Reduced adenosine A2a receptor–mediated efferent arteriolar vasodilation contributes to diabetes-induced glomerular hyperfiltration
Authors: Patrik Persson, Peter Hansell, Fredrik Palm
Journal: Kidney International (2014)

Diabetes is associated with increased risk for development of kidney disease, and an increased glomerular filtration rate is an early indication of altered kidney function. Here we determine whether reduced adenosine A2a receptor–mediated vasodilation of the efferent arteriole contributes to the increased glomerular filtration rate in diabetes. The glomerular filtration rate, renal blood flow, and proximal tubular stop flow pressure were investigated in control and streptozotocin-diabetic rats during baseline and after administration of the adenosine A2a receptor antagonist ZM241385 or the adenosine A2a receptor agonist CGS21680. The diabetes-induced glomerular hyperfiltration was reduced by 24% following A2a receptor stimulation but was unaffected by A2a receptor inhibition. Contrarily, glomerular filtration rate in controls increased by 22% after A2a receptor inhibition and was unaffected by A2a stimulation. The increased glomerular filtration rate after A2a receptor inhibition in controls and decreased glomerular filtration rate after A2a receptor activation in diabetics were caused by increased and decreased stop flow pressure, respectively. None of the interventions affected renal blood flow. Thus, the normal adenosine A2a receptor–mediated tonic vasodilation of efferent arterioles is abolished in the diabetic kidney. This causes increased efferent arteriolar resistance resulting in increased filtration fraction and hyperfiltration. Diabetes is associated with increased risk for development of kidney disease, and an increased glomerular filtration rate is an early indication of altered kidney function. Here we determine whether reduced adenosine A2a receptor–mediated vasodilation of the efferent arteriole contributes to the increased glomerular filtration rate in diabetes. The glomerular filtration rate, renal blood flow, and proximal tubular stop flow pressure were investigated in control and streptozotocin-diabetic rats during baseline and after administration of the adenosine A2a receptor antagonist ZM241385 or the adenosine A2a receptor agonist CGS21680. The diabetes-induced glomerular hyperfiltration was reduced by 24% following A2a receptor stimulation but was unaffected by A2a receptor inhibition. Contrarily, glomerular filtration rate in controls increased by 22% after A2a receptor inhibition and was unaffected by A2a stimulation. The increased glomerular filtration rate after A2a receptor inhibition in controls and decreased glomerular filtration rate after A2a receptor activation in diabetics were caused by increased and decreased stop flow pressure, respectively. None of the interventions affected renal blood flow. Thus, the normal adenosine A2a receptor–mediated tonic vasodilation of efferent arterioles is abolished in the diabetic kidney. This causes increased efferent arteriolar resistance resulting in increased filtration fraction and hyperfiltration. Diabetes is associated with several complications including nephropathy, which is a major cause for end-stage renal disease requiring renal replacement therapy. One of the earliest clinical signs of altered kidney function in diabetes is increased glomerular filtration rate (GFR), which is considered to be an early risk factor for disease progression. Recent studies suggest that glomerular hyperfiltration is associated with a progressive decline in renal function,1Moriya T. Tsuchiya A. Okizaki S. et al.Glomerular hyperfiltration and increased glomerular filtration surface are associated with renal function decline in normo- and microalbuminuric type 2 diabetes.Kidney Int. 2012; 81: 486-493Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar leading to a shorter time until initiation of dialysis, although microalbuminuria may not develop.2Thomas M.C. Moran J.L. Harjutsalo V. et al.Hyperfiltration in type 1 diabetes: does it exist and does it matter for nephropathy?.Diabetologia. 