Dipeptidyl peptidase 4 (DPP-4) inhibitors exert pleiotropic effects beyond glycemic control. We investigated the renoprotective effects of DPP-4 inhibitors on aging mice mediated by the renin-angiotensin system (RAS). C57BL/6 mice were divided into three groups: the two-month-old mice (YM group), the eighteen-month-old mice (AM group) and the eighteen-month-old, linagliptin-treated mice (AM + LIN group). Renal function was improved, based on serum creatinine and cystatin-C levels (p < 0.05 compared with the AM group for both parameters). Fibrotic areas and the levels of proteins related to fibrosis improved in the AM + LIN group (p < 0.001 compared with the AM group for all parameters). In the AM + LIN group, the DPP-4-positive area and activity and expressions of DPP-4 were decreased (p < 0.05 compared with the AM group for all parameters). The levels of proteins related to the RAS, including prorenin receptor, angiotensin-converting enzyme, angiotensin II and angiotensin 1 receptor, were decreased in the AM + LIN group (p < 0.05, p < 0.01, p < 0.05, and p < 0.01 compared with the AM group, respectively). NADPH oxidase 2 and NADPH oxidase 4 levels decreased in the AM + LIN group (p < 0.001 compared with the AM group for both proteins), whereas the levels of endothelial nitric oxide synthase (eNOS) phosphorylated at serine1177 and superoxide dismutase 1 were increased (p < 0.01 compared with the AM group for both proteins). DPP-4 inhibitors may exert renoprotective effects via prorenin receptor/angiotensin-converting enzyme/angiotensin II/angiotensin 1 receptor axis.
Figure 1. Effects of linagliptin on renal function and aging-related renal injury histology. (A) Serum creatinine levels were increased in the AM group compared to the AM group (p < 0.001), and decreased in the AM + LIN group compared to the AM group (p < 0.05). (B) Albuminuria was increased in the AM group, but it was not different in the AM and AM + LIN groups. (C) Creatinine clearance was decreased in the AM group, but it was similar in the AM and AM + LIN groups. (D) Serum cystatin-C levels were increased in the AM group compared to the AM group (p < 0.001) and decreased in the AM + LIN group compared to the AM group (p < 0.05). (E) The expansion of the mesangial area of PAS-stained kidneys was significantly reduced in the AM + LIN group (original magnification ×400). (F) Decreased tubulointerstitial fibrosis was observed in Masson’s trichrome-stained kidney sections from the AM + LIN group (original magnification ×400). (G) The areas of extracellular matrix in the glomerulus and (H) the areas of tubulointerstitial fibrosis determined using a quantitative assessment were definitely decreased in the AM + LIN group (p < 0.001 for both). (*p < 0.05 and ‡p < 0.001).
Figure 2. Effects of linagliptin on fibrosis and inflammation in renal tissue. (A) The numbers of collagen IV-, (B) TGF-β- and (C) MCP-1-positive cells were decreased in the AM + LIN group compared to the AM group. (D), (E) and (F) The areas positive for collagen IV, TGF-β and MCP-1 were markedly increased in the AM + LIN group. (G) Levels of markers fibrosis and inflammation were analyzed using western blotting. (H) and (I) Comparison of fibronectin and collagen IV levels between the three groups. (J) and (K) Comparison of TGF-β and CTGF levels between the three groups. (*p < 0.05, †p < 0.01, and ‡p < 0.001).
Figure 3. Effects of linagliptin on DPP-4 levels in renal tissues and serum DPP-4 activity. (A) Representative images of immunohistochemical staining for DPP-4 showing positively stained areas in the renal tissue. (B) The DPP-4-positive area was markedly decreased in the AM + LIN group compared with the AM group (‡p < 0.001). (C) Representative western blots showing DPP-4 levels in the renal tissue. (D) Lower levels of the DPP-4 protein were detected in the AM + LIN group than in the AM group (p < 0.05) (E) Lower DPP-4 activity was observed in the AM + LIN group than in the AM group (p < 0.05). (F) The serum DPP-4 activity was increased in the AM group compared with the YM group (p < 0.001), but it was not different between the AM group and the AM + LIN group. (*p < 0.05 and ‡p < 0.001).
