology remains unknown.
Here, using transcriptome profiling and RTEC-specific gene ablation studies, we report that Vgf is a stress inducible gene that plays a protective role during the development of AKI.

Mouse models of acute kidney injury.
To identify common stress-induced cellular transcriptome changes linked to the pathogenesis of acute kidney injury, we sought to perform bulk RNA sequencing of renal tissues from distinct murine models of AKI. To this end, we utilized the well-characterized mouse models of ischemia-reperfusion injury (IRI), drug-induced nephrotoxicity (cisplatin), and rhabdomyolysismediated kidney injury. IRI-associated AKI results from a generalized or localized impairment of oxygen and nutrient delivery to the kidneys (6). Cisplatin nephrotoxicity results from specific drug uptake (24) and direct toxicity to tubular epithelial cells. Rhabdomyolysis-associated AKI results from skeletal muscle injury and the subsequent myoglobin release into the systemic circulation, which causes renal dysfunction (3).
In these mouse models, bilateral ischemic surgery, intra-peritoneal cisplatin injection, and intramuscular glycerol injection trigger AKI within 24-72 hours. The development and progression of AKI was determined by accumulation of nitrogenous waste (blood urea nitrogen and serum creatinine) and histological analysis of tissue damage (H&E staining and renal damage score).
During ischemia (Fig. 1A-C) and rhabdomyolysis-associated ( Fig. 1D-F) kidney injury, onset of renal impairment occurs 24 hours post-surgery or -injection, while in the cisplatin-associated kidney injury models, renal impairment is observed 72 hours post-injection ( Fig. 1G-I).

Transcriptome profiling of AKI-associated differentially expressed genes.
Due to temporal differences in the onset of kidney injury, we chose to compare gene expression signals at time-points where the extent of kidney injury is similar in the three groups.
To this end, we isolated renal tissues from control (mock and vehicle, n=8), IRI (24 hours, n=4), rhabdomyolysis (24 hours, n=4), and cisplatin (72 hours, n=4) treated mice and then performed RNA sequencing (4-8 biological replicates). Principal component analysis (PCA) showed that the biological replicates clustered together across groups, signifying a high degree of similarity ( Fig.   2A). Hierarchical clustering (Fig. 2B) revealed both divergent and convergent gene signatures between control and the three AKI groups. In the three AKI conditions (FDR<0.05 and fold change2), we identified a common set of 1501 differentially (771 genes were downregulated and 709 genes were upregulated) expressed genes (Fig. 2C). In Supplementary Files 1 and 2, we have provided the complete list and normalized expression levels of all detected and differentially expressed genes. Enrichment of genes related to glutathione, nicotinamide, and fatty acid metabolism was observed upon gene ontology (GO) and KEGG pathway analysis (Suppl. File 3). These pathways have been recently probed for their role in renal dysfunction (25,26).
To identify previously unexplored genes, we initially focused our attention on the top differentially expressed genes (DEGs) in the AKI conditions. The top common upregulated genes in AKI mice were Fosl1, Krt20, Mmp10, 1700001F09Rik, Sprr2f, Lcn2, Sprr2g, Havcr1, Nptx2, and Vgf (Fig. 2D). On the other hand, the top common downregulated genes were Ccdc169, Pvalb, Egf, Trdn, Inmt, Col6a6, Wfdc16, Pde6a, Slc7a13, and Gm6300. The molecular functions of some of these DEGs including the widely studied injury biomarkers Lcn2 and Havcr1 has been explored previously (27,28). We found that similar to Havcr1 and Lcn2 upregulation, 200-2000 fold induction of Vgf gene expression is observed in the three AKI conditions (Fig. 2E-G). These results indicated that Vgf is transcriptionally upregulated in response to wide-ranging forms of renal injury.

Stress-induced Vgf upregulation in RTECs during AKI.
Vgf (nonacronymic) was first identified as a nerve growth factor (Ngf) induced gene in a neuroendocrine cell line (18). The Vgf gene encodes a highly conserved precursor polypeptide of 615 (human) and 617 (rat and mice) amino acids. The precursor polypeptide contains several cleavage sites and protease action at these locations results in the generation of a number of peptides, which exert pleotropic biological activities (19), including promotion of pro-survival signaling in an autocrine and paracrine fashion (29,30). While Vgf plays critical roles in neuronal and endocrine tissues, its role in the kidneys remains unknown.
