Takako Nagai

Linagliptin-Mediated DPP-4 Inhibition Ameliorates Kidney Fibrosis in Streptozotocin-Induced Diabetic Mice by Inhibiting Endothelial-to-Mesenchymal Transition in a Therapeutic Regimen

Kidney fibrosis is the final common pathway of all progressive chronic kidney diseases, of which diabetic nephropathy is the leading cause. Endothelial-to-mesenchymal transition (EndMT) has emerged as one of the most important origins of matrix-producing fibroblasts. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been introduced into the market as antidiabetes drugs. Here, we found that the DPP-4 inhibitor linagliptin ameliorated kidney fibrosis in diabetic mice without altering the blood glucose levels associated with the inhibition of EndMT and the restoration of microRNA 29s. Streptozotocin-induced diabetic CD-1 mice exhibited kidney fibrosis and strong immunoreactivity for DPP-4 by 24 weeks after the onset of diabetes. At 20 weeks after the onset of diabetes, mice were treated with linagliptin for 4 weeks. Linagliptin-treated diabetic mice exhibited a suppression of DPP-4 activity/protein expression and an amelioration of kidney fibrosis associated with the inhibition of EndMT. The therapeutic effects of linagliptin on diabetic kidneys were associated with the suppression of profibrotic programs, as assessed by mRNA microarray analysis. We found that the induction of DPP-4 observed in diabetic kidneys may be associated with suppressed levels of microRNA 29s in diabetic mice; linagliptin restored microRNA 29s and suppressed DPP-4 protein levels. Using cultured endothelial cells, we found that linagliptin inhibited TGF-β2–induced EndMT, and such anti-EndMT effects of linagliptin were mediated through microRNA 29 induction. These results indicate the possible novel pleiotropic action of linagliptin to restore normal kidney function in diabetic patients with renal impairment.

Dietary restriction ameliorates diabetic nephropathy through anti-inflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes.

Aim. Despite the beneficial effects of dietary restriction (DR) on lifespan, age-related diseases, including diabetes and cardiovascular diseases, its effects on type 2 diabetic nephropathy remain unknown. This study examined the renoprotective effects of DR in Wistar fatty (fa/fa) rats (WFRs). Methods. WFRs were treated with DR (40% restriction) for 24 weeks. Urinary albumin excretion, creatinine clearance, renal histologies, acetylated-NF-κB (p65), Sirt1 protein expression, and p62/Sqstm 1 accumulation in the renal cortex, as well as electron microscopic observation of mitochondrial morphology and autophagosomes in proximal tubular cells were estimated. Results. DR ameliorated renal abnormalities including inflammation in WFRs. The decrease in Sirt1 levels, increase in acetylated-NF-κB, and impaired autophagy in WFRs were improved by DR. Conclusions. DR exerted anti-inflammatory effects and improved the dysregulation of autophagy through the restoration of Sirt1 in the kidneys of WFRs, which resulted in the amelioration of renal injuries in type 2 diabetes.

Interactions of DPP-4 and integrin β1 influences endothelial-to-mesenchymal transition

Integrin β1 and dipeptidyl peptidase (DPP)-4 play roles in endothelial cell biology. Vascular endothelial growth factor (VEGF)-A inhibits endothelial-to-mesenchymal transition (EndMT) through VEGF-R2, but through VEGF-R1 promotes EndMT by reducing the bioavailability of VEGF-A. Here we tested whether DPP-4-integrin β1 interactions have a role in EndMT in the renal fibrosis of diabetic nephropathy. In streptozotocin-induced fibrotic kidneys in diabetic CD-1 mice, levels of endothelial DPP-4, integrin β1, and phospho-integrin β1 were all higher and associated with plasma cystatin C elevation. The DPP-4 inhibitor linagliptin ameliorated kidney fibrosis, reduced plasma cystatin C levels, and suppressed endothelial levels of DPP-4, integrin β1, and phospho-integrin β1. In cultured endothelial cells, DPP-4 and integrin β1 physically interacted. Suppression of DPP-4 by siRNA was associated with suppression of integrin β1 and vice versa. Knockdown of either integrin β1 or DPP-4 resulted in the silencing of TGF-β2-induced TGF-β receptor heterodimer formation, smad3 phosphorylation, and EndMT. DPP-4 negatively regulated endothelial viability signaling by VEGF-R2 suppression and VEGF-R1 induction in endothelial cells. Thus, DPP-4 and integrin β1 interactions regulate key endothelial cell signal transduction in both physiological and pathological conditions including EndMT. Hence, inhibiting DPP-4 may be a therapeutic target for treating kidney fibrosis in diabetes.

