The Renal YY1-KIM1-DR5 Axis Regulates The Progression Of Acute Kidney Injury

Acute kidney injury (acute kidney injury, AKI) is diagnosed in more than 10 million people worldwide every year, causing approximately 1.7 million deaths and seriously threatening people's lives and health [1, 2]. Ischemia-reperfusion and urethral obstruction caused by clinical operation are important causes of acute kidney injury[3]; Acute kidney injury may also be induced by the use of multiple drugs, such as anti-inflammatory drugs [4]. Acute kidney injury often leads to increased extracellular matrix, renal tubular atrophy, and cell death, leading to renal dysfunction and irreversible damage to the kidney, which induces severe chronic kidney disease CKD [5] and eventually develops into end-stage renal disease. The risk of developing CKD in patients with acute kidney injury is 8 times that of healthy people, and the risk of all-cause death is 2 times that of healthy people[6]. Acute kidney injury has a high morbidity rate and high mortality rate, which seriously threatens human health. However, the pathogenesis is complex and there is no effective drug treatment strategy[7, 8]. To conduct research on its key therapeutic targets and design therapeutic drugs accordingly, It has important basic research and clinical transformation significance.

Click to cistanche tubulosa extract for kidney disease

On July 17, 2023, Professor Zheng Ling from Wuhan University, together with Professor Huang Kun and Associate Professor Chen Hong from Tongji School of Pharmacy, Huazhong University of Science and Technology, published a paper entitled "A renal YY1-KIM1-DR5 axis regulates the progression of acute kidney" in Nature Communications. Injury” research reveals the mechanism of action of the kidney injury molecule KIM1 in acute kidney injury and designs antagonistic peptides through intervention targets.

Kidney injury molecule KIM1 is a transmembrane glycoprotein that is mainly expressed in the kidney, and its expression level is low under physiological conditions[9]. Therefore, it has been approved by the FDA as an early biomarker for various kidney diseases [10]. In addition, because KIM1 is expressed in epithelial cells, it can mediate a variety of extracellular signal transmission and play a variety of effects and has been reported to participate in various physiological and pathological activities such as virus invasion, immune response, tissue damage, and repair, and tumor development [11, 12]. What role does the KIM1 molecule play in the development of acute kidney injury? What is the specific molecular mechanism of action? Can inhibition of KIM1 and its downstream effector molecules be used as a treatment for acute kidney injury? How to design acute kidney injury treatment drugs accordingly? This series of important scientific issues still need further research.

A simplified diagram of the structure of KIM1 is shown below (Fig. 1a). The researchers found that KIM1 was significantly upregulated in the AKI model (Fig. 1b–d). In renal tubular epithelial cells overexpressing KIM1, stimulation with cisplatin can promote cisplatin-induced apoptosis and upregulation of inflammatory factors; KIM1 knockout can alleviate cisplatin-induced apoptosis and inhibit the expression of inflammatory factors ( Figure 1e–h). Further studies found that overexpression of KIM1 promoted cisplatin-induced poly-ADP-ribose polymerase 1 (poly-ADP-ribose polymerase 1, PARP1) cleavage and phosphorylation of p53, and aggravated apoptosis; KIM1 knockout inhibits the cleavage of PARP1 and the phosphorylation of p53 induced by cisplatin (Figure 1i-j). The above results collectively indicate that KIM1 may affect cisplatin-induced cell damage by promoting apoptosis.

Figure 1 KIM1 was significantly upregulated and aggravated inflammatory and apoptotic responses after AKI

Co-IP and FRET experiments proved that there is a binding between KIM1 and DR5, and the binding between KIM1 and DR5 tends to be enhanced under damage conditions (Fig. 2a-d). DR5 was significantly upregulated after kidney injury and had a clear co-localization with KIM1 (Fig. 2e,f). Knockdown of DR5 protected against cisplatin-induced apoptosis exacerbated by KIM1 (Fig. 2g). KIM1 overexpression promotes cisplatin-induced DR5 oligomerization, which is manifested as enhanced FRET signal; while KIM1 knockdown inhibits cisplatin-induced DR5 oligomerization, which is manifested by decreased FRET signal (Fig. h, i). The results of non-denaturing gel electrophoresis showed that under cisplatin stimulation, KIM1 overexpression promoted the formation of DR5 high-molecular-weight oligomers (High-ordered oligomers), and KIM1 knockdown inhibited the formation of DR5 high-molecular-weight oligomers (Figure j, k). Further studies found that KIM1 can bind to the full length of DR5, DR5ΔTMH, and DR5ΔCytD, but not to DR5ΔECD, indicating that ECD is the key segment for DR5 to bind KIM1 (Figure 2l). At the same time, KIM1-related truncations (Ig V, Mucin, TM+CytD) and the full length of DR5 were overexpressed, and the KIM1 Ig V segment was found to be the key segment for its binding to DR5. It is suggested that KIM1 promotes DR5 oligomerization under cisplatin stimulation.