We initially sought to validate the RNAseq data and investigate the cellular origin of Vgf mRNA upregulation. To do so, we utilized a reporter mouse (31) that express membrane-localized green fluorescent protein (GFP) in the tubular epithelial cells (Fig. 3A). These mice were challenged with ischemia, cisplatin, and rhabdomyolysis (Suppl. Fig. 1) followed by isolation of GFP-positive cells from the kidneys and subsequent examination of Vgf gene expression. We found that Vgf mRNA upregulation occurs in RTECs (GFP-positive cells) early during the development of AKI (Fig. 3B-D). A similar increase in Vgf expression was observed when human and murine RTEC cell lines (HK-2 and BUMPT cells) as well as primary murine RTECs were challenged with cisplatin under in vitro conditions (Fig. 3E). Based on these results we concluded that Vgf upregulation in RTECs is a common response to stress in vitro and in vivo.

Vgf gene deletion in renal tubular epithelial cells aggravates AKI.
To probe the functional role of Vgf in the pathogenesis of AKI, we examined the effect of Vgf gene ablation on the severity of AKI. To this end, we generated Vgf conditional knockout (Vgf PT-/-) mice by crossing the Vgf floxed mice with the Ggt1-Cre mice. In Ggt1-Cre mice, Cre recombinase is expressed in RTECs 7-10 days after birth and as a result Cre-mediated gene ablation is unlikely to influence normal renal development (32). Vgf PT-/mice were indistinguishable from wild-type littermates and normal renal function was not evidently influenced by Vgf deficiency in RTECs (Suppl. Fig.2). However, when the control and Vgf PT-/littermates were challenged with ischemia, cisplatin-and rhabdomyolysis, we observed that Vgf gene deletion markedly exacerbates renal injury ( Fig. 4A-I). Immunoblot analysis of renal tissues confirmed Vgf ablation in the conditional knockout mice (Fig. 4J). To further corroborate these results, we cultured primary RTECs from the wild type and Vgf PT-/mice, challenged them with cisplatin and then carried out viability assays. Cell survival and caspase assays (Fig 4K and Suppl. Fig. 3) showed that Vgf gene deletion results in increased cisplatin-induced cell death.
Thus, we propose that Vgf plays a cytoprotective role in RTECs under stress conditions associated with AKI.
We found that TLQP-21 levels were increased in the renal tissues of wild type mice challenged with ischemia, cisplatin and rhabdomyolysis-associated AKI (Fig. 5A). Additionally, wild type primary murine RTECs secreted TLQP-21 in the medium when challenged with cisplatin ( Fig. 5B). Interestingly, tissue distribution studies in mice have shown that intravenously injected TLQP-21 markedly accumulates in the kidney (37). This prompted us to carry out in vivo 'addback' experiments to determine if the TLQP-21 administration can reverse the aggravated renal impairment phenotype observed in the Vgf PT-/mice. To this end, we administered a scrambled peptide (Scr) or TLQP-21 to control and Vgf PT-/mice, 24 and 48 hours after challenging them with cisplatin ( Fig. 5C). Remarkably, we found that TLQP-21 administration mitigates cisplatinassociated AKI in the Vgf PT-/mice ( Fig. 5D-F), indicating that loss of TLQP-21 might be partly responsible for the increased sensitivity to renal injury. Complementary studies in primary murine RTECs showed that TLQP-21 treatment can protect Vgf deficient RTECs from cisplatin-induced cell death ( Fig. 5G-I). These results indicate that the loss of TLQP-21 is partly responsible for the aggravated renal impairment phenotype seen in the Vgf deficient mice.

VGF regulation by Sox9 in the early acute phase of renal injury.
