PACS-2 Ameliorates Tubular Injury In Diabetic Nephropathy By Maintaining MAM Integrity And Promoting Mitophagy

Background:Diabetic nephropathy（DN）is among the most common and severe microvascular complications of diabetes mellitus,and the primary cause of end-stage renal disease in China.Current therapeutic options for DN remain limited.Renal tubulointerstitial lesions are prominent histopathological changes in DN.Prevention and treatment targeting tubulointerstitial injury has been shown to be critical for retarding the progression of DN.However,mechanisms underlying tubulointerstitial injury in DN have not been fully elucidated.Mitophagy deficiency plays a key role in tubular injury in early DN.Recent studies suggest that mitophagosomes form at mitochondria-associated endoplasmic reticulum membranes（MAM）,and MAM disruption prevents initiation of autophagosome formation.Moreover,we have previously demonstrated that MAM integrity disruption is closely related to tubular injury in DN.However,the regulation of MAM integrity under states of hyperglycemia in tubules and the mechanism links MAM to tubular pathobiology in DN remain unknown.Phosphofurin acidic cluster sorting protein 2（PACS-2）,an integral protein in MAM,is involved in the translocation of its cargo proteins containing acid clusters and also identified as one of the key regulators of MAM formation.PACS-2 has been linked to the pathogenesis of metabolic disease,such as obesity and insulin resistance.We found previously that PACS-2 was downregulated in the kidneys from patients with DN.Thus,we hypothesize that PACS-2may be implicated in the pathogenesis of tubular injury in DN through regulating MAM integrity and mitophagy.Objective:To investigate the role of PACS-2 in tubular injury in DN and potential mechanisms.Methods:1.Immunohistochemistry was applied to detect the expression of PACS-2 in kidneys from patients with DN and control subjects.Pearson’s correlation analyses were performed to test the correlation between PACS-2 expression levels and renal function or the degree of tubulointerstitial injury.2.Pacs-2 flox mice were crossed with Ggt1-Cre mice that carried a Cre allele under the control of a proximal tubule（PT）-specific promoter to generate mice with PT-specific knockout of Pacs-2(Pacs-2^ptKO).Littermates without carrying a Cre allele(Pacs-2^ctrl)were used as controls.Mice were induced with streptozocin（STZ）to establish the type 1diabetic animal model.The mice were divided into four group as below:Pacs-2^ctrl,Pacs-2^ptKO,Pacs-2^ctrl+STZ,Pacs-2^ptKO+STZ.Mice were followed up for an additional 12 weeks before collecting urine and sacrifice.Albumin in the urine and serum creatinine were tested.HE（hematoxylin-eosin）,PAS（periodic acid-schiff）and Masson staining were performed to detect the histopathological changes.Transmission electronic microscopy was used to examine the mitochondrial morphology and MAM integrity.Immunofluorescence and Western blot were applied to detect the expression of proteins involved in mitochondrial dynamics or mitophagy.3.In vitro experiments were conducted with human proximal tubular cell line（HK-2 cell）.HK-2 cells were transfected with the PACS-2overexpressing plasmid or empty vector,and treated with normal glucose（NG）or high glucose（HG）for 24 hours.Immunofluorescence was used to examine mitochondrial morphology,MAM amount and different phases of mitophagy.Western blot experiments were performed to detect the expression of proteins implicated in mitochondrial dynamics and mitophagy,and cell apoptosis.Immunoprecipitation was applied to validate the interaction between PACS-2 and its client protein,BECN1.Results:1.PACS-2 is significantly downregulated in the kidneys from patients with DN comparing to control subjects.Moreover,PACS-2expression level is positively correlated with estimated glomerular filtration rate（eGFR）,and negatively correlated with the degree of renal tubulointerstitial injury.2.Comparing to Pacs-2^Ctrlmice,Pacs-2^Ctrl+STZ mice exhibit markedly increased albuminuria and tubulointerstitial injury,accompanied by mitochondrial fragmentation,MAM integrity disruption and defective mitophagy in proximal tubular cells.These changes are more pronounced in Pacs-2^ptKO+STZ mice.3.High glucose induces mitochondria to fragment and uncouple from endoplasmic reticulum in HK-2 cells,leading to MAM integrity disruption and mitophagy deficiency,which further cause mitochondrial dysfunction and mitochondrial ROS overproduction,ultimately resulting in cell apoptosis.PACS-2 overexpression can partially reverse above changes.Mechanistically,we find an acid cluster within the protein sequence of BECN1 that can potentially be identified by PACS-2,and validate the interaction between PACS-2 and BECN1.Thus,PACS-2 binds to BECN1and mediates the relocalization of BECN1 to MAM where they promote the formation of mitophagosome.Conclusion:PACS-2 alleviates tubular injury in DN through promoting mitophagy by increasing the formation of MAM and transporting BECN1 towards MAM.