The Role of SGLT2 Inhibitors and DPP4 Inhibitors in Preventing Diabetic Nephropathy

Diabetic nephropathy is the most common cause of end stage kidney disease in the world. Newer diabetic medications have arrived over the last few years. However there is lack of experimental and clinical evidence in favour of better outcomes. The two most important drug categories that have emerged in this century are the dipeptidyl peptidase-4 inhibitors DPP4 inhibitors (DPP4i) and sodium glucose cotransporter inhibitors (SGLT2i). Their role in preventing diabetic nephropathy irrespective of their glycaemic benefits is unknown. The aim of our project was to demonstrate the renoprotective benefits of these medications. In this thesis we used endothelial nitric oxide synthase knock out (eNOS -/-) mice and induced type 1 diabetes using streptozotocin (STZ) injection. We studied the changes of diabetic nephropathy in these mice including clinical outcomes, biochemical changes, inflammatory and fibrotic pathways. Our study with empagliflozin showed that SGLT2 inhibitors might not have a beneficial role in preventing diabetic nephropathy when blood glucose levels were high. We studied the role of linagliptin, a DPP4i, in preventing the interaction between DPP4 and cation independent mannose-6-phosphate receptor (CIM6PR) in the setting of high glucose in an in vitro model using kidney proximal tubular cells. Our results showed that linagliptin reduced the interaction between DPP4 and CIM6PR possibly resulting in the prevention of activation of latent TGFß. We proved this subsequently in an in vivo model of STZ induced type 1diabetes in eNOS -/- mice using linagliptin and another DPP4i, saxagliptin. We demonstrated that these DPP4i were able to reduce tubulointerstitial fibronectin deposition and demonstrated reduced expression of pSmad2/3, a downstream marker of TGFß activation. Hence our studies have helped in partly identifying the puzzle of diabetic nephropathy and provide some answers on the role of newer anti diabetic agents in preventing it

Linagliptin

<p>WB Images and data analysis for linagliptin project</p

sglt2

<p>sglt2</p

SGLT2 inhibition and prevention of diabetic nephropathy

<p>To look at in vivo eefct of empagliflozin in preventing diabetic nephropathy in the presence of high blood glucose. This study is aimed at looking at renoprotection of SGLT2 inhibition independent of its hypoglycaemic effect.</p

Linagliptin Limits High Glucose Induced Conversion of Latent to Active TGFß through Interaction with CIM6PR and Limits Renal Tubulointerstitial Fibronectin.

In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4) inhibitors have been shown to be antifibrotic. We have previously shown that cation independent mannose-6-phosphate receptor (CIM6PR) facilitates the conversion of latent to active transforming growth factor β1 (GFß1) in renal proximal tubular cells (PTCs) and linagliptin (a DPP4 inhibitor) reduced this conversion with downstream reduction in fibronectin transcription.We wanted to demonstrate that linagliptin reduces high glucose induced interaction between membrane bound DPP4 and CIM6PR in vitro and demonstrate reduction in active TGFß mediated downstream effects in a rodent model of type 1 diabetic nephropathy independent of high glycaemic levels.We used human kidney 2 (HK2) cells and endothelial nitric oxide synthase knock out mice to explore the mechanism and antifibrotic potential of linagliptin independent of glucose lowering. Using a proximity ligation assay, we show that CIM6PR and DPP4 interaction was increased by high glucose and reduced by linagliptin and excess mannose-6-phosphate (M6P) confirming that linagliptin is operating through an M6P-dependent mechanism. In vivo studies confirmed these TGFß1 pathway related changes and showed reduced fibronectin, phosphorylated smad2 and phosphorylated smad2/3 (pSmad2/3) with an associated trend towards reduction in tubular atrophy, which was independent of glucose lowering. No reduction in albuminuria, glomerulosclerotic index or cortical collagen deposition was observed.Linagliptin inhibits activation of TGFß1 through a M6P dependent mechanism. However this in isolation is not sufficient to reverse the multifactorial nature of diabetic nephropathy

Investigating the Regulatory Process, Safety, Efficacy and Product Transparency for Nutraceuticals in the USA, Europe and Australia

Increased numbers of patients with chronic conditions use nutraceuticals or food-based therapeutics. However, to date, there is no global consensus on the regulatory processes for nutraceuticals. With the increased use, issues of quality and safety have also arisen. This review summarises the current regulations held for nutraceuticals in the USA, European and Australian jurisdictions using regulatory authority sites and databases. The efficacy and safety concerns, product development, gaps in regulation and challenges in ensuring product authenticity are also summarised. The data highlight the complexity that the globalisation of nutraceuticals brings with respect to challenges in regulation and associated claims regarding efficacy and safety. The development of an effective system with integrity is needed to increase vertical collaboration between consumers, healthcare practitioners, and government agencies and the development of international risk assessment criteria and botanical compendia. This will help in greater transparency and improved trust in the process and products. Emerging technologies could play a role in improving systems engineering by information sharing and leveraging the strengths of different countries. In conclusion, nutraceuticals have been poorly regulated leading to spurious claims based on little or no real evidence. This makes it difficult to separate meaningful results from poor data. More stringent regulation and an effective system of integrity are required to ensure efficacy and safety and enable the adequate monitoring and increase consumer and healthcare professionals’ confidence

Linagliptin reduced tubular pSmad 2/3 expression (a marker of transforming growth factor beta activation) in diabetic mice and showed a trend towards reduced cortical pSmad 2 expression on western blot analysis in diabetic mice.

<p>A) Diabetic mice demonstrated increased pSmad2/3 nuclear expression with immunohistochemistry, which was reduced by linagliptin and telmisartan. Representative photographs for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X 200). Quantification was done by counting the number of positive nuclei at X200 magnification. Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, ## = P<0.01 vs dm. B) Diabetic mice showed a trend towards increase in pSmad 2/total smad2 expression compared to control mice and a reduction with both linagliptin and telmisartan. This trend was consistent with the immunohistochemistry findings but did not reach statistical significance. Quantification was done using Image J. Data are expressed as mean ± SEM, n = 6.</p

Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3

Metabolic and physical parameters of mice.

<p>Values are shown as mean ± SEM.</p><p>* = P<0.05 vs control</p><p>** = P<0.01 vs control</p><p>^## = P<0.01 vs diabetic</p><p>Metabolic and physical parameters of mice.</p