2.1 BLI exerts nephroprotective effects in mouse AKI–CKD transition models

Folic acid (FA)-induced and unilateral ureteral obstruction (UUO) kidney injury mouse models are widely used in the study of AKI–CKD transition or CKD.²¹ We first investigated the effect of BLI on the FA-induced model (Figure 1A). FA significantly increased the mouse kidney indices,²² whereas these changes were reversed by BLI (Figure 1B). In clinical settings, the most commonly utilized parameters for detecting kidney injury include blood urea nitrogen (BUN), creatinine (Cr), and microalbuminuria (MAU) levels.²³ We found that FA challenge significantly increased the serum BUN, Cr, urinary Cr, and MAU levels in mice, while the serum and urinary Cr and urinary MAU levels in mice treated with BLI did not increase (Figure 1C,D). We subsequently investigated the mRNA expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), two known tubule injury biomarkers for early kidney injury.²⁴ Treatment with BLI significantly attenuated the increase in KIM-1 and NGAL levels induced by FA (Figure 1E). Moreover, hematoxylin and eosin (H&E) staining of the kidneys also demonstrated the protective effects of BLI on kidney damage. Mice in the FA-induced model exhibited severe tubular dilatation, obvious tubular epithelial cell edema, and significant cast formation, which were not observed in the BLI treatment group (Figure 1F,G).

To further confirm these findings, we conducted studies using the UUO model (Figure 1H). Consistently, BLI had protective effects against functional kidney injury (Figure 1I–L) and structural organ damage (Figure 1M,N) in mice. Taken together, these data demonstrated that BLI exerts nephroprotective effects on AKI–CKD transition models. As the cornerstone of kidney-preserving pharmacologic therapy in the clinic is RAS blockade, we used telmisartan as a positive control.³ Notably, in terms of multiple kidney function indicators, the nephroprotective effect of BLI was no worse than or even better than that of the positive control telmisartan (Tel). Especially for the reduction in serum BUN, the effect of BLI was significantly greater than that of telmisartan in both models; in the FA model, the advantages of BLI over telmisartan were also reflected in the improvements in the kidney index and MAU.

2.2 BLI inhibits the inflammatory response in mouse AKI–CKD transition models

A dramatic increase in inflammatory cytokine levels is one of the major clinical features of both AKI and CKD, and low-grade systemic inflammation is also a substantial contributor to the AKI–CKD transition.²⁵ We subsequently examined the serum and mRNA expression levels of proinflammatory cytokines in kidney tissues. The levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and C-C motif chemokine ligand 2 (CCL2) were significantly increased in the FA- or UUO-treated mice; however, these increases were not observed in the BLI-treated mice (Figure 2A,D). Consistently, BLI significantly inhibited the upregulation of IL-1β, IL-6, TGF-β, TNF-α, and CCL2 in FA or UUO-induced kidney tissue, both at the transcriptional and translational levels (Figure 2G and Figure S4). Macrophages have been shown to account for the majority of inflammatory infiltrates in various kidney diseases, including AKI and CKD.²⁶ We next investigated the kidney infiltration of macrophages by immunohistochemical staining for CD68 and F4/80. Compared with those in control or BLI-treated mice, the number of CD68- or F4/80-positive cells significantly increased in mice after FA treatment or UUO surgery (Figure 2B,C,E,F), indicating that BLI treatment markedly decreased the kidney infiltration of macrophages. These results suggest that BLI substantially suppressed the inflammatory response in AKI–CKD transition models. Notably, BLI demonstrated significantly stronger anti-inflammatory effects than telmisartan in both models.

2.3 BLI blocks kidney fibrosis in mouse AKI–CKD transition models

Without treatment, AKI will lead to the continued deterioration of kidney function, and the kidney is in a long-term proinflammatory, profibrotic state and gradually becomes a fibrotic kidney,^{27, 28} which is characterized primarily by the accumulation of extracellular matrix (ECM) components. To evaluate the effectiveness of BLI on kidney fibrosis, the levels of ECM and fibrosis markers were examined. The results from Masson's trichrome staining of the kidneys demonstrated that BLI mitigated ECM accumulation in both AKI–CKD transition models (Figure 3A,C). Collagen I (Col-I), collagen III (Col-III), collagen IV (Col-IV), fibronectin (Fn), and α-smooth muscle actin (α-SMA) are the main components of the ECM. Then, immunohistochemical staining and immunoblotting analysis were employed to assess the expression of this protein. The results showed that the expressions of profibrotic proteins such as Col-I, Col-III, Col-IV, Fn, and α-SMA were significantly increased in mice after FA challenge or UUO surgery, while these changes were not observed in the BLI-treated mice (Figure 3B,D). These results suggested that BLI blocks the AKI–CKD transition by inhibiting kidney fibrosis.