2012; 55: 1505-1513Crossref PubMed Scopus (36) Google Scholar The mechanisms for diabetes-induced glomerular hyperfiltration have been the subject of extensive research, but are still largely unclear. GFR is determined by the hydrostatic pressure in the glomerular capillaries (PGC) resulting from the relative vascular resistances of the afferent and efferent arterioles. PGC will be opposed by the hydrostatic pressure in Bowman's space (PBow) and the plasma colloid osmotic pressure in the glomerular capillaries (πGC) resulting in a net filtration pressure (PNET), which is the driving force for filtration. Finally, the filtration coefficient (Kf), which is dependent on the filtration area and permeability of the glomerular membrane, determines the actual filtration for each mmHg of PNET. Hence, GFR increases if PGC increases, either by a dilation of the afferent arteriole with a subsequent increase in renal blood flow (RBF) or by constriction of the efferent arteriole with minimal effects on RBF. Thus, diabetes-induced glomerular hyperfiltration can occur independently of altered RBF. Increased RBF is compatible with dilation of the afferent arteriole by enhanced nitric oxide (NO) production or reduced tubuloglomerular feedback (TGF) activity secondary to increased proximal tubular reabsorption of Na+.3Ito A. Uriu K. Inada Y. et al.Inhibition of neuronal nitric oxide synthase ameliorates renal hyperfiltration in streptozotocin-induced diabetic rat.J Lab Clin Med. 2001; 138: 177-185Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,4Vallon V. Richter K. Blantz R.C. et al.Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.J Am Soc Nephrol. 1999; 10: 2569-2576PubMed Google Scholar Unchanged RBF is compatible with a pressure-mediated increase in GFR leading to increased filtration fraction (FF),5Ditzel J. Junker K. Abnormal glomerular filtration rate, renal plasma flow, and renal protein excretion in recent and short-term diabetics.Br Med J. 1972; 2: 13-19Crossref PubMed Scopus (122) Google Scholar, 6Nordquist L. Brown R. Fasching A. et al.Proinsulin C-peptide reduces diabetes-induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption.Am J Physiol Renal Physiol. 2009; 297: F1265-F1272Crossref PubMed Scopus (49) Google Scholar, 7Persson P. Hansell P. Palm F. 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Blantz R.C. et al.Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.J Am Soc Nephrol. 1999; 10: 2569-2576PubMed Google Scholar,11Jensen P.K. Christiansen J.S. Steven K. et al.Renal function in streptozotocin-diabetic rats.Diabetologia. 1981; 21: 409-414Crossref PubMed Scopus (150) Google Scholar or by increased efferent arteriolar resistance.12Hostetter T.H. Troy J.L. Brenner B.M. Glomerular hemodynamics in experimental diabetes mellitus.Kidney Int. 1981; 19: 410-415Abstract Full Text PDF PubMed Scopus (707) Google Scholar Decreased proximal tubular pressure in diabetes is in part related to increased glucose-linked Na+ reabsorption via sodium-glucose-linked transporters, as inhibition results in increased proximal tubular pressure and decreased GFR.4Vallon V. Richter K. Blantz R.C. et al.Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.J Am Soc Nephrol. 1999; 10: 2569-2576PubMed Google Scholar,13Sallstrom J. Eriksson T. Fredholm B.B. et al.Inhibition of sodium-linked glucose reabsorption normalizes diabetes-induced glomerular hyperfiltration in conscious adenosine A -receptor deficient mice.Acta Physiol (Oxf). 2014; 210: 440-445Crossref PubMed Scopus (17) Google Scholar Decreased tubular hydraulic resistance, especially in the loop of Henle due to tubular hypertrophy, has also been reported to contribute.14Seyer-Hansen K. Hansen J. Gundersen H.J. Renal hypertrophy in experimental diabetes. A morphometric study.Diabetologia. 1980; 18: 501-505Crossref PubMed Scopus (184) Google Scholar Increased efferent arteriolar resistance can be mediated by angiotensin II—e.g., to maintain GFR when blood pressure is reduced. Interestingly, intra-renal angiotensin II concentration is increased in the diabetic kidney,15Singh R. Singh A.K. Leehey D.J. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat glomeruli.Am J Physiol Renal Physiol. 2005; 288: F1183-F1190Crossref PubMed Scopus (83) Google Scholar and angiotensin-converting enzyme inhibition effectively reduces diabetes-induced glomerular hyperfiltration.16Sochett E.B. Cherney D.Z. Curtis J.R. et al.Impact of renin angiotensin system modulation on the hyperfiltration state in type 1 diabetes.J Am Soc Nephrol. 