Next, we sought to identify the transcriptional mechanisms underlying stress-induced Vgf upregulation in RTECs. While exploring the transcription factor binding sites in the Vgf promoter, we noticed the presence of a putative Sox9 binding site (Fig. 6A). Sox9 is upregulated in RTECs in response to injury and is a critical transcriptional regulator of epithelial cell fate during AKI (31,38-41). To test the hypothesis that Sox9 is involved in Vgf upregulation during AKI, we performed promoter-driven luciferase-based reporter assays (Fig. 6B) in HEK293 cells, which have low endogenous Sox9 expression. To this end, we used HEK293 cells with stable vector transfection (low Sox9) and Sox9 overexpression (high Sox9) for Vgf promoter driven luciferase reporter assays as described in our recent study (31). We found that Sox9 increases Vgf promoter activity (Fig. 6C). Importantly, site-directed mutagenesis of Sox9 binding site within the Vgf promoter suppressed promoter activity. To substantiate these findings, we performed chromatin immunoprecipitation analysis, which confirmed Sox9 binding at the Vgf promoter in vivo (Fig. 6D).
We next asked if RTEC-specific Sox9 deficiency influences stress responsive Vgf upregulation.
Our recent study (31) revealed a protective role of Sox9 during ischemic and nephrotoxic AKI.
We also found that RTEC-specific Sox9 gene deletion aggravates rhabdomyolysis-associated AKI (Suppl. Fig. 4). When we carried out gene expression analysis of renal tissues from control and Sox9 PT-/mice, we found that stress-induced Vgf upregulation is Sox9 dependent (Fig. 6E-G). Taken together, these data indicate that Sox9 controls Vgf gene transcription in RTECs during AKI.

Discussion
Here we have mapped the transcriptome changes accompanying the development of ischemic, nephrotoxic, and rhabdomyolysis associated acute kidney injury. We find that these diverse stress conditions trigger transcriptional upregulation of Vgf gene in renal tubular epithelial cells. Importantly, we provide functional evidence that Sox9-mediated Vgf upregulation protects RTECs from cell death and dysfunction linked with AKI. These findings identify Vgf as an essential stress-responsive and protective gene in kidney epithelial cells.
Spatial and temporal changes in gene expression in response to ischemia reperfusion associated kidney injury has been comprehensively explored (39,42). Since multiple etiologies can contribute to the development of AKI, in the current study, we aimed to identify common transcriptional changes that occur in the acute phase of three distinct murine models of AKI.
Consistent with previous studies (39,42), we observed that genes such as Sprr2f and Krt20 and well-characterized renal injury biomarkers such as Lcn2 and Havcr1 were significantly upregulated during ischemic, nephrotoxic, and rhabdomyolysis associated acute kidney injury.
Furthermore, pathway enrichment analysis revealed that genes linked to cell death and survival, wound healing, small molecule and fatty acid metabolism, and molecular transport were differentially expressed during AKI.
Vgf was among the top upregulated genes in the renal tissues of mice undergoing ischemic, nephrotoxic, and rhabdomyolysis-associated AKI. The Vgf gene is known to be expressed in a subset of cells in the central and peripheral nervous system as well as endocrine cells in the adrenal gland, gastrointestinal tract, and pancreas (18). Within the nervous system, Vgf expression is rapidly induced by neurotrophins, synaptic activity, nerve injury, inflammation, and other stimuli (21). Consistent with its expression in the central and peripheral nervous system, Vgf has been implicated in the regulation of neuroplasticity associated with learning, memory, depression, and chronic pain (21,43,44). Additionally, Vgf plays a critical role in energy homeostasis and metabolism (23,34,45). Mice with germline Vgf deletion are lean, hypermetabolic, and resistant to diet-, lesion-, and genetically induced obesity and diabetes (20).
Interestingly, the role of Vgf in renal physiology and pathology has remained unexplored.
