Role of HSP60 (HSPD1) in diabetes‐induced renal tubular dysfunction: regulation of intracellular protein aggregation, ATP production, and oxidative stress

Abstract

Because underlying mechanisms of diabetic nephropathy/tubulopathy remained poorly understood, we aimed to define a key protein involving in hyperglycemia-induced renal tubular dysfunction. All altered renal proteins identified from previous large-scale proteome studies were subjected to global protein network analysis, which revealed heat shock protein 60 (HSP60, also known as HSPD1) as the central node of protein-protein interactions. Functional validation was performed using small interfering RNA (siRNA) to knock down HSP60 (siHSP60). At 48 h after exposure to high glucose (HG) (25 mM), Madin-Darby canine kidney (MDCK) renal tubular cells transfected with controlled siRNA (siControl) had significantly increased level of HSP60 compared to normal glucose (NG) (5.5 mM), whereas siHSP60-transfected cells showed a dramatically decreased HSP60 level. siHSP60 modestly increased intracellular protein aggregates in both NG and HG conditions. Luciferin-luciferase assay showed that HG modestly increased intracellular ATP, and siHSP60 further enhanced such an increase. OxyBlot assay showed significantly increased level of oxidized proteins in HG-treated siControl-transfected cells, whereas siHSP60 caused marked increase of oxidized proteins under the NG condition. However, the siHSP60-induced accumulation of oxidized proteins was abolished by HG. In summary, our data demonstrated that HSP60 plays roles in regulation of intracellular protein aggregation, ATP production, and oxidative stress in renal tubular cells. Its involvement in HG-induced tubular cell dysfunction was most likely regulation of intracellular ATP production.-Aluksanasuwan, S., Sueksakit, K., Fong-ngern, K., Thongboonkerd, V. Role of HSP60 (HSPD1) in diabetes-induced renal tubular dysfunction: regulation of intracellular protein aggregation, ATP production, and oxidative stress.

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal‐Induced Cytotoxicity in Renal Tubular Epithelial Cells

“…Levels of oxidized proteins were measured by Oxyblot assay using OxyBlot Protein Oxidation Detection Kit (S7150; Chemicon, Temecula, CA) as previously described. [9,10] Briefly, at 6, 12, 24, and 48 h posttreatment, the controlled and COM-treated MDCK cells were harvested and washed with PBS. Thereafter, an equal amount of proteins derived from each sample was derivatized with 2,4-dinitrophenylhydrazine (DNPH).…”

Protective Cellular Mechanism of Estrogen Against Kidney Stone Formation: A Proteomics Approach and Functional Validation

“…Our findings demonstrated that two major enzymes in the glycolysis pathway (α‐enolase and GAPDH) were both decreased in the estrogen‐treated cells in consistent with the decreased level of intracellular ATP. Previous studies have reported that ATP overproduction is associated with increased oxidative stress, increased levels of reactive oxygen species, and tissue injury . On the other hand, the decreased level of intracellular ATP is associated with reduced risk of oxidative stress and cell/tissue injury .…”

“…Previous studies have reported that ATP overproduction is associated with increased oxidative stress, increased levels of reactive oxygen species, and tissue injury. [46][47][48] On the other hand, the decreased level of intracellular ATP is associated with reduced risk of oxidative stress and cell/tissue injury. [49,50] It was thus plausible that the decreased intracellular ATP level observed in our present study was one of the protective mechanisms of estrogen to prevent renal cells/tissue from CaOx-induced oxidative stress and renal injury frequently observed in kidney stone disease.…”

Pre-inhalation of Hydrogen-rich Gases Protect Against Caerulein-induced Mouse Acute Pancreatitis While Enhance the Pancreatic Hsp60 Protein Expression

“…Studies have shown that siHsp60 lead to the increase of oxidized protein in renal tubular cells [21]. Ren`s work ever show that hydrogen-rich saline inhibit pyrindomain-containing 3 (NLRP3) in ammasome activation and attenuates experimental mice AP [7], whereas report also show that Hsp60 can stimulate NLRP3 in ammasome pathway [22].…”