Figure 4. Effects of linagliptin on angiotensin II levels and PRR, ACE and AT1R protein expression in kidneys. (A) The levels of Ang II in the kidneys were decreased in the AM + LIN group compared with the AM group (p < 0.05). (B) Representative western blots of PRR, ACE and AT1R protein expression. (C) Lower levels of the PRR protein were detected in the AM + LIN group than in the AM group (p < 0.05). (D) Lower levels of the ACE protein were observed in the AM + LIN group than in the AM group (p < 0.01). (E) Lower levels of the AT1R protein were observed in the AM + LIN group than in the AM group (p < 0.01). (*p < 0.05, †p < 0.01, and ‡p < 0.001).
Figure 5. Effects of linagliptin on angiotensin (1-7) concentrations and ACEII, AT2R and MasR protein expression in kidneys. (A) Representative western blots of ACEII, AT2R and MasR protein expression. (B), (C) and (D) Levels of the ACEII, AT2R and MasR proteins were decreased in the AM group compared to the YM group (p < 0.05 for all), but the levels were similar in the AM and AM + LIN groups. (E) Lower levels of Ang (1-7) were detected in the kidneys from the AM group than in the YM group (p < 0.001), but similar levels were observed in the AM and AM+LIN groups. (*p < 0.05 and ‡p < 0.001).
Figure 6. Effects of linagliptin on phospho-Ser1177eNOS/eNOS, NOX2 and NOX4 in renal tissue. (A) Representative western blots of phospho-Ser1177eNOS/eNOS expression. (B) The ratio of phospho-Ser1177 eNOS/eNOS was significantly increased in the AM + LIN group (p < 0.05). (C) Representative western blots of Nox2 and Nox4 levels. (D) Nox2 levels were decreased in the AM + LIN group compared with the AM group (p < 0.001). (E) Nox4 levels were significantly decreased in the AM + LIN group (p < 0.001). (*p < 0.05, †p < 0.01, and ‡p < 0.001).
Figure 7. Effects of linagliptin on SOD1 and SOD2 in renal tissue. (A) Representative western blots of SOD1 and SOD2 levels. (B) SOD1 levels were increased in the AM + LIN group compared with the AM group (p < 0.01). (C) SOD2 levels were decreased in the AM group compared to the YM group (p < 0.05), but the levels were not significantly different between the AM group and the AM + LIN group. (*p < 0.05 and †p < 0.01).
Figure 8. Effects of linagliptin on renal tubular cell injury in HRPTEpiCs. (A) The Ang II-treated group showed an increased number of SA β-gal-positive cells compared to the Cont group. Treatment with LIN decreased the number of SA β-gal-positive cells in the Ang II group, whereas LIN treatment did not affect that in the Cont group. (B) Representative western blots of expression of the renin-angiotensin system in HRPTEpiCs. (C) and (D) Ang II increased the expression of ACE and AT1R compared to the control (p < 0.05 for both), but those effects diminished after adding linagliptin to the Ang II-treated cells (p < 0.01 and p < 0.05, respectively). (E) and (F) The expression levels of AT2R and MasR in the Ang II groups were not different regardless of LIN treatment. (G) Representative western blots of expression of the markers of anti-inflammatory and antioxidant systems in HRPTEpiCs. (H) and (I) The levels of fibronectin and collagen IV were increased in HRPTEpiCs treated with Ang II (p < 0.05 and p < 0.001, respectively), but those effects were stabilized after the administration of linagliptin to Ang II-treated cells (p < 0.05 and p < 0.01, respectively). (J) and (K) The expression levels of SOD1 and SOD2 were not significantly changed in HRPTEpiCs treated with Ang II. The expression of SOD1 improved after adding linagliptin to Ang II-treated cells (p < 0.05), but the expression of SOD2 did not change even after the addition of linagliptin. (L) Representative western blots showing decreased levels of DPP-4 and AT1R in HRPTEpiCs transfected with siRNAs targeting DPP-4. (M) and (N) DPP-4 and AT1R levels were significantly decreased in cells transfected with the siRNA targeting DPP-4 (p < 0.001 and p < 0.05, respectively). (*p < 0.05, †p < 0.01, and ‡p < 0.001).