2006; 17: 1703-1709Crossref PubMed Scopus (108) Google Scholar However, a TGF-mediated increase in GFR is not supported by recent reports as glomerular hyperfiltration is present in adenosine A1 knockout mice lacking a functional TGF mechanism,13Sallstrom J. Eriksson T. Fredholm B.B. et al.Inhibition of sodium-linked glucose reabsorption normalizes diabetes-induced glomerular hyperfiltration in conscious adenosine A -receptor deficient mice.Acta Physiol (Oxf). 2014; 210: 440-445Crossref PubMed Scopus (17) Google Scholar,17Sallstrom J. Carlsson P.O. Fredholm B.B. et al.Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism.Acta Physiol (Oxf). 2007; 190: 253-259Crossref PubMed Scopus (60) Google Scholar,18Faulhaber-Walter R. Chen L. Oppermann M. et al.Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury.J Am Soc Nephrol. 2008; 19: 722-730Crossref PubMed Scopus (83) Google Scholar although conflicting data exist.19Vallon V. Schroth J. Satriano J. et al.Adenosine A(1) receptors determine glomerular hyperfiltration and the salt paradox in early streptozotocin diabetes mellitus.Nephron Physiol. 2009; 111: p30-p38Crossref PubMed Scopus (69) Google Scholar In addition to vascular adenosine A1 and angiotensin II AT1 receptors mediating vasoconstriction, adenosine A2 receptors are abundant in both renal vasculature and tubules.20Vitzthum H. Weiss B. Bachleitner W. et al.Gene expression of adenosine receptors along the nephron.Kidney Int. 2004; 65: 1180-1190Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Adenosine A2 receptors consist of two subtypes—A2a and A2b—with adenosine having higher affinity for A2a compared with A2b.21Fredholm B.B. Irenius E. Kull B. et al.Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells.Biochem Pharmacol. 2001; 61: 443-448Crossref PubMed Scopus (356) Google Scholar Stimulation of either subtype results in increased adenylate cyclase activity and vasodilation.22Nakamoto H. Ogasawara Y. Kajiya F. Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.Hypertens Res. 2008; 31: 315-324Crossref PubMed Scopus (15) Google Scholar,23Freissmuth M. Nanoff C. Tuisl E. et al.Stimulation of adenylate cyclase activity via A2-adenosine receptors in isolated tubules of the rabbit renal cortex.Eur J Pharmacol. 1987; 138: 137-140Crossref PubMed Scopus (9) Google Scholar Expression studies suggest a predominant protein expression of A2b receptors in pre-glomerular vessels,24Jackson E.K. Zhu C. Tofovic S.P. Expression of adenosine receptors in the preglomerular microcirculation.Am J Physiol Renal Physiol. 2002; 283: F41-F51Crossref PubMed Scopus (88) Google Scholar which is also supported by functional studies in rats.25Feng M.G. Navar L.G. Afferent arteriolar vasodilator effect of adenosine predominantly involves adenosine A2B receptor activation.Am J Physiol Renal Physiol. 2010; 299: F310-F315Crossref PubMed Scopus (37) Google Scholar However, mRNAs for both A2b and A2a receptors have been found in mouse efferent arterioles.26Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009; 75: 793-799Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar This would imply that adenosine dilates efferent arterioles preferentially through the high-affinity A2a receptor. This is supported by the finding that an adenosine A2 receptor antagonist increased GFR despite reducing RBF in normoglycemic control mice.27Persson P. Hansell P. Palm F. Adenosine A2 receptor-mediated regulation of renal hemodynamics and glomerular filtration rate is abolished in diabetes.Adv Exp Med Biol. 2013; 765: 225-230Crossref PubMed Scopus (10) Google Scholar Interestingly, A2 receptor inhibition had no effect in diabetic mice. We therefore hypothesize that diabetes-induced glomerular hyperfiltration is caused by elevated FF because of defective vasodilation through adenosine A2a signaling in the efferent arterioles. All diabetic animals had increased blood glucose concentration and left kidney weight compared with controls (Table 1), whereas plasma protein concentration was similar in both groups (controls 52±3g/l, n=9 vs. diabetics 51±2g/l, n=11; not