Combined treatment with empagliflozin, caloric restriction, mesenchymal stem cells and Klotho for halting the progression of diabetic kidney disease and ameliorate acute kidney injury

Abstract

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD) around the world. It affects people of all ages, sex and ethnicity. The occurrence of acute kidney injury (AKI) predisposes to CKD and can worsen renal function in individuals with CKD under conservative treatment. Blockade of the renin-angiotensin-aldosterone system (RAAS) is the gold standard for slowing the progression of DKD, in addition to changes in lifestyle. Recently, inhibitors of the sodium-glucose co-transporter-2 (iSGLT2) have been shown to halt the progression of DKD through glycemic control, restoration of tubule-glomerular feedback and also modulation of signaling pathways that is unregulated in DKD, with stress oxidative and fibrosis. However, in other CKDs, such as glomerulopathies and hypertensive nephrosclerosis, iSGLT2 demonstrated a reduction in the progression of CKD, suggesting that the effects of these medications are independent of glycemic control. Thus, we intend to study the effect of iSGLT2 using the BTBRob/ob mouse, homozygous for the knockout of the leptin gene, which is a reversible preclinical model of type 2 diabetes mellitus (DM2) and DKD, obesity, hyperphagia, insulin resistance and hyperglycemia (6th week of age), followed by the establishment of DKD with increasing levels of albuminuria, dependent on time (8th week of age), similar to changes in DKD in humans, such as mesangial expansion, podocyturia and macrophage infiltration. A subject also emerging from the literature is the existence of specific renal tissue stem/progenitor cells and their renal regenerative capacity. Our group previously documented the existence of a population of stem/progenitor cells specific for renal tissue identified with the c-Kit marker (CD117) and capable of repairing renal tissue after acute insults, such as ischemia-reperfusion injury and syndrome nephrotic. Thus, in this project we are proposing to evaluate a combination of therapies, such as the use of empagliflozin (iSGLT2), caloric restriction, mesenchymal stem cells (MSCs), gene therapy with the klotho to enable the recovery from kidney injury and curtail the progression of DKD through the modulation of oxidative stress, cell death and cell senescence, so that we also seek to understand the role of kidney-specific stem/progenitor cells identified by c-Kit in acute and chronic kidney injury setting using lineage tracing. Project 1: We will use the BTBRob/ob mouse, which will be preconditioned with empagliflozin (25 mg/kg/feed) and caloric restriction (30%) and treated with MSCs and klotho. We will verify general clinical outcomes (weight, blood glucose and plasma insulin) and renal functional parameters (glomerular filtration rate, volume of diuresis, glucosuria and albuminuria) and renal structural (number of podocytes, mesangial expansion, oxidative stress, cell death due to apoptosis, cell senescence and fibrosis). In co-culture experiments with podocytes or proximal tubule cells with MSCs in the conditioning media associated with Angiotensin-II, we will evaluate the mechanism of action of klotho through NOX4-TRPC6 interaction, as well as oxidative stress, apoptosis and cellular senescence. Project 2: In the models of acute kidney injury (AKI) due to ischemia-reperfusion and glycerol-induced rhabdomyolysis, we will evaluate the effectiveness of preconditioning with empagliflozin (25 mg/kg/feed) and the impact of the treatment of MSCs and klotho on outcomes functional (plasma creatinine and urea) and structural (AKI score, oxidative stress, cell death due to apoptosis, cell senescence and fibrosis). Using the mouse c-KitCreERT2/+; mTmG, in which, after the administration of Tamoxifen, we will perform lineage tracing of EGFP-c-Kit progenitor/stem cells and analyze the impact of those therapeutic strategies on the maintenance and proliferation of the c-Kit cell pool, as well as their differentiation capacity in vivo. (AU)