Centers for Disease Control and Prevention (2013). The State of Aging and Healthy in America. Atlanta, GA: Createspace Independent Publisher.
European Commission (2015). The 2015 Ageing Report: Economic and Budgetary Projections for the 28 EU Member States (2013-2060). Brussels: European Economy Series 3/2015, 1-424.
Statistics Korea (2015). Results of the 2015 Population and Housing Census (Population, Household and Housing). Daejeon: Statistics Korea.
Jin DC, Yun SR, Lee SW, Han SW, Kim W, Park J (2016). Current characteristics of dialysis therapy in Korea: 2015 registry data focusing on elderly patients. Kidney Res Clin Pract, 35:204-11.
Weiss JW, Thorp ML, O'Hare AM (2010). Renin-angiotensin system blockade in older adults with chronic kidney disease: a review of the literature. Curr Opin Nephrol Hypertens, 19:413-9.
Kanasaki K, Kitada M, Koya D (2012). Pathophysiology of the aging kidney and therapeutic interventions. Hypertens Res, 35:1121-8.
Nitta K, Okada K, Yanai M, Takahashi S (2013). Aging and chronic kidney disease. Kidney Blood Press Res, 38:109-20.
Bolignano D, Mattace-Raso F, Sijbrands EJ, Zoccali C (2014). The aging kidney revisited: a systematic review. Ageing Res Rev, 14:65-80.
O'Sullivan ED, Hughes J, Ferenbach DA (2017). Renal aging: causes and consequences. J Am Soc Nephrol, 28:407-20.
Coresh J, Astor BC, Greene T, Eknoyan G, Levey AS (2003). Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third National Health and Nutrition Examination Survey. Am J Kidney Dis, 41:1-12.
Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, et al (2007). Prevalence of chronic kidney disease in the United States. JAMA, 298:2038-47.
Levey AS, De Jong PE, Coresh J, El Nahas M, Astor BC, Matsushita K, et al (2011). The definition, classification, and prognosis of chronic kidney disease: a KDIGO controversies conference report. Kidney Int, 80:17-28.
Siragy HM, Carey RM (2010). Role of the intrarenal renin-angiotensin-aldosterone system in chronic kidney disease. Am J Nephrol, 31:541-50.
Kim Y, Park CW (2016). Adenosine monophosphate-activated protein kinase in diabetic nephropathy. Kidney Res Clin Pract, 35:69-77.
Kim Y, Park CW (2017). New therapeutic agents in diabetic nephropathy. Korean J Intern Med, 32:11-25.
Aroor AR, Sowers JR, Jia G, DeMarco VG (2014). Pleiotropic effects of the dipeptidylpeptidase-4 inhibitors on the cardiovascular system. Am J Physiol Heart Circ Physiol, 307:H477-H92.
Glorie LL, Verhulst A, Matheeussen V, Baerts L, Magielse J, Hermans N, et al (2012). DPP4 inhibition improves functional outcome after renal ischemia-reperfusion injury. Am J Physiol Renal Physiol, 303:F681-F8.
Joo KW, Kim S, Ahn SY, Chin HJ, Chae DW, Lee J, et al (2013). Dipeptidyl peptidase IV inhibitor attenuates kidney injury in rat remnant kidney. BMC Nephrol, 14:98.
Katagiri D, Hamasaki Y, Doi K, Okamoto K, Negishi K, Nangaku M, et al (2013). Protection of glucagon-like peptide-1 in cisplatin-induced renal injury elucidates gut-kidney connection. J Am Soc Nephrol, 24:2034-43.
Min HS, Kim JE, Lee MH, Song HK, Kang YS, Lee MJ, et al (2014). Dipeptidyl peptidase IV inhibitor protects against renal interstitial fibrosis in a mouse model of ureteral obstruction. Lab Invest, 94:598-607.
Lim SW, Jin L, Piao SG, Chung BH, Yang CW (2015). Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury. Lab Invest, 95:1174-85.