significant (NS)). In the cohort treated with the adenosine A2a antagonist ZM241385, mean arterial pressure (MAP) was not significantly different between groups and not affected by the adenosine A2a antagonist. In the cohort treated with the adenosine A2a agonist CGS21680, MAP was lower in diabetics compared with controls, and in both groups MAP decreased in response to the adenosine A2a agonist (Table 1). All diabetic animals developed glomerular hyperfiltration.Table 1Body weight (Bw), left kidney weight (Kw), blood glucose, mean arterial pressure (MAP), hematocrit (Hct), urine flow (Vu), renal vascular resistance (RVR), vascular resistance of afferent arterioles (RA), and vascular resistance of efferent arterioles (RE)Bw (g)Kw (g)Blood glucose (mmol/l)MAP (mmHg)HctVu (μl/min/kidney)RVRRA (mmHg/ml/min)REControl (n=8) Baseline401±151.4±0.15.0±0.2100±40.48±0.012.1±0.111.5±1.54.7±0.75.1±0.4 CGS21680 3nmol/l94±3aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.48±0.012.0±0.210.3±1.2aP<0.05 compared with baseline in the same group. Values are means±s.e.m.3.9±0.35.1±0.7 CGS21680 6nmol/l88±2aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.47±0.012.0±0.29.7±0.9aP<0.05 compared with baseline in the same group. Values are means±s.e.m.Diabetes (n=11) Baseline329±8bP<0.05 compared with control.1.8±0.1bP<0.05 compared with control.18.1±0.6bP<0.05 compared with control.90±30.47±0.0127.8±4.29.0±0.43.4±0.34.8±0.3 CGS21680 3nmol/l81±3aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.47±0.0120.0±3.6aP<0.05 compared with baseline in the same group. Values are means±s.e.m.8.4±0.43.3±0.34.1±0.3aP<0.05 compared with baseline in the same group. Values are means±s.e.m. CGS21680 6nmol/l78±3aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.46±0.01aP<0.05 compared with baseline in the same group. Values are means±s.e.m.19.1±3.9aP<0.05 compared with baseline in the same group. Values are means±s.e.m.8.3±0.4aP<0.05 compared with baseline in the same group. Values are means±s.e.m.Control (n=8) Baseline367±101.2±0.05.3±0.2105±30.48±0.011.3±0.215±1.17.4±0.78.0±1.0 ZM241385107±20.48±0.012.4±0.414.7±1.36.6±0.79.2±1.2Diabetes (n=10) Baseline330±8bP<0.05 compared with control.1.8±0.0bP<0.05 compared with control.24.3±0.9bP<0.05 compared with control.98±30.48±0.0121.5±2.911.4±1.16.6±0.96.6±1.2 ZM241385101±30.49±0.01aP<0.05 compared with baseline in the same group. Values are means±s.e.m.19.8±3.111.6±1.07.1±0.76.2±0.9Control (n=6) Baseline330±101.3±0.05.8±0.4100±20.47±0.008.2±2.311.3±1.2 L-NAME106±30.47±0.017.0±2.015.4±1.6aP<0.05 compared with baseline in the same group. Values are means±s.e.m. L-NAME+CGS2168090±2aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.46±0.014.6±1.717.0±1.5aP<0.05 compared with baseline in the same group. Values are means±s.e.m.Diabetes (n=10) Baseline340±91.9±0.1bP<0.05 compared with control.22.9±1.2bP<0.05 compared with control.89±30.47±0.0130.0±4.311.3±0.8 L-NAME96±4aP<0.05 compared with baseline in the same group. Values are means±s.e.m.0.47±0.0125.9±4.1aP<0.05 compared with baseline in the same group. Values are means±s.e.m.15.1±1.6aP<0.05 compared with baseline in the same group. Values are means±s.e.m. L-NAME+CGS2168083±30.47±0.0116.6±±3.2aP<0.05 compared with baseline in the same group. Values are means±s.e.m.15.8±1.1aP<0.05 compared with baseline in the same group. Values are means±s.e.m.Abbreviation: L-NAME, Nω-nitro-L-arginine methyl ester hydrochloride.a P<0.05 compared with baseline in the same group. Values are means±s.e.m.b P<0.05 compared with control. Open table in a new tab Abbreviation: L-NAME, Nω-nitro-L-arginine methyl ester hydrochloride. A2a inhibition increased GFR in controls, but had no effect in diabetics, whereas A2a stimulation had no significant effect in controls but decreased GFR in diabetics (Figure 1). A2a inhibition and low-dose A2a stimulation had no effects on RBF in any of the groups, compared with baseline. However, the highest dose of the A2a agonist decreased RBF in diabetics (Figure 2). As a consequence, A2a inhibition increased FF in controls but had no significant effect in diabetics. Furthermore, A2a stimulation had no effect on FF in controls but decreased FF in diabetics (Figure 3). Calculated renal vascular resistance (RVR) was not