2.4 BLI inhibits kidney ferroptosis in both mouse and cell models

The above results illustrate the nephroprotective effect of BLI on the whole body or at the kidney level. To explore the mechanism of action of BLI, we conducted RNA sequencing analysis using the kidney tissues from the FA-induced mouse model as indicated in Figure 1A. The results showed that genes altered in the BLI-treated group were enriched in pathways relevant to ferroptosis, such as fatty acid metabolism, glutathione (GSH) metabolism, and ferroptosis (Figure 4A). Ferroptosis is a nonapoptotic form of cell death that is driven by substantial iron accumulation and lipid peroxidation.²⁹ According to previous reports, ferroptosis is an important driver of FA- or UUO-induced kidney fibrosis.^{30, 31} Therefore, we investigated the mRNA expression of critical genes involved in the signaling pathways that regulate ferroptosis. As depicted in Figure 4B, we observed changes in the expression of multiple genes associated with ferroptosis in the kidneys of the FA-treated mice compared to those of the control group and found that the degree of changes decreased after BLI treatment. Lipid peroxidation, indicated by the accumulation of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as well as GSH depletion and iron accumulation, is the main biochemical characteristic of ferroptosis.³² We found that the serum MDA concentration increased and the GSH concentration decreased in both AKI–CKD transition models compared with those in normal mice, but the levels were almost unchanged in BLI-treated mice (Figure 4C). Immunostaining of kidney specimens revealed that 4-HNE, the levels of iron, and the levels of proteins involved in iron storage, such as ferritin heavy chain-1 (FTH1) and transferrin receptor-1 (TfR1),^{33, 34} also increased significantly in both models, but these changes were not observed in the BLI treatment group (Figure 4D and Figure S5). In particular, the level of 4-HNE in the BLI-treated group was close to that in the normal group and was significantly greater than that in the positive control group. As ferroptosis coincided with morphologic changes,³⁵ transmission electron microscopy studies revealed that BLI significantly protect FA-induced mitochondrial cristae disappearance and outer membrane rupture in mouse kidney (Figure 4E). Moreover, the immunoblotting results also showed that the expressions of proteins negatively associated with ferroptosis, such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and ferroptosis suppressor protein 1 (FSP1), significantly decreased in both models but were basically similar to that in the normal control group in the BLI-treated group. Consistent with the immunostaining results, the expressions of proteins positively associated with ferroptosis (FTH1, FTL, and TfR1) were significantly increased in both models, but BLI treatment blocked this process (Figure 4F). These results indicate that BLI blocks the AKI–CKD transition by inhibiting kidney ferroptosis in mice.

We also found that, similar to ferrostatin-1 (Fer-1), a selective inhibitor of ferroptosis, BLI abolished ferroptosis induced by erastin (Era)³⁶ in both NRK-52E and HK-2 cells (Figure 5A). Then, we used RhoNox-1, a fluorescent probe specific for divalent iron ions, to detect changes in the concentration of cellular labile iron. Era treatment significantly increased the fluorescence signals, while BLI or Fer-1 treatment did not, consistent with the results of the in vivo experiment (Figure 5B and Figure S6A). Free iron can interact with reactive oxygen species (ROS) to form lipid radicals that react with oxygen to induce lipid peroxidation. We next investigated intracellular ROS levels in cells, and the results revealed that Era treatment induced ROS generation, but BLI or Fer-1 treatment did not (Figure 5B and Figure S6A,B). In addition, Era induced significant GSH downregulation and MDA upregulation in both NRK-52E and HK-2 cells, but BLI or Fer-1 treatment blocked these changes (Figure 5C and Figure S6C). Subsequently, we examined biomarkers of ferroptosis. Both the immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR) results showed that BLI or Fer-1 treatment significantly reversed the changes in the expression of GPX4, SLC7A11, FSP1, FTH1, FTL, and TfR1 induced by Era (Figure 5D,E). Ferroptosis and ROS production are closely related, and mitochondria are important organelles for ROS production.³⁷ We found that exposure of HK2 and NRK-52E cells to Era disrupted the mitochondrial membrane potential (MMP), as indicated by increased green fluorescence, which indicated depolarization of the mitochondrial membrane. However, the cells treated with BLI or Fer-1 exhibited a normal MMP (Figure S6D). Taken together, these data indicated that BLI mitigates kidney ferroptosis in both mouse and cell models.

