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

The Role of the Gut Microbiome in Diabetes and Obesity-Related Kidney Disease

Diabetic kidney disease (DKD) is a progressive disorder, which is increasing globally in prevalence due to the increased incidence of obesity and diabetes mellitus. Despite optimal clinical management, a significant number of patients with diabetes develop DKD. Hence, hitherto unrecognized factors are likely to be involved in the initiation and progression of DKD. An extensive number of studies have demonstrated the role of microbiota in health and disease. Dysregulation in the microbiota resulting in a deficiency of short chain fatty acids (SCFAs) such as propionate, acetate, and butyrate, by-products of healthy gut microbiota metabolism, have been demonstrated in obesity, type 1 and type 2 diabetes. However, it is not clear to date whether such changes in the microbiota are causative or merely associated with the diseases. It is also not clear which microbiota have protective effects on humans. Few studies have investigated the centrality of reduced SCFA in DKD development and progression or the potential therapeutic effects of supplemental SCFAs on insulin resistance, inflammation, and metabolic changes. SCFA receptors are expressed in the kidneys, and emerging data have demonstrated that intestinal dysbiosis activates the renal renin-angiotensin system, which contributes to the development of DKD. In this review, we will summarize the complex relationship between the gut microbiota and the kidney, examine the evidence for the role of gut dysbiosis in diabetes and obesity-related kidney disease, and explore the mechanisms involved. In addition, we will describe the role of potential therapies that modulate the gut microbiota to prevent or reduce kidney disease progression

Linagliptin reduced fibronectin transcription and expression in diabetic mice.

<p><b>(A)</b> Diabetic mice demonstrated increased cortical fibronectin mRNA transcription by real time PCR. This was significantly reduced by linagliptin and telmisartan. <b>(B)</b> There was a significant increase in tubulointerstitial FN expression measured by immunohistochemistry in the diabetic animals and this was reduced with linagliptin. 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). Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, # = P<0.05 vs dm, ## = P<0.01 vs dm.</p

M6P and linagliptin reduced high glucose induced CIM6PR:DPP4 interaction in HK2 cells.

<p><b>(A)</b> Proximity ligation assay demonstrating endogenous protein-protein interaction between membranous DPP4 and CIM6PR in HK2 cells visualised as individual fluorescent dots. This is increased in 30mM high glucose (HG) environment compared to control 5mM glucose (ctrl). M6P at 1μM and linagliptin at 30nM reduced the high glucose induced interaction. A quantitation of this is shown in <b>(B)</b>. Data is represented as mean ± standard error, n = 3. * = P<0.05 compared to 5mM glucose, # = P<0.05 compared to 30mM glucose.</p

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

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

Linagliptin partially reduced tubular atrophy in diabetic mice.

<p>Diabetic mice demonstrated tubular atrophy, which was partially reduced by linagliptin and telmisartan. Representative photographs of PAS stained sections of tubulointerstitium 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 of tubular atrophy in all groups was done by counting the number of atrophic tubules per 400 tubule count (Data are expressed as mean ± SEM with * = P<0.05 vs ctrl).</p

Linagliptin did not reduce glomerulosclerosis in diabetic mice.

<p>Diabetic mice demonstrated increased glomerulosclerosis, which was improved by telmisartan but not by linagliptin as demonstrated by quantification of glomerulosclerosis by glomerulosclerotic index. Representative photographs of PAS stained sections for control (ctrl), control + linagliptin (ctrl + lina), diabetic (dm), diabetic + linagliptin (dm + lina) and diabetic + telmisartan (dm + tel) groups are shown (Magnification = original X400). Data are expressed as mean ± SEM with ** = P<0.01 vs ctrl, ## = P<0.01 vs dm.</p