different between controls and diabetics and was unaffected by A2a inhibition. However, A2a stimulation decreased RVR in both groups (Table 1). Calculation of relative vascular resistance of afferent (RA) and efferent (RE) arterioles demonstrated that adenosine A2a agonist decreased RE in diabetics without significant effect in controls or on RA (Table 1).Figure 2Adenosine A2a receptors do not control renal blood flow. Renal blood flow in control and diabetic rats during baseline (BL) and after treatment with either the adenosine A2a antagonist ZM241385 (a) or the adenosine A2a agonist CGS21680 (b). Compared with baseline in controls and #P<0.05 compared with baseline in diabetics analyzed by Bonferroni's multiple comparisons test. Result from two-way analysis of variance: (a) interaction: not significant (NS), diabetes: P<0.05, ZM241385: NS. (b) Interaction: P<0.05, diabetes: NS, CGS21680: NS.View Large Image Figure ViewerDownload (PPT)Figure 3Filtration fraction is regulated by adenosine A2a receptors in controls but this regulation is absent in diabetes. Filtration fraction in control and diabetic rats during baseline (BL) and after treatment with either the adenosine A2a antagonist ZM241385 (a) or the adenosine A2a agonist CGS21680 (b). *P<0.05 compared with baseline in controls and #P<0.05 compared with baseline in diabetics analyzed by Bonferroni's multiple comparisons test. Result from two-way analysis of variance: (a) interaction: not significant (NS), diabetes: NS, ZM241385: P<0.05. (b) Interaction: P<0.05, diabetes: NS, CGS21680: NS.View Large Image Figure ViewerDownload (PPT) PFF was lower in diabetics compared with controls, and neither A2a inhibition nor stimulation affected PFF. However, A2a inhibition increased PSF in controls but was without significant effect in diabetics. A2a stimulation had no effect in controls but decreased PSF in diabetics. As a consequence, A2a inhibition increased PNET in controls but was without significant effect in diabetics, whereas A2a stimulation did not alter PNET in controls but reduced it in diabetics (Figure 4). The nonselective NO synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) did not affect GFR or FF but decreased RBF in both controls and diabetics (Figure 5). Combined renal artery infusion of L-NAME and CGS21680 was without effect on GFR in controls but decreased GFR in diabetics, together with a further reduction in RBF leaving FF not significantly changed from baseline (Figure 5).Figure 5Adenosine A2a receptor mediated decrease in filtration fraction is nitric oxide dependent. Glomerular filtration rate (a), renal blood flow (b), and filtration fraction (c) in control and diabetic rats during baseline (BL), renal artery infusion of unselective nitric oxide synthase inhibitor (L-NAME), and renal artery infusion of L-NAME in combination with the selective adenosine A2a agonist CGS21680 (L-NAME+CGS21680). *P<0.05 compared with baseline in controls, #P<0.05 compared with baseline in diabetics, †P<0.05 compared with L-NAME in controls, and ‡P<0.05 compared with L-NAME in diabetics analyzed by Bonferroni's multiple comparisons test. Result from two-way analysis of variance: (a) interaction: not significant (NS), diabetes: P<0.05, treatment: P<0.05. (b) Interaction: NS, diabetes: NS, treatment: P<0.05. (c) Interaction: NS, diabetes: P<0.05, treatment: NS.View Large Image Figure ViewerDownload (PPT) The main finding from the present study is that adenosine subtype A2a receptors maintain normal GFR in the control kidney by determining efferent arteriolar tone, and thereby FF. This mechanism is abolished in the diabetic kidney, causing relative efferent arteriolar vasoconstriction, elevating FF and subsequently causing glomerular hyperfiltration. However, our results demonstrate that A2a receptors are functional and that stimulation of these receptors is an effective way to normalize GFR in diabetes. These findings not only describe a novel mechanism mediating diabetes-induced glomerular hyperfiltration but also demonstrate an effective therapeutic approach to normalize it, which may prevent the decline in renal function normally occurring as the disease progresses.1Moriya T. Tsuchiya A. Okizaki S. et al.Glomerular hyperfiltration and