We found that Vgf expression is low in the normal adult kidneys. Moreover, renal epithelialcell-specific Vgf deficiency did not have any deleterious effect on the normal kidney structure or function and did not alter the overall body weight. Importantly, Vgf expression increased by more than 500 fold in RTECs during ischemic, nephrotoxic, and rhabdomyolysis associated AKI. A previous study (39) also described Vgf gene induction during IRI, however, its functional role in the pathogenesis remained unknown. We find that RTEC-specific Vgf deletion markedly aggravated renal impairment linked with ischemic, nephrotoxic, and rhabdomyolysis-associated AKI. Notably, stress-responsive Vgf upregulation was recapitulated in human and murine cell culture models of cisplatin associated cellular injury. Functional studies also showed that Vgf deficiency sensitizes RTECs to cisplatin-mediated cell death. These studies reveal that Vgf protects RTECs from cell death and dysfunction.
The neuro-endocrine functions attributed to the Vgf gene are dependent on the posttranslational processing of Vgf polypeptide into various bioactive peptides, such as TLQP-21, TLQP-62, AQEE-30, LQEQ-19, and NERP2. Among these, TLQP-21 is known to control regulatory processes involved in energy expenditure, lipolysis, glucose-stimulated insulin secretion, gastric acid secretion and pain (34,44,46). We found that along with Vgf mRNA, TLQP-21 levels also increase in the renal tissues during AKI. Strikingly, systemic TLQP-21 administration partly reversed the injury-induced aggravation of renal impairment observed in the RTEC-specific Vgf-deficient mice. These findings suggest that Vgf-derived TLQP-21 plays a protective role during AKI. A critical feature of Vgf peptides is their cell type specific diversity in tissues studied so far and their selective modulation in response to organ or cell type relevant stimuli (19). Future studies are thus necessary to comprehensively profile Vgf derived peptides in renal tissues under normal and stress conditions. In the current study, we find that Vgf deficiency exacerbates AKI, a phenotype that is similar to the RTEC-specific Sox9 deficient mice (31). However, Sox9 is a crucial transcriptional regulator of not only the early pathogenic phase (31), but also the later recovery phase of AKI (38,40). Sox9 expressing RTECs are involved in the repair and regeneration processes post-AKI (38,40). Based on our findings that Vgf is a downstream Sox9 target gene, it will be interesting to examine if Vgf contributes to repair and regeneration. It would also be interesting to examine if systemic TLQP-21 administration can accelerate the recovery and repair processes post-AKI.
Collectively, our study has revealed Vgf as an essential Sox9 target gene that protects RTECs under stress conditions associated with acute kidney injury. Primary murine tubular cell culture. Murine renal cortical tissues were minced and digested with 0.75 mg/ml collagenase IV (Thermo-Fisher Scientific). Cells were centrifuged at 2000 g for 10 min in DMEM/F-12 medium with 32% Percoll (Amersham). After two washes with serum-free media, the cells were plated in collagen-coated dishes and cultured in DMEM/F-12 medium supplemented with 5 μg/ml transferrin, 5 μg/ml insulin, 0.05 μM hydrocortisone, and 50 μM vitamin C (Sigma-Aldrich). Fresh media was supplemented every alternate day, and after 5-7 days of growth, the isolated proximal tubular cells were trypsinized and re-plated at 1 × 10 5 cells per well in 24-well plates. To induce cell death, primary RTECs were incubated with 50 μM cisplatin (Sigma-Aldrich) in fresh culture medium for 24 h, followed by viability and caspase assays.

Cell viability and caspase assays.
Trypan blue staining and MTT assay was used to determine cellular viability as reported in our previous study (31). BUMPT, HK-2 cells or RTECs were seeded in 6-well, 24-well, or 96-well plates, followed by cisplatin treatment for 24-48 h. At the end of the incubation period, cells from 6-well plates were harvested, followed by trypan blue staining and manual cell counting with a hemocytometer and/or by using Countess Automated Cell Counter (Thermo Fisher); translucent cells were considered as viable and blue-stained cells were counted as dead. Cellular viability was calculated by dividing the number of viable cells by the total cell number and each sample was done in triplicate. For MTT assays, after cisplatin treatment, 10 μL of MTT reagent (5 mg/mL MTT in PBS) was added to each well, and plates were incubated at 37 °C with 5% CO2 for 4 h, followed by addition of 100 μl of acidified isopropanol (Sigma-Aldrich) and measurement of absorbance at 590 nm. The half-maximal inhibitory concentration (IC50) was calculated by nonlinear regression analysis using GraphPad Prism.
For caspase assays (58), RTECs were lysed in a buffer containing 1% Triton X-100, and 10 μg of protein from cell lysates was added to an enzymatic assay buffer containing 50 μM DEVD-AFC for 60 min at 37 °C. Fluorescence at excitation 360 nm/emission 535 nm was measured, and free AFC was used to plot a standard curve. Subsequently, the standard curve was used to convert the fluorescence reading from the enzymatic reaction into the nM AFC liberated per mg protein per hour as a measure of caspase activity. PCR products were analyzed by electrophoresis using 1.5% agarose gels.

Animal models of acute Kidney injury.
We carried out all the studies presented here in agematched male mice at 8-12 weeks of age using methods described in our recent studies (31,59,60). In all the studies with conditional Vgf and Sox9 knockout mice, we used male littermates from mice bred in-house. Experiments were carried out in a blinded fashion where the investigators assessing, measuring or quantifying experimental outcomes were blinded to the genotype or treatment of the mice. For ischemia-reperfusion experiments, mice were anesthetized by isoflurane and placed on a surgical platform where the body temperature was monitored throughout the procedure. The skin was disinfected, kidneys were exposed and bilateral renal pedicles were clamped for 30 minutes. Consequently, the clamps were removed to initiate reperfusion followed by suturing to close the muscle and skin around the incision. To compensate for the fluid loss, 0.5 ml warm sterile saline was administered via intraperitoneal injection. Blood was collected on day 1 via cardiac puncture after carbon dioxide asphyxiation.
Renal tissues were collected and processed for RNA-seq, qPCR, and histological analysis as described previously. For nephrotoxicity experiments, cisplatin (30 mg/kg) was administered by i.p. injection as described previously. After cisplatin injection, blood was collected on days 0-3 by submandibular vein bleed or on day 3 via cardiac puncture after carbon dioxide asphyxiation.
Renal tissues were collected and processed for RNA-seq, qPCR, and histological analysis. To induce rhabdomyolysis, 8-12 weeks old male mice were injected with 7.5 ml/kg 50% glycerol intramuscularly to the two hind-legs or injected with saline as a control, followed by tissue collection at 24 hours and RNA-seq, qPCR, and histological analysis

Data Availability
The RNA-Seq data have been deposited in the Gene Expression Omnibus (GSE153625). And the rest of data are contained within the manuscript.

29.
Takeuchi, H., Inagaki, S., Morozumi, W., Nakano, Y., Inoue, Y., Kuse, Y., Mizoguchi, T.,   The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Dunnett's (E-G) was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.  as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey's multiple-comparison test (A-I) was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. was indicated as statistically significant. One-way ANOVA followed by Dunnett's was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.     In the three AKI groups, a common set of 1501 genes were found to differentially expressed as compared to control group. (E-G) Gene expression analysis of Havcr1, Lcn2, and Vgf genes showed injury induced upregulation. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Dunnett's (E-G) was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.   RTEC-specific Vgf knockout mice were generated by crossing Ggt1-Cre mice with Vgf-floxed mice. 8-12 weeks old littermate control and Vgf conditional knockout male mice (indicated by Vgf PT−/-) were then challenged with bilateral renal ischemia (30 minutes), cisplatin (30 mg/kg, single intraperitoneal injection) treatment, or glycerol-induce rhabdomyolysis (7.5 ml/kg 50% glycerol in the hind-leg muscles) followed by examination of renal structure and function. Blood urea nitrogen, serum creatinine, and renal histological analysis (H&E) showed that tubular epithelial-specific VGF deficiency results in aggravated renal impairment in the IRI (A-C), Cisplatin (D-F), and rhabdomyolysis (G-I) associated mouse models of AKI. Data presented (A-I) are cumulative of two out of four independent experiments (n = 5) that showed similar results (J) Immunoblot analysis of Vgf protein levels was performed using renal cortical tissues from the control and Vgf deficient mice followed by densitometric analysis (normalized to β-actin levels) using ImageJ. The graph depicts relative Vgf protein levels and the upper panel is a representative western blots showing successful gene knockout in the renal tissues. Blots are representative of two independent experiments. (K) Primary renal tubular cells isolated from mice with indicated genotypes were treated with 50 µM cisplatin, followed by cell viability assessment using trypan blue staining. Vgf deficiency increased the sensitivity to cisplatin-induced cell death in vitro. Data are presented as individual data points (n = 5 biologically independent samples), from one out of three independent experiments, all producing similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey's multiple-comparison test (A-I) was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.  