The protective role of Wfdc-2 in acute kidney injury

Acute kidney injury (AKI) is a prevalent clinical syndrome, associated with high morbidity, mortality, and healthcare costs. Owing to an incomplete understanding of the underlying mechanisms modulating AKI progression, and the multifactorial AKI etiology, no therapeutic interventions can reliably improve survival, attenuate injury, or expedite recovery, other than dialysis and kidney replacement therapies. Although animal models of AKI are very effective in recapitulating pathological and biomarker features that have been found in humans, the models have not been predicted about its clinical effectiveness of potential therapeutic agents, which work to limit injury in the animal but not in humans. Moreover, several biomarkers of renal injury have been identified but the utility of these biomarkers is largely confined to research studies, whereas widespread clinical application is limited. Thus, the clearance of the pathophysiology of AKI is the only way to find the effective approach to attenuate injury or for early diagnosis of AKI. 

This study used Madin-Darby canine kidney (MDCK) cells to construct an in vitro three-dimensional (3D) renal tubule model to simulate the tubular injury repair process. Flow cytometry was used to sort cells at different cell cycles, and transcriptome sequencing was performed to screen for repair-related factors. CRISPR/Cas9 was used to prepare the WFDC-2 gene knockout cell line. An ischemia-reperfusion model (IRI-AKI) was established: 8-week-old C57/BL mice underwent bilateral renal pedicle clamping for 35 minutes for reperfusion, and blood and kidney tissue samples were collected 24 hours later. Kidneys from fetal mice at E13.5 days were used for RNA-ISH staining. A drug intervention model was established: 12 hours after IRI-AKI modeling, drugs were injected via the tail vein, and blood and kidney tissue samples were collected 12 hours after injection.

A total of 162 genes were selected as the significantly different expressed genes during in vitro 3D renal tubular repairing. RNA in situ hybridization (RNA-ISH) was used to explore the expression pattern and location of each gene in kidney. Firstly, we found that the location of WFDC-2 gene in ISH was complemented with kidney injury molecular 1 (KIM-1), and WFDC-2 expression was significantly increased from 12 h to 24 h after I/R injury. Secondly, in 3D renal tubule models, Wfdc-2 was secreted into the lumen of Cyst which was formed by single layer of polarized MDCK cells. It is suggested that Wfdc-2 was a classical secretary protein. Overexpression of WFDC-2 in MDCK cells significantly increased the rates of single luminal Cyst in Day 6, while the rates of single lumen were significantly decreased in WFDC-2 knockout group. Re-expression of WFDC-2 could ameliorate the rates of single lumen to normal levels. Moreover, supplementing recombined human Wfdc-2 protein (rhWfdc-2) during Cyst formation could also increase the rates of single lumen. It demonstrated that Wfdc-2 plays a key role in cystic lumen formation as a secreted protein. Thirdly, WFDC-2 was highly expressed in kidney and co-located with renal tubules in E13.5 fetal mouse. WFDC-2 was also expressed in human specimens, and the expression levels of WFDC-2 were associated with the outcomes of patients. Finally, rhWfdc-2 was injected into mouse through tail vein at 12 h after I/R injury. The expression of KIM-1 was significantly decreased in both mRNA and protein levels in kidney, and the levels of serum creatinine was also decreased when compared with BSA injection mice. 

In summary, Wfdc-2 significantly promotes IRI-AKI-induced kidney damage, possibly mediated by promoting renal tubular lumen stability and KIM-1 expression. Here we forecast that Wfdc-2 is expected to become the first protein drug specifically for AKI.