Antiapoptotic effect of ouabain in renal cells exposed to toxic glucose levels

Diabetes mellitus is a group of metabolic diseases that causes enormous social and economic burden. The kidney is the filtration organ that produces urine and is the primary target of diabetic complications, a condition called diabetic nephropathy (DN). DN is a form of chronic kidney disease (CKD) and CKD is characterized by a progressive decrease in renal function which results in renal failure. Diabetes mellitus is characterized by hyperglycemia which induces renal cell apoptosis and causes tissue loss in the kidney. Together, these hallmarks are the major pathophysiological events observed in DN. Bcl-2 family proteins play an important role in the intrinsic apoptosis pathway and the family member, BAD, has been shown to be proapototic. Ouabain is a cardiotonic steroid and a highly specific Na, K-ATPase ligand. The binding of ouabain to Na, K-ATPase has been suggested to activate downstream signaling. Ouabain-bound Na, K-ATPase can interact with the 1, 4, 5-trisphosphate receptor, generating a calcium signal that has a protective effect. In this thesis we have studied the global signaling pathways generated by ouabain-bound Na, K-ATPase, and the mechanism responsible for hyperglycemia induced renal cell apoptosis. In addition, we have investigated the molecular mechanism behind ouabain’s protective effect against apoptosis.

In study I, we investigated changes in the cellular phosphoproteome after ouabain treatment. A total of 2580 ouabain-regulated phosphorylation events were identified. Among the proteins that were phospho-regulated, a large proportion were involved in the regulation of cell adhesion and proliferation. Multiple protein kinases, including Ca2+/calmodulin-dependent protein kinase were also regulated. This study revealed that Na, K-ATPase is a versatile signal transducer of its ligand, ouabain.

In study II, we used primary cell cultures of proximal tubular cells (PTCs), podocytes and mesangial cells to study their sensitivity to high glucose treatment. We found that PTCs and mesangial cells were more vulnerable to glucose toxicity than podocytes due to their expression of sodium-dependent glucose cotransporters (SGLTs). SGLT inhibition or down-regulation protected renal cells from high glucose induced apoptosis and ouabain treatment had the same protective effect.

In study III, we applied super resolution microscopy to follow the apoptotic process triggered by high glucose. After two hours of high glucose treatment, the proapoptotic protein, BAD, had translocated to the mitochondria and initiated apoptosis. BAD translocation to mitochondria was abolished in the presence of ouabain. This study highlighted BAD’s role as an important player in glucose induced apoptosis and demonstrated the protective effect of ouabain which intervenes early in the apoptotic process, by preventing BAD activation.

List of scientific papers

I. Elena Panizza, Liang Zhang, Jacopo Maria Fontana, Kozo Hamada, Daniel Svensson, Evgeny E. Akkuratov, Lena Scott, Katsuhiko Mikoshiba, Hjalmar Brismar, Janne Lehtio, and Anita Aperia. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival. FASEB journal. 2019; 33(9):10193-206.
https://doi.org/10.1096/fj.201900445R

II. Linnéa M. Nilsson, Liang Zhang, Alexander Bondar, Daniel Svensson, Annika Wernerson, Hjalmar Brismar, Lena Scott, and Anita Aperia. Prompt apoptotic response to high glucose in SGLT-expressing renal cells. American journal of physiology Renal physiology. 2019;316(5):F1078-F89.
https://doi.org/10.1152/ajprenal.00615.2018

III. Liang Zhang*, Daniel Svensson*, Jacopo Maria Fontana, Kristoffer Bernhem, Linnea M. Nilsson, Lena Scott, Hans Blom, Hjalmar Brismar and Anita Aperia. Mapping of glucose induced apoptosis in kidney epithelial cells and the anti-apoptotic effect of ouabain - a BAD story. *Contributed equally. [Manuscript]