Figure 2 KIM1 binds to DR5 and promotes its oligomerization

In the cisplatin-induced AKI mouse model, the renal tubule-specific Kim1 knockout (Kim1Ksp-KO) mice had lower serum creatinine and blood urea nitrogen levels than WT mice, and the shedding of renal tubular epithelial cells and lumen dilation were reduced. showed that Kim1Ksp-KO mice had less damage (Fig. 3a–c). Kidney-specific knockout of Kim1 inhibited the activation of Caspase 3, 8, and 9 (Fig. 3d), indicating that the signaling pathway downstream of DR5 was inhibited. Kidney TUNEL-positive staining of Kim1Ksp-KO mice was significantly reduced compared with that of WT mice (Fig. 3e), indicating that Kim1 knockdown alleviated cisplatin-induced apoptosis at the mouse level. Concurrently, tubule-specific knockout of Kim1 inhibited the formation of DR5 high-molecular-weight oligomers (Fig. 3f). Furthermore, the same conclusion was obtained in the bIRI-induced AKI mouse model, that is, tubule-specific knockout of Kim1 alleviated bIRI-induced kidney injury by inhibiting DR5 downstream apoptotic signaling (Fig. 3g–l). These results suggest that tubule-specific knockout of Kim1 attenuates cisplatin- and ischemia-reperfusion-induced AKI.

Figure 3 Kidney tubule-specific knockout of Kim1 relieves AKI induced by cisplatin and ischemia-reperfusion

For the binding site between KIM1 and DR5, AlphaFold2 and Human KIM1-DR5 PPI were used to screen antagonistic peptides and combined with cell verification, antagonistic peptide P2 was obtained (Fig. 4a, b). P2 can inhibit the expression of damage-related molecules and the activation of Caspase3, 8, and 9 induced by cisplatin, and promote the expression of anti-apoptotic molecules (Fig. 4c, d). Further studies demonstrated that 5'(6) FAM-tagged P2 co-localized well with KIM1 and DR5 in a cisplatin-induced AKI mouse model (Fig. 4e). In an animal model of cisplatin-induced AKI, tail vein injection of P2 significantly improved renal function and at the same time ameliorated renal pathological damage (Fig. 4f–h). P2 significantly inhibited cisplatin-induced activation of Caspase3, 8, 9 and improved cisplatin-induced apoptosis (Fig. 4i, j). The results of Co-IP in kidney tissue showed that P2 could block the binding of KIM1 to DR5 under cisplatin stimulation (Fig. 4k). The above results indicated that under cisplatin injury, P2 can reach the interaction site between KIM1 and DR5, block the combination of KIM1 and DR5, thereby inhibiting apoptosis and improving AKI.

Figure 4 Antagonist peptide P2 interferes with KIM1-DR5 interaction to alleviate acute kidney injury

Figure 5 YY1-KIM1-DR5 axis promotes the development of AKI and potential treatment strategies

Article conclusion and discussion, inspiration, and outlook

In summary, this study used the transcription factor prediction database, combined with proteomics methods, to reveal the upstream regulatory network and downstream effector molecules of KIM1 in the state of kidney injury, and proposed for the first time that the "YY1-KIM1-DR5" axis may develop in AKI play an important role (Figure 5). Under physiological conditions, YY1 maintains a high level and acts as a transcriptional repressor for KIM1. At this time, the expression level of KIM1 is low. In the state of kidney injury, YY1 is down-regulated, its transcriptional repression of KIM1 is partially released, and the level of KIM1 is up-regulated. Up-regulated KIM1 binds to DR5, a key molecule that regulates apoptosis, promotes its oligomerization, activates the caspase cascade reaction, induces apoptosis, and aggravates AKI. 

This study provides a new idea and a new target for the treatment of AKI. By constructing a kidney tubule-specific knockout Kim1 mouse (Kim1Ksp-KO), it was found that knockout of Kim1 could inhibit the oligomerization of DR5, inhibit apoptosis, and improve AKI; an antagonistic peptide screening system was constructed using AlphaFold2, and specific inhibitory peptides were obtained. Antagonist peptide P2 blocks the interaction of KIM1-DR5, and P2 can block the combination of KIM1-DR5 at the animal level and improve AKI.