Abstract
With the diabetes pandemic, chronic kidney disease is a burden, with diabetic nephropathy (DN) being the primary leading cause of end-stage renal disease. Antidiabetic drugs have shown renoprotective effects via different mechanisms, and among those are SGLT2 inhibitors and DPP-4 inhibitors. Hypoxia-inducible factor (HIF) pathway, a significant contributor to inflammation development in the kidneys, is a major pathway that regulates erythropoietin synthesis. Prolyl hydroxylase domain (PHD) decreases erythropoietin synthesis by inhibiting HIFs. PHD inhibitors are anticipated to have additional effects, such as protection against metabolic diseases, due to HIF's impact on many genes. The diabetic kidney is characterized by enhanced HIF-1α activation but decreased HIF-2α activity. These actions may significantly contribute to glomerular and renal tubular dysfunction, renal inflammation and fibrosis, and decreased erythropoietin synthesis observed in DN. This work aims to test the renoprotective effect of SGLT2 inhibitors and DPP-4 inhibitors by controlling the proinflammatory and anti-inflammatory domains of the HIF pathway.
Type 1 DM rat model was induced in 24-hour-fasted Sprague Dawley rats by a single intraperitoneal injection of 45 mg/kg streptozotocin (STZ). DM was verified by measuring blood glucose seven days following STZ injection, and only animals with glucose levels of ≥15 mM were included (n = 24), randomly and equally distributed, then received either saline or vildagliptin (3 mg/Kg) or empagliflozin (30 mg/Kg) once daily by oral gavage for three months. An additional group was used as a non-diabetic control. At the end of the experiment, rats were placed in metabolic cages for 24-h urine collection, then sacrificed. Blood samples and kidney tissue were collected. Kidney function was assessed by measuring serum creatinine and urinary albumin: creatinine ratio. HIF1α, HIF2α, and PHD3 mRNA expression in kidney tissues were assessed using quantitative real-time PCR, and to indirectly assess HIF2α protein stability, immunohistochemical staining for PHD3 was also performed.
Vildagliptin and empagliflozin could significantly ameliorate the diabetic disturbance of renal function, as demonstrated in serum creatinine (F = 23.518, p<0.001) with a non-significant difference between both treated groups, and in the significantly reduced albumin/creatinine ratio (F = 14.453, p<0.001), with a significantly lower ratio for empagliflozin (4.73±0.54) than vildagliptin-treated group (9.09±1.04). Regarding the HIF pathway as a potential mechanism, empagliflozin-treated groups showed a significantly higher HIF2α gene expression in kidney tissue samples (0.98±0.10) than the vildagliptin-treated group (0.58±0.05). For the expression of its regulator, PHD3, there was a significant difference between groups (F = 5.694, p = 0.005). However, the vildagliptin-treated group (0.65±0.06) didn′t differ significantly from the untreated group (0.60±0.03), in contrast to the empagliflozin-treated group (2.01±0.50), which showed a significantly higher expression than the other groups (Figure 1).
Immunohistochemical staining of PHD3 in renal tubules. (A) Represents diabetic group with strong staining in about 80% of proximal tubules. (B): Vildagliptin treated diabetic group with moderate staining in about 30% of proximal tubules. (C): empagliflozin treated diabetic group with weak staining in about 20% of proximal tubules. (PHD3×400).
Empagliflozin significantly increased the level of HIF-2α in renal tissue. In addition, it nearly neutralizes the tubular expression of PHD3 compared to non-treated and vildagliptin-treated rats. SGLT2 inhibition is effective for correcting the proinflammatory/anti-inflammatory imbalance in DN.
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