In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Dunnett's was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.  and Sox9 (+) expressing cells were transiently co-transfected with reporter renila luciferase constructs (empty, Vgf or Vgf mut ) and reference cypridina luciferase (normalizing control), followed by measurement of luciferase activity at 24 hours. The normalized luciferase activity of mock (-) group was then compared with the Sox9 (+) group. The results show that the Sox9 can activate transcription from the Vgf promoter. In the Vgf mut construct, the Sox9 binding site was mutated from ATTGTT to AACAAT. (D) Sox9 Chromatin immunoprecipitations (ChIP) were carried out from the renal tissues of control and Vgf PT-/-mice undergoing Ischemia, cisplatin nephrotoxicity, and rhabdomyolysis associated AKI. Subsequent qPCR analysis using primers specific for the murine Vgf promoter region showed that Sox9 can bind to Vgf promoter in vivo. (E-G) q-PCR based gene expression analysis was carried out in the renal tissues from littermate control and Sox9 PT-/-mice under at indicated time-points after induction of kidney injury. The injury induced Vgf mRNA upregulation was suppressed in the Sox9 PT-/-mice. Data (C-G) are presented as individual data points (n = 4-5 biologically independent samples), from one out of three independent experiments, all producing similar results. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed Tukey's (C) or Dunnett's (D-G) multiple-comparison test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.  Figure S1 A B Supplementary Information Figure S1. Characterization of the RTEC-specific EGFP expressing reporter mice. Ggt1-Cre mice were crossed with ROSAmT/mG mice to generate transgenic mice that express membrane-localized EGFP in RTECs. 8-12 weeks male mice were then challenged with bilateral renal ischemia (30 minutes), cisplatin (30 mg/kg, single intraperitoneal injection) treatment, or glycerol-induced rhabdomyolysis (7.5 ml/kg 50% glycerol in the hind-leg muscles) followed by examination of renal structure and function. The mock/vehicle groups represent respective control groups (with no injury). (A-B) Representative graphs depicting injury-induced increase in blood urea nitrogen and serum creatinine levels (IRI and Rhabdo at 24 hours and Cisplatin at 72 hours). The graphs (n = 5) are representative of three independent experiments. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. Student's t test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Figure S2 Supplementary Information Figure S2. Characterization of the RTEC-specific Vgf deficient mice. Ggt1-Cre mice were crossed with Vgf-floxed mice to generate RTEC-specific Vgf deficient mice. (A) Body weight measurements showed no differences between the control and Vgf deficient mice up to 12 weeks of age. (B-C) Renal function (BUN and Creatinine) was examined in littermates with indicated genotypes at 12 weeks of age under baseline conditions. These results show that RTEC-specific Vgf knockout does not affect kidney function under normal conditions. Data are presented as individual data points (n = 10), from a single long-term experiment. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. Student's t test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ns= not significant. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Dunnett's was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 ✱✱✱ ✱✱✱ A B Supplementary Information Figure S4 Supplementary Information Figure S4. Sox9 plays a protective role during Rhabdomyolysis-associated AKI. To generate mice with renal tubule-specific Sox9 knockout, Ggt1-Cre mice were crossed with Sox9-floxed mice. Control and Sox9 PT-/-male litermates (8-12 weeks age) were challenged with glycerol-induced rhabdomyolysis (7.5 ml/kg 50% glycerol in the hind-leg muscles) followed by examination of renal impairment. (A-B) Blood urea nitrogen and serum creatinine measurements showed that RTEC-specific Sox9 deficiency exacerbates rhabdomyolysis-associated AKI. Data are presented as individual data points (n = 4), from one out of 3-4 independent experiments. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e. and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey's multiple-comparison test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001.