N-acetyl-seryl-aspartyl-lysyl-proline Inhibits Diabetes-Associated Kidney Fibrosis and Endothelial-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) emerges as an important source of fibroblasts. MicroRNA let-7 exhibits anti-EndMT effects and fibroblast growth factor (FGF) receptor has been shown to be an important in microRNA let-7 expression. The endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a substrate of angiotensin-converting enzyme (ACE). Here, we found that AcSDKP inhibited the EndMT and exhibited fibrotic effects that were associated with FGF receptor-mediated anti-fibrotic program. Conventional ACE inhibitor plus AcSDKP ameliorated kidney fibrosis and inhibited EndMT compared to therapy with the ACE inhibitor alone in diabetic CD-1 mice. The endogenous AcSDKP levels were suppressed in diabetic animals. Cytokines induced cultured endothelial cells into EndMT; coincubation with AcSDKP inhibited EndMT. Expression of microRNA let-7 family was suppressed in the diabetic kidney; antifibrotic and anti-EndMT effects of AcSDKP were associated with the restoration of microRNA let-7 levels. AcSDKP restored diabetes- or cytokines-suppressed FGF receptor expression/phosphorylation into normal levels both in vivo and in vitro. These results suggest that AcSDKP is an endogenous antifibrotic molecule that has the potential to cure diabetic kidney fibrosis via an inhibition of the EndMT associated with the restoration of FGF receptor and microRNA let-7.

Elevation of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline: a blood pressure-independent beneficial effect of angiotensin I-converting enzyme inhibitors

Blockade of the renin-angiotensin system (RAS) is well recognized as an essential therapy in hypertensive, heart, and kidney diseases. There are several classes of drugs that block the RAS; these drugs are known to exhibit antifibrotic action. An analysis of the molecular mechanisms of action for these drugs can reveal potential differences in their antifibrotic roles. In this review, we discuss the antifibrotic action of RAS blockade with an emphasis on the potential importance of angiotensin I-converting enzyme (ACE) inhibition associated with the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP).

Effect of Antifibrotic MicroRNAs Crosstalk on the Action of N-acetyl-seryl-aspartyl-lysyl-proline in Diabetes-related Kidney Fibrosis

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous antifibrotic peptide. We found that suppression of AcSDKP and induction of dipeptidyl peptidase-4 (DPP-4), which is associated with insufficient levels of antifibrotic microRNA (miR)s in kidneys, were imperative to understand the mechanisms of fibrosis in the diabetic kidneys. Analyzing streptozotocin (STZ)-induced diabetic mouse strains, diabetic CD-1 mice with fibrotic kidneys could be differentiated from less-fibrotic diabetic 129Sv mice by suppressing AcSDKP and antifibrotic miRs (miR-29s and miR-let-7s), as well as by the prominent induction of DPP-4 protein expression/activity and endothelial to mesenchymal transition. In diabetic CD-1 mice, these alterations were all reversed by AcSDKP treatment. Transfection studies in culture endothelial cells demonstrated crosstalk regulation of miR-29s and miR-let-7s against mesenchymal activation program; such bidirectional regulation could play an essential role in maintaining the antifibrotic program of AcSDKP. Finally, we observed that AcSDKP suppression in fibrotic mice was associated with induction of both interferon-γ and transforming growth factor-β signaling, crucial molecular pathways that disrupt antifibrotic miRs crosstalk. The present study provides insight into the physiologically relevant antifibrotic actions of AcSDKP via antifibrotic miRs; restoring such antifibrotic programs could demonstrate potential utility in combating kidney fibrosis in diabetes.

N-acetyl-seryl-aspartyl-lysyl-proline: a valuable endogenous anti-fibrotic peptide for combating kidney fibrosis in diabetes

Fibroproliferative diseases are responsible for 45% of deaths in the developed world. Curing organ fibrosis is essential for fibroproliferative diseases. Diabetic nephropathy is a common fibroproliferative disease of the kidney and is associated with multiorgan dysfunction. However, therapy to combat diabetic nephropathy has not yet been established. In this review, we discuss the novel therapeutic possibilities for kidney fibrosis in diabetes focusing on the endogenous anti-fibrotic peptide, N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), which is the substrate for angiotensin-converting enzyme and exhibits meaningful anti-fibrotic effects in various experimental models of fibrotic disease.

