The Effect of Albumin on Podocyte Apoptosis: the Role of PKC δ, p38 MAPK and Endoplasmic Reticulum Stress

Abstract

Chronic kidney disease (CKD) is a public health problem characterized by high morbidity and mortality and with enormous social and economic impact on the health system. The high prevalence of patients maintained in dialysis programs, and the perspective of increasing the number of cases of CKD is worrisome for health professionals worldwide. One of the most serious consequences of CKD progression is albuminuria, which is mainly due to severe lesions in the glomerular basement membrane and tubular injury. Increased permeability of the glomerular ultrafiltration barrier to albumin is directly related to podocyte injury, mainly due to loss of foot processes, oxidative stress and apoptotic events. However, the cellular mechanisms involved in such processes have not yet been elucidated. Thus, the aim of this study is to investigate the mechanisms by which albumin participates in podocyte injury, considering the contribution of cubilin receptors, PKC δ (delta), p38 MAPK and the endoplasmic reticulum stress.MethodsDifferentiated mouse podocytes were distributed in four experimental groups: control and treated with albumin at the concentrations of 1, 5 and 10 mg/ml, in serum‐free RPMI 1640, for 24 hours. After the treatment, apoptosis was assessed by flow cytometry. For immunofluorescence experiments, cells were treated with FITC albumin 1 mg/mL for 30 min, 1h, 3h and 24h. The images were obtained by confocal microscopy, objective of 63x. Protein expression was analyzed by immunoblotting; GRP78, PKC δ, p38 MAPK and Caspase‐12 were evaluated 30 min, 1h, 3h and 24h after the treatment of the cells with albumin 1 mg/ml. GAPDH was used as endogenous control. All results are presented as the mean ± standard error. Statistical analysis was assessed by one‐way ANOVA followed by the Bonferroni's test. Significance was assumed at p<0.05.ResultsAlbumin treatment at the concentration of 1 mg/ml for 24h resulted in a significant increase on the total apoptosis rate compared to the control. In immunofluorescence assay, colocalizations between FITC albumin and cubilin were observed in almost all the time periods tested and mainly after 1 hour of the treatment. Regarding protein expression, a significant increase on GRP78 was observed 1 hour after the albumin treatment. In addition, a significant increase on the expression of phosphorylated PKC δ, p38 MAPK and cleaved Caspase‐12 were also observed in the periods of 1h, 3h and 24h after the albumin treatment. These last two parameters were reduced by SB203580, a p38 MAPK specific inhibitor (100 nM)ConclusionsThese results suggest that the protein cubilin has an important role on the internalization of albumin in the podocytes. In addition, albumin induces apoptosis by activation of Caspase‐12 in these cells, mainly via the endoplasmic reticulum stress/PKC δ/p38 MAPK pathway.Support or Funding InformationFinancial support: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).