Theiss HD, Brenner C, Engelmann MG, Zaruba MM, Huber B, Henschel V, et al (2010). Safety and efficacy of SITAgliptin plus GRanulocyte-colony-stimulating factor in patients suffering from Acute Myocardial Infarction (SITAGRAMI-Trial)--rationale, design and first interim analysis. Int J Cardiol, 145:282-4.
Shah Z, Kampfrath T, Deiuliis JA, Zhong J, Pineda C, Ying Z, et al (2011). Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis. Circulation, 124:2338-49.
Mentlein R (1999). Dipeptidyl-peptidase IV (CD26)--role in the inactivation of regulatory peptides. Regul Pept, 85:9-24.
Von Websky K, Reichetzeder C, Hocher B (2014). Physiology and pathophysiology of incretins in the kidney. Curr Opin Nephrol Hypertens, 23:54-60.
Chaykovska L, Alter ML, von Websky K, Hohmann M, Tsuprykov O, Reichetzeder C, et al (2013). Effects of telmisartan and linagliptin when used in combination on blood pressure and oxidative stress in rats with 2-kidney-1-clip hypertension. J Hypertens, 31:2290-8; discussion 9.
Nakashima S, Matsui T, Takeuchi M, Yamagishi SI (2014). Linagliptin blocks renal damage in type 1 diabetic rats by suppressing advanced glycation end products-receptor axis. Horm Metab Res, 46:717-21.
Lim JH, Kim EN, Kim MY, Chung S, Shin SJ, Kim HW, et al (2012). Age-associated molecular changes in the kidney in aged mice. Oxid Med Cell Longev, 2012:171383.
Paneni F, Costantino S, Cosentino F (2015). Molecular pathways of arterial aging. Clin Sci (Lond), 128:69-79.
Czypiorski P, Rabanter LL, Altschmied J, Haendeler J (2013). Redox balance in the aged endothelium. Z Gerontol Geriatr, 46:635-8.
Elinder CG, Barany P, Heimburger O (2014). The use of estimated glomerular filtration rate for dose adjustment of medications in the elderly. Drugs Aging, 31:493-9.
Muskiet MH, Smits MM, Morsink LM, Diamant M (2014). The gut-renal axis: do incretin-based agents confer renoprotection in diabetes? Nat Rev Nephrol, 10:88-103.
Tanaka T, Higashijima Y, Wada T, Nangaku M (2014). The potential for renoprotection with incretin-based drugs. Kidney Int, 86:701-11.
Nistala R, Savin V (2017). Diabetes, hypertension, and chronic kidney disease progression: role of DPP4. Am J Physiol Renal Physiol, 312:F661-F70.
Kanasaki K, Shi S, Kanasaki M, He J, Nagai T, Nakamura Y, et al (2014). Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen. Diabetes, 63:2120-31.
Sharkovska Y, Reichetzeder C, Alter M, Tsuprykov O, Bachmann S, Secher T, et al (2014). Blood pressure and glucose independent renoprotective effects of dipeptidyl peptidase-4 inhibition in a mouse model of type-2 diabetic nephropathy. J Hypertens, 32:2211-23; discussion 23.
Srivastava SP, Shi S, Kanasaki M, Nagai T, Kitada M, He J, et al (2016). Effect of Antifibrotic MicroRNAs Crosstalk on the Action of N-acetyl-seryl-aspartyl-lysyl-proline in Diabetes-related Kidney Fibrosis. Sci Rep, 6:29884.
Tsai TH, Sun CK, Su CH, Sung PH, Chua S, Zhen YY, et al (2015). Sitagliptin attenuated brain damage and cognitive impairment in mice with chronic cerebral hypo-perfusion through suppressing oxidative stress and inflammatory reaction. J Hypertens, 33:1001-13.
Zheng T, Qin L, Chen B, Hu X, Zhang X, Liu Y, et al (2016). Association of plasma DPP4 activity with mild cognitive impairment in elderly patients with type 2 diabetes: results from the GDMD study in China. Diabetes Care, 39:1594-601.