Oral Administration of N-Acetyl-seryl-aspartyl-lysyl-proline Ameliorates Kidney Disease in Both Type 1 and Type 2 Diabetic Mice via a Therapeutic Regimen

Kidney fibrosis is the final common pathway of progressive kidney diseases including diabetic nephropathy. Here, we report that the endogenous antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), the substrate of angiotensin-converting enzyme (ACE), is an orally available peptide drug used to cure kidney fibrosis in diabetic mice. We utilized two mouse models of diabetic nephropathy, streptozotocin- (STZ-) induced type 1 diabetic CD-1 mice and type 2 diabetic nephropathy model db/db mice. Intervention with the ACE inhibitor imidapril, oral AcSDKP, or imidapril + oral AcSDKP combination therapy increased urine AcSDKP levels. AcSDKP levels were significantly higher in the combination group compared to those of the other groups. AcSDKP oral administration, either AcSDKP alone or in addition to imidapril, ameliorated glomerulosclerosis and tubulointerstitial fibrosis. Plasma cystatin C levels were higher in both models, at euthanasia, and were restored by all the treatment groups. The levels of antifibrotic miRs, such as miR-29 or let-7, were suppressed in the kidneys of both models; all treatments, especially the combination of imidapril + oral AcSDKP, restored the antifibrotic miR levels to a normal value or even higher. AcSDKP may be an oral antifibrotic peptide drug that would be relevant to combating fibroproliferative kidney diseases such as diabetic nephropathy.

The biological significance of angiotensin-converting enzyme inhibition to combat kidney fibrosis.

Both angiotensin-converting enzyme inhibitor (ACE-I) and angiotensin II receptor blocker have been recognized as renin–angiotensin system (RAS) inhibitors. These two RAS inhibitors are rarely recognized as drugs with distinct pharmacological effects in the clinic or most clinical trials. Some preclinical basic research and clinical trials indicate that ACE-I might display superior organ-protective effects, especially anti-fibrotic effects. Such anti-fibrotic effects of ACE-I could be associated with an endogenous anti-fibrotic peptide, N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). In this review, we focused on the anti-fibrotic effects of RAS inhibition and the endogenous anti-fibrotic peptide AcSDKP.

FGFR1 is critical for the anti-endothelial mesenchymal transition effect of N -acetyl-seryl-aspartyl-lysyl-proline via induction of the MAP4K4 pathway

Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to organ fibrogenesis, and we have reported that the anti-EndMT effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is associated with restoring expression of diabetes-suppressed fibroblast growth factor receptor (FGFR), the key anti-EndMT molecule. FGFR1 is the key inhibitor of EndMT via the suppression of the transforming growth factor β (TGFβ) signaling pathway, and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) inhibits integrin β1, a key factor in activating TGFβ signaling and EndMT. Here, we showed that the close proximity between AcSDKP and FGFR1 was essential for the suppression of TGFβ/smad signaling and EndMT associated with MAP4K4 phosphorylation (P-MAP4K4) in endothelial cells. In cultured human dermal microvascular endothelial cells (HMVECs), the anti-EndMT and anti-TGFβ/smad effects of AcSDKP were lost following treatment with a neutralizing FGFR1 antibody (N-FGFR1) or transfection of FRS2 siRNA. The physical interaction between FGFR1 and P-MAP4K4 in HMVECs was confirmed by proximity ligation analysis and an immunoprecipitation assay. AcSDKP induced P-MAP4K4 in HMVECs, which was significantly inhibited by treatment with either N-FGFR1 or FRS2 siRNA. Furthermore, MAP4K4 knockdown using specific siRNAs induced smad3 phosphorylation and EndMT in HMVECs, which was not suppressed by AcSDKP. Streptozotocin-induced diabetic CD-1 mice exhibited suppression of both FGFR1 and P-MAP4K4 expression levels associated with the induction of TGFβ/smad3 signaling and EndMT in their hearts and kidneys; those were restored by AcSDKP treatment. These data demonstrate that the AcSDKP–FGFR1–MAP4K4 axis has an important role in combating EndMT-associated fibrotic disorders.