increased glomerular filtration surface are associated with renal function decline in normo- and microalbuminuric type 2 diabetes.Kidney Int. 2012; 81: 486-493Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Adenosine A2 receptors are expressed on both afferent and efferent arterioles,26Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009; 75: 793-799Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar with a subtype distribution of mainly low-affinity A2b receptors localized to the afferent arteriole and high-affinity A2a receptors expressed in the efferent arteriole.24Jackson E.K. Zhu C. Tofovic S.P. Expression of adenosine receptors in the preglomerular microcirculation.Am J Physiol Renal Physiol. 2002; 283: F41-F51Crossref PubMed Scopus (88) Google Scholar, 25Feng M.G. Navar L.G. Afferent arteriolar vasodilator effect of adenosine predominantly involves adenosine A2B receptor activation.Am J Physiol Renal Physiol. 2010; 299: F310-F315Crossref PubMed Scopus (37) Google Scholar, 26Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009; 75: 793-799Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Increased expression of both A1 and A2 receptors has been reported in diabetes, which provides an explanation for the increased vascular sensitivity to exogenously administered adenosine in diabetes.28Pawelczyk T. Grden M. Rzepko R. et al.Region-specific alterations of adenosine receptors expression level in kidney of diabetic rat.Am J Pathol. 2005; 167: 315-325Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar The involvement of A2a receptors in controlling FF and GFR via a preferential efferent arteriole dilation, as well as that this mechanism is abolished in diabetes, is supported by the results from the micropuncture experiments. A2a receptor inhibition increased PSF only in controls, whereas A2a receptor stimulation had an effect only in diabetes. As PFF was unaffected by any of the treatments in both groups, calculation of PNET revealed that A2a receptors contribute to maintain normal filtration pressure in controls, whereas PNET can be significantly reduced if stimulating A2a receptors in diabetes. Taken together with the results from whole-kidney RBF and GFR, these results strongly suggest a crucial involvement of A2a receptors in controlling efferent arteriolar tone. Infusion of adenosine has previously been shown to reduce FF and GFR with minimal effect on RBF,29Tagawa H. Vander A.J. Effects of adenosine compounds on renal function and renin secretion in dogs.Circ Res. 1970; 26: 327-338Crossref PubMed Scopus (130) Google Scholar,30Osswald H. Spielman W.S. Knox F.G. Mechanism of adenosine-mediated decreases in glomerular filtration rate in dogs.Circ Res. 1978; 43: 465-469Crossref PubMed Scopus (129) Google Scholar which is compatible with A2 receptor–mediated dilation of post-glomerular resistance vessels.22Nakamoto H. Ogasawara Y. Kajiya F. Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.Hypertens Res. 2008; 31: 315-324Crossref PubMed Scopus (15) Google Scholar,26Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009; 75: 793-799Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Indeed, efferent arterioles express A2 receptors.26Al-Mashhadi R.H. Skott O. Vanhoutte P.M. et al.Activation of A(2) adenosine receptors dilates cortical efferent arterioles in mouse.Kidney Int. 2009; 75: 793-799Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar It should be noted that adenosines can reduce whole-kidney FF but still maintain FF in superficial nephrons because of blood flow being redistributed from superficial to juxtamedullary nephrons.31Agmon Y. Dinour D. Brezis M. Disparate effects of adenosine A1- and A2-receptor agonists on intrarenal blood flow.Am J Physiol. 1993; 265: F802-F806PubMed Google Scholar,32Zou A.P. Nithipatikom K. Li P.L. et al.Role of renal medullary adenosine in the control of blood flow and sodium excretion.Am J Physiol. 1999; 276: R790-R798PubMed Google Scholar The present study also demonstrates that A2a receptors control vascular tonus of the efferent arterioles in the control kidney and that a superimposed stimulation on top of the endogenous stimulus does not have any additional effect. This could be due to the relatively low dose of the A2a agonist (CGS21680) used in the present study, which was fourfold lower compared with the reported Ki for the substance.33Klotz K.N. Adenosine receptors and their ligands.Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 382-391Crossref PubMed Scopus (297) Google Scholar This is in line with a previous report that concludes diabetes is associated with increased sensitivity to exogenous adenosine.34Pflueger A.C. Schenk F. Osswald H. Increased sensitivity of the renal vasculature to adenosine in streptozotocin-induced diabetes mellitus rats.Am J Physiol. 1995; 269: F529-F535PubMed Google Scholar It is also possible that the system is already fully activated in controls and therefore a superimposed stimulation is without effect. Interestingly, the normal vasodilation of the efferent arterioles through A2a receptors was abolished in diabetes, but exogenous stimulation of these receptors restored FF and GFR. Adenosine concentration in the renal interstitium of rat kidney has been estimated to be 50nmol/l in the renal cortex and 200nmol/l in the medulla in control animals,32Zou A.P. Nithipatikom K. Li P.L. et al.Role of renal medullary adenosine in the control of blood flow and sodium excretion.Am J Physiol. 1999; 276: R790-R798PubMed Google Scholar and increases during high Na+ intake.35Siragy H.M. Linden J. Sodium intake markedly alters renal interstitial fluid adenosine.Hypertension. 1996; 27: 404-407Crossref PubMed Scopus (78) Google Scholar Adenosine concentration is mainly determined by the concentration of extracellular ATP, ADP, and AMP and the dephosphorylating enzymes ecto-5′-nucleotidase (5′-NT, CD73) and ecto-nucleoside triphosphate diphosphohydrolase (NTPD, CD39).36Grenz A. Zhang H. Hermes M. et al.Contribution of E-NTPDase1 (CD39) to renal protection from ischemia-reperfusion injury.FASEB J. 2007; 21: 2863-2873Crossref PubMed Scopus (135) Google Scholar However, adenosine is rapidly cleared by cellular reuptake mediated by basolateral equilibrative nucleoside transporters on the renal epithelia.37Grenz A. Bauerle J.D. Dalton J.H. et al.Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice.J Clin Invest. 2012; 122: 693-710Crossref PubMed Scopus (94) Google Scholar A potential explanation to the finding in the present study is that diabetes is associated with reduced intra-renal tissue concentrations of adenosine, and it therefore is a 'shortage of ligand' issue rather than a direct dysfunctional cellular signaling pathway. However, direct measurements of adenosine concentration in diabetics are missing but this speculation is supported by decreased 5′-NT activity in mesangial cells exposed to high glucose and increased adenosine deaminase activity in type 2 diabetic patients38Kocic G. Djordjevic V. Vlahovic P. et al.Antioxidants modulate adenosine metabolism in rat mesangial cells cultured under high glucose conditions.Ren Fail. 2002; 24: 691-701Crossref PubMed Scopus (5) Google Scholar,39Lee J.G. Kang D.G. Yu J.R. et al.Changes in adenosine deaminase activity in patients with type 2 diabetes mellitus and effect of DPP-4 inhibitor treatment on ADA activity.Diabetes Metab J. 2011; 35: 149-158Crossref PubMed Scopus (35) Google Scholar Furthermore, because of increased proximal tubular reabsorption through sodium-glucose linked transporters, distal tubular Na+ K+ and Cl- concentrations are decreased, attenuating macula densa ATP release and vascular adenosine signaling through the normal function of the TGF mechanism.4Vallon V. Richter K. Blantz R.C. et al.Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.J Am Soc Nephrol. 1999; 10: 2569-2576PubMed Google Scholar,40Pollock C.A. Lawrence J.R. Field M.J. Tubular sodium handling and tubuloglomerular feedback in experimental diabetes mellitus.Am J Physiol. 1991; 260: F946-F952PubMed Google Scholar Indeed, this is suggested to cause an error signal eventually contributing to glomerular hyperfiltration in diabetes via decreased adenosine A1 receptor signaling,19Vallon V. Schroth J. Satriano J. et al.Adenosine A(1) receptors determine glomerular hyperfiltration and the salt paradox in early streptozotocin diabetes mellitus.Nephron Physiol. 2009; 111: p30-p38Crossref PubMed Scopus (69) Google Scholar although conflicting results exist.17Sallstrom J. Carlsson P.O. Fredholm B.B. et al.Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism.Acta Physiol (Oxf). 2007; 190: 253-259Crossref PubMed Scopus (60) Google Scholar,18Faulhaber-Walter R. Chen L. Oppermann M. et al.Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury.J Am Soc Nephrol. 2008; 19: 722-730Crossref PubMed Scopus (83) Google Scholar However, an attenuated TGF signal can also decrease adenosine signaling via the adenosine A2 receptors, supported by this study and also previously suggested to occur in the isolated juxtaglomerular preparation.41Ren Y. Garvin J.L. Carretero O.A. Efferent arteriole tubuloglomerular feedback in the renal nephron.Kidney Int. 2001; 59: 222-229Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Vascular sensitivity to exogenous adenosine is increased in diabetic rats supporting the conclusion of a generally decreased adenosine signaling.34Pflueger A.C. Schenk F. Osswald H. Increased sensitivity of the renal vasculature to adenosine in streptozotocin-induced diabetes mellitus rats.Am J Physiol. 1995; 269: F529-F535PubMed Google Scholar Furthermore, the previous report by Vallon and Osswald42Vallon V. Osswald H. Dipyridamole prevents diabetes-induced alterations of kidney function in rats.Naunyn Schmiedebergs Arch Pharmacol. 1994; 349: 217-222Crossref PubMed Scopus (39) Google Scholar demonstrated that dipyridamole, an inhibitor of equilibrative nucleoside transporter known to increase tissue adenosine levels, completely normalized diabetes-induced glomerular hyperfiltration. Unfortunately, RBF was not measured, but intra-renal infusion of adenosine for 6 days also decreased GFR but with maintained RBF, suggesting that the main target is the efferent arteriole. NO has been reported to increase GFR in diabetes in several studies.3Ito A. Uriu K. Inada Y. et al.Inhibition of neuronal nitric oxide synthase ameliorates renal hyperfiltration in streptozotocin-induced diabetic rat.J Lab Clin Med. 2001; 138: 177-185Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,43Hiragushi K. Sugimoto H. Shikata K. et al.Nitric oxide system is involved in glomerular hyperfiltration in Japanese normo- and micro-albuminuric patients with type 2 diabetes.Diabetes Res Clin Pract. 2001; 53: 149-159Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar However, direct measurement of NO bioavailability in early experimental diabetes with NO-sensitive microelectrodes clearly showed reduced bioavailability,44Palm F. Friederich M. Carlsson P.O. et al.Reduced nitric oxide in diabetic kidneys due to increased hepatic arginine metabolism: implications for renomedullary oxygen availability.Am J Physiol Renal Physiol. 2008; 294: F30-F37Crossref PubMed Scopus (50) Google Scholar and NOS3 expression has recently been shown to inversely correlate to increased GFR and albuminuria in diabetes independently of blood pressure.45Wang C.H. Li F. Hiller S. et al.A modest decrease in endothelial NOS in mice comparable to that associated with human NOS3 variants exacerbates diabetic nephropathy.Proc Natl Acad Sci USA. 2011; 108: 2070-2075Crossref PubMed Scopus (49) Google Scholar Furthermore, the antioxidant coenzyme Q10, which reduces oxidative stress and thereby improves NO bioavailability, prevents glomerular hyperfiltration and proteinuria in experimental diabetes.46Persson M.F. Franzen S. Catrina S.B. et al.Coenzyme Q10 prevents GDP-sensitive mitochondrial uncoupling, glomerular hyperfiltration and proteinuria in kidneys from db/db mice as a model of type 2 diabetes.Diabetologia. 2012; 55: 1535-1543Crossref PubMed Scopus (80) Google Scholar Together, these results suggest that reduced NO bioavailability can contribute to diabetes-induced glomerular hyperfiltration. This may be secondary to reduced adenosine A2 receptor signaling with a subsequent reduction in efferent arteriole NOS3 activity. Indeed, adenosine A2a receptor stimulation did not decrease FF in diabetics during NOS inhibition, demonstrating that A2a-mediated vasodilation of efferent arterioles is NO dependent. Furthermore, it also demonstrates that adenosine A2 receptor–mediated vasodilation through NO counteracts the vasoconstriction mediated by adenosine A1 receptor in the afferent arterioles. In conclