In the current study, we found that inhibition of EHHADH expression led to a loss of peroxisomes in RTECs through pexophagy, which is relative to the increased ROS levels. Additionally, the autophagic receptor NBR1 was required for EHHADH-mediated pexophagy. We observed that downregulation of renal tubular EHHADH expression occurred in patients with DN and showed that genetic loss of Ehhadh expression aggravated tubulointerstitial injury under diabetic conditions accompanied by increased pexophagy in vivo.
Tubular injury is a key factor in the development and progression of DN 31. Abnormal lipid metabolism and oxidative stress are one of the main causes of tubular injury mediated in high glucose condition 32, 33, 34. Peroxisomes are intimately associated with lipid metabolism and oxidative stress, and their ability to carry out fatty acid oxidation and lipid synthesis, especially the production of ether lipids, may be critical for generating cellular signals required for normal physiology. Peroxisomes are highly enriched in kidneys, and a reduced abundance of functional peroxisomes has been observed in RTECs from both human patient kidney samples and in several experimental models of kidney diseases 7, 8, 35, 36. Previous studies have also found that peroxisome is downregulated in a variety of kidney diseases. PEX5 and PEX11a are both the essential functional proteins of peroxisomes. Renal tubular conditional deletion of Pex5 (Pex5cKO) 37, or knockout Pex11a [45] in mice resulted in dramatic loss of peroxisomes, but not affect the kidney survival. However, integrated transcriptomic and metabolomic analyses showed profound reprogramming in the metabolic, antioxidant and lipid synthesis pathways of the kidneys of Pex5cKO mice. The result was in line with our findings in Ehhadh KO mice. We speculate that loss of EHHADH expression increases susceptibility to the development of DN, and analysis from the public database showed that EHHADH expression was specifically downregulated in damaged tubules of patients with DN 25. As expected, although the weight and blood glucose of Ehhadh KO mice were slightly lower than those of WT mice under diabetic conditions, they still displayed more severe tubulointerstitial injury, indicating the importance of EHHADH in tubulointerstitial injury under hyperglycemia, which also is in agreement with previous findings that PEX11a deficiency aggravated renal interstitial lesions in mice 38.
A previous study established the association between EHHADH expression and renal tubular injury in the kidneys of male mice, but the underlying mechanism is not well explained 20. The p.E3K mutation in EHHADH was identified in five generations of a family with isolated autosomal dominant Fanconi renal syndrome. The p.E3K mutation caused mistargeting of peroxisomal EHHADH to mitochondria, leading to impaired mitochondrial fatty acid β-oxidation and respiration and decreased ATP production, but peroxisomal deficiency and dysfunction were not observed 18, 19.
Based on our above findings, we confirmed that a lack of peroxisomal EHHADH expression causes peroxisomal deficiency and dysfunction. Similar to mitophagy, peroxisomes can be targeted for selective autophagic degradation in lysosomes, which is called pexophagy 39, 40. This is the first study to present the relationship between EHHADH expression and pexophagy. EHHADH is one of the rate-limiting enzymes of peroxisomal FAO metabolism; indeed, we observed that Ehhadh knockdown in RTECs increased ROS levels, which served as the inducer of pexophagy. Peroxisomes damage leads to increased intracellular ROS, which can also induce increased pexophagy. Our study also observed increased intra cell ROS in Ehhadh knockdown RTECs. In addition, the increased pexophagy caused by EHHADH downregulation was significantly restored after ROS inhibition, indicating that EHHADH-mediated pexophagy is partially dependent on ROS production.
The ubiquitination of PMPs is required for pexophagy 41, 42, 43, and NBR1 was shown to play an important role in EHHADH-mediated pexophagy. Although we observed increased ubiquitination of PMP70 and enhanced binding between PMP70 and NBR1 in EHHADH-knockdown RTECs, the precise binding sites and other PMPs that underwent ubiquitination and coordinated with NBR1 still need further study.
In summary, our study has provided novel insights into the regulation of pexophagy by EHHADH deficiency. We have identified that downregulation of EHHADH can lead to peroxisomal deficiency, which in turn increases the susceptibility to tubular injury and fibrosis under hyperglycemia. These findings highlight the potential of targeting pexophagy as a therapeutic strategy for the treatment and prevention of DN in the future.