2.5 BLI exerts its nephroprotective effect by targeting JAK1

To further explore the molecular mechanisms of action of BLI, we conducted a transcription factor enrichment analysis using transcriptomic data. As shown in Figure 6A, the transcription factor STAT was the second most significantly altered factor. We also found that BLI significantly suppressed FA- or UUO-induced phosphorylation of STAT1 and STAT3 (Figure 6B and Figure S7A). Furthermore, gene set enrichment analysis of transcriptomic data revealed that BLI significantly downregulated the IL6-JAK-STAT signaling pathway (Figure S7B). Therefore, we speculated that BLI might exert its effects by binding to STAT proteins or their upstream proteins. To explore the interaction between BLI and proteins, molecular dynamics stimulation was used. Figure 6C shows that the lowest binding energies occurred between BLI and STAT1, STAT3, JAK1, JAK2, and JAK3. We found that when BLI had the lowest binding energy with JAK1, it could bind to JAK1 via hydrogen bond interactions with Gly1020, Arg1007, and Gly884 (Figure 6D). Moreover, BLI inhibited JAK1 phosphorylation in mouse kidney tissues (Figure 6E and Figure S7C). Therefore, we hypothesized that BLI exerts a nephroprotective effect by binding to JAK1 and inhibiting its phosphorylation.

Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) are biophysical technologies used to assess the binding of a drug to its target protein.^{38, 39} The binding of a ligand to a target protein leads to an increase in thermal and protease stabilization. The DARTS results also indicated that BLI stabilized JAK1 in cell lysates after treatment with pronase (Figure 6F). The results of CETSA demonstrated that, compared with DMSO, BLI substantially enhanced the thermal stabilization of JAK1 at 54°C and 57°C in HK2 and NRK-52E cells (Figure 6G). Next, we synthesized biotinylated BLI (Bio-BLI, Scheme S1) and confirmed that its activity was similar to that of BLI (Figure 6H). A subsequent pull-down assay using Bio-BLI further confirmed the binding of BLI to JAK1 (Figure 6I). Finally, the effect of BLI on JAK1 kinase was evaluated,⁴⁰ the results indicated that BLI is a potent inhibitor of JAK1 with an IC₅₀ of 0.376 ± 0.0311 µM. Taken together, these results demonstrated that BLI can directly target and inhibit JAK1.

2.6 BLI inhibits ferroptosis by targeting JAK1 and inhibiting the JAK-STAT signaling pathway

Numerous reports have provided compelling evidence for the regulatory role of the JAK-STAT pathway in ferroptosis.⁴¹ Therefore, we hypothesized that BLI inhibits ferroptosis by modulating the JAK-STAT signaling pathway. To further test this hypothesis, we used the JAK1 inhibitor Jak1-In8 and the JAK1 agonist RO8191 in subsequent research. With respect to the Era-induced ferroptosis model, we found that the JAK1 inhibitor Jak1-In8 inhibited ferroptosis in a manner similar to that of BLI. However, when we combined the JAK1 agonist RO8191 with BLI, BLI was not effective (Figure S8A). Other indicators related to ferroptosis, such as iron ion levels, ROS, GSH, and MDA, were also measured. BLI and Jak1-In8 significantly downregulated iron overload induced by Era, reducing cellular ROS and MDA levels while upregulating GSH levels (Figures S8B–D and S9A–C). Moreover, as shown in Figure S8E, BLI effectively suppressed the phosphorylation of JAK1, STAT1, and STAT3, and these effects were abolished by RO8191. We further examined hepcidin (HAMP), a major regulator of systemic iron metabolism,⁴² ferroportin1 (FPN), the only recognized iron-exporting transporter in mammals,^{43, 44} and SLC7A11, a ferroptosis suppressor.^{45, 46} The results showed that Era treatment could increase the expression of HAMP and decrease the expression of FPN and SLC7A11 at the mRNA and protein levels, while BLI or Jak1-In8 treatment could block this process, and these effects could be completely reversed by RO8191. These results suggest that the inhibition of ferroptosis by BLI is JAK1 dependent (Figures S8E and S9D).

BLI was reported to be a cyclin-dependent kinase (CDK) inhibitor or peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist.^{47, 48} Therefore, we used a CDK inhibitor (20-223) and a PPAR-γ agonist (pioglitazone) to mimic the possible mechanism of BLI. We found that 20–223 and pioglitazone were unable to block Era-induced ferroptosis in HK2 and NRK-52E cells (Figure S8F), indicating that CDK inhibition and PPAR-γ activation could not inhibit ferroptosis. Therefore, BLI is unlikely to function through these two targets, further suggesting that BLI may exert its nephroprotective effect by targeting JAK1 rather than through CDK or PPAR-γ. Taken together, these results demonstrated that BLI inhibits ferroptosis by targeting the JAK-STAT signaling pathway.

2.7 JAK1 knockdown attenuated the nephroprotective effect of BLI

To verify the role of JAK1 in the nephroprotective effect of BLI, we generated a gene-edited mouse model in which JAK1 was knocked down (JAK1-KD). JAK1-KD mice were confirmed by immunohistochemistry and treated with FA and BLI as described (Figure 7A,B). We found that FA challenge increased kidney indices, serum Cr and BUN levels, and urinary Cr and MAU levels in JAK1-KD mice. However, BLI treatment did not reduce the levels of these indicators (Figure 7C,D). Moreover, BLI treatment did not influence the mRNA expression of KIM-1 or NGAL (Figure 7E). Histological analysis also indicated that JAK1 knockdown attenuated the beneficial effects of BLI on pathological kidney injury, fibrosis, and iron concentration (Figure 7F,G). Finally, the serum levels of inflammatory cytokines and kidney tissue oxidative stress were also measured, and the results indicated that the anti-inflammatory and antioxidant effects of BLI were diminished by JAK1 knockdown (Figure 7H,I). These results strongly suggest that BLI exerts its function dependent on JAK1 and exerts its nephroprotective effects by targeting JAK1.

2.8 BLI is as effective and safe as ivarmacitinib in AKI–CKD transition mouse model

Previous studies have confirmed that the JAK-STAT signaling pathway is activated in CKD, especially in diabetic kidney disease (DKD), but JAK2 and STAT3 are the main concerns, and there are few studies on JAK1.^49-51 To further demonstrate the possibility of alleviating CKD progression via targeted inhibition of JAK1, we used another specific JAK1 inhibitor, ivarmacitinib (Ivarm),⁵² in parallel with BLI (Figure 8A). We found that ivarmacitinib also effectively reduced kidney dysfunction, as well as the expression levels of ferroptosis biomarkers and serum inflammatory cytokines, similar to what was observed with BLI in the FA-induced AKI–CKD transition model (Figure 8B–H). These results suggest that blocking AKI–CKD transition by targeting JAK1 is feasible and provide indirect evidence for the mechanism of nephroprotective effect of BLI.

We also preliminarily evaluated the toxicity of BLI in the FA-induced AKI–CKD transition mouse model. The results of HE staining showed that mice in the untreated group and the high-dose (100 mg/kg) BLI treatment group both exhibited clear tissue structures and intact morphology in the heart, liver, spleen, and lung, with no apparent damage. These results showed that BLI is safe and has almost no toxicity while playing a nephroprotective role in vivo (Figure 8I).

Taken together, our data strongly demonstrated the mechanism by which BLI blocks the AKI–CKD transition. For detail, BLI targets JAK1 and inhibits its phosphorylation and activation of the JAK-STAT, subsequently regulating multiple downstream events: (1) promotes the transcription and translation of SLC7A11 to regulate the levels of intracellular GSH and GPX4, thereby maintaining the balance of the glutamate/cystine transport system and reducing ROS production; (2) inhibits the transcription and translation of HAMP, maintain iron homeostasis, and further inhibits iron deposition, mitochondrial damage, ROS production, lipid peroxidation, and ferroptosis; (3) inhibits the release of CCL2 and other factors, and reduces the number of macrophages recruited to the kidney tissue, thus inhibiting the inflammatory response characterized by increased levels of IL-1β, IL-6, TNF-α, TGF-β, and other factors. Finally, the AKI–CKD transition process is blocked, and the occurrence of kidney fibrosis is prevented (Figure 8J).

4.1 Animal experiments

Male C57BL/6J mice (8 weeks, 22–24 g) were purchased from Vital River (Beijing, China, Certificate No.: SCXK2022-0030). JAK1-KO C57BL/6 mice were purchased from Gempharmatech Co., Ltd. (Shanghai, China, Certificate No.: SCXK 2023-0009). The mice were housed under specific pathogen-free conditions and were provided free access to tap water and rodent food and were fed under controlled conditions. After 3 days of adaptation to the housing conditions, the mice (C57BL/6J) were randomly divided into five groups as follows (n ≥ 10/group): Con (0.3 mM NaHCO₃), model (250 mg/kg FA), BLI-L (250 mg/kg FA + 50 mg/kg BLI), BLI-H (250 mg/kg FA + 100 mg/kg BLI), and Tel (5 mg/kg Tel) groups. The mice were sacrificed 14 days after FA treatment, and kidney, blood, and urine specimens were collected for further experiments.

4.2 Detection of kidney function parameters

Whole-blood samples were allowed to clot for 1 h at 4°C before centrifugation at 3500 × g for 15 min for collection of serum. Cr, BUN, urine Cr, and MAU were measured using a specific reagent kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

4.3 RNA-seq profiling

Total RNA was extracted from kidney tissues with TRIzol reagent (Biosharp). Three biological replicates for the Con group, Model group, and BLI group were sequenced using the BGISEQ-500 platform. The DEGs were screened out if they had a q value ≤ 0.001 and a fold change ≥2. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analysis was subsequently performed to reveal the pathways influenced by BLI. Transcription factor analysis was performed to identify the transcription factors influenced by BLI.

4.4 Cell lines and culture

HK2 and NRK-52E cells were obtained from Procell Life Science & Technology Co., Ltd. HK2 cells were maintained in minimum essential medium (HyClone) supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids, 100 U/mL streptomycin, and 100 U/mL penicillin (Gibco). NRK-52E cells were maintained in Dulbecco's modified Eagle's medium (containing 4.5 g/L d-glucose) supplemented with 5% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin. Both cell lines were cultured in a 37°C incubator with 5% CO₂.

4.5 JAK1-BLI binding assays

The binding of BLI to JAK1 was determined by multiple assays. HK2 and NRK-52E cells were seeded in 10-cm culture dishes until they reached 80%–90% confluence. For the CETSA, prior to cell collection, the cells were treated with BLI or the same volume of vehicle for 90 min. Then, the cells were collected and lysed in RIPA lysis buffer for 20 min, and the supernatant was collected by centrifugation at 10,000 × g at 4°C for 10 min. The lysates were divided into PCR tubes and heated at a designated temperature (42–57°C) for 3 min on a heating block. After heating, the tubes were placed on ice for 3 min and boiled in loading buffer at 95°C for 10 min for immunoblotting analysis.

For the DARTS assay, the cells were collected and lysed in RIPA lysis buffer for 20 min. After that, the supernatant was collected by centrifugation at 10,000 × g at 4°C for 10 min. The lysates were divided into PCR tubes in equivalent volumes containing 100 µg of protein and incubated with BLI or the same volume of vehicle for 20 min at 4°C. Then, pronase (Sigma) was added to the samples. The samples were incubated at 25 °C for 10 min. Finally, the lysates were boiled in loading buffer at 95°C for 10 min for immunoblotting analysis.

For the pull-down assay, biotinylated BLI was added to streptavidin-agarose beads (Thermo Fisher Scientific) and incubated at 4°C for 8 h. Biotin alone, unbiotinylated BLI, and untreated beads were used as controls. Lysates prepared from HK2 and NRK-52E cells were then added to streptavidin-agarose beads with Bio-BLI. The mixture was incubated at 4°C for 1 h with gentle rocking. The samples were washed three times and boiled in loading buffer at 95°C for 10 min for immunoblotting analysis.

4.6 JAK1 kinase activity assay

JAK1 kinase was obtained from Invitrogen, and assay was performed using HTRF (Homogenous Time Resolved Fluorescence) detection technology.⁴⁰ Briefly, the enzyme reaction was conducted in reaction buffer consisting of 1 mM DTT, 5 mM MgCl₂, 50 mM HEPES (pH7.0), 0.05% BSA, and 0.1% NaN₃. The assay was performed in a 384-well plate (10 µL) assay format. The final concentrations of the enzyme, TK-substrate-biotin, and ATP were 1 ng/µL, 1 µM, and 3.92 µM, respectively. Compounds were screened at serial diluted concentrations in the presence of 2% DMSO with a 5 min pre-incubation of the kinase and compounds. All reactions were initiated by the addition of ATP and TK-substrate-biotin, incubated at 30°C for 30 min, and quenched with the stop buffer containing 25 nM Strep-XL665 and TK Ab-Cryptate. Time-resolved fluorescence was monitored using a Synergy H1 (Biotek) by excitation at 330 nm and emission donor at 620 nm or emission acceptor at 665 nm, respectively. The files recorded by the Synergy H1 were analyzed in Excel and contained the acceptor and donor counts for each sample. IC₅₀ values were determined using the Graphpad Prism 10 Software.

4.7 Statistical analysis

The data are expressed as the mean ± SEM. For comparisons between two groups, statistical significance was determined using Student's t test. When comparing more than two groups, one-way analysis of variance was used to assess statistical significance. A p value less than 0.05 was considered to indicate statistical significance. Other methods and materials related to the fermentation, isolation, identification of BLI, and synthesis of biotinylated BLI are included in the Supporting Information.