Lei Y, Yang G, Hu L, Piao L, Inoue A, Jiang H, et al (2017). Increased dipeptidyl peptidase-4 accelerates diet-related vascular aging and atherosclerosis in ApoE-deficient mice under chronic stress. Int J Cardiol, 243:413-20.
Korosi J, McIntosh CH, Pederson RA, Demuth HU, Habener JF, Gingerich R, et al (2001). Effect of aging and diabetes on the enteroinsular axis. J Gerontol A Biol Sci Med Sci, 56:M575-9.
Vedantham S, Kluever AK, Deindl E (2018). Is there a chance to promote arteriogenesis by DPP4 inhibitors even in type 2 diabetes? A critical review. Cells, 7:181.
Yoon HE, Choi BS (2014). The renin-angiotensin system and aging in the kidney. Korean J Intern Med, 29:291-5.
Carey RM (2007). Angiotensin receptors and aging. Hypertension, 50:33-4.
Schulman IH, Zhou MS, Treuer AV, Chadipiralla K, Hare JM, Raij L (2010). Altered renal expression of angiotensin II receptors, renin receptor, and ACE-2 precede the development of renal fibrosis in aging rats. Am J Nephrol, 32:249-61.
Yoon HE, Kim EN, Kim MY, Lim JH, Jang IA, Ban TH, et al (2016). Age-associated changes in the vascular renin-angiotensin system in mice. Oxid Med Cell Longev, 2016:14.
Miyata N, Park F, Li XF, Cowley AW (1999). Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney. Am J Physiol, 277:F437-F46.
Mann JF, Schmieder RE, Dyal L, McQueen MJ, Schumacher H, Pogue J, et al (2009). Effect of telmisartan on renal outcomes: a randomized trial. Ann Intern Med, 151:1-10, w1-2.
Saruta T, Hayashi K, Ogihara T, Nakao K, Fukui T, Fukiyama K (2009). Effects of candesartan and amlodipine on cardiovascular events in hypertensive patients with chronic kidney disease: subanalysis of the CASE-J Study. Hypertens Res, 32:505-12.
Marney A, Kunchakarra S, Byrne L, Brown NJ (2010). Interactive hemodynamic effects of dipeptidyl peptidase-IV inhibition and angiotensin-converting enzyme inhibition in humans. Hypertension, 56:728-33.
Chang MW, Chen CH, Chen YC, Wu YC, Zhen YY, Leu S, et al (2015). Sitagliptin protects rat kidneys from acute ischemia-reperfusion injury via upregulation of GLP-1 and GLP-1 receptors. Acta Pharmacol Sin, 36:119-30.
Montezano AC, Nguyen Dinh Cat A, Rios FJ, Touyz RM (2014). Angiotensin II and vascular injury. Curr Hypertens Rep, 16:431.
Zhang X, Yang J, Yu X, Cheng S, Gan H, Xia Y (2017). Angiotensin II-induced early and late inflammatory responses through NOXs and MAPK pathways. Inflammation, 40:154-65.
Somanna NK, Valente AJ, Krenz M, Fay WP, Delafontaine P, Chandrasekar B (2016). The Nox1/4 dual inhibitor GKT137831 or Nox4 knockdown inhibits angiotensin-II-induced adult mouse cardiac fibroblast proliferation and migration. AT1 physically associates with Nox4. J Cell Physiol, 231:1130-41.
Robin Gourmelon, Sandrine Donadio-Andréi, Karim Chikh, Muriel Rabilloud, Elisabetta Kuczewski, Anne-Sophie Gauchez, Anne Charrié, Pierre-Yves Brard, Raphaëlle Andréani, Jean-Cyril Bourre, Christine Waterlot, Domitille Guédel, Anne Mayer, Emmanuel Disse, Charles Thivolet, Hélène Du Boullay, Claire Falandry, Thomas Gilbert, Anne François-Joubert, Antoine Vignoles, Catherine Ronin, Marc Bonnefoy. Subclinical Hypothyroidism: is it Really Subclinical with Aging?[J]. Aging and disease, 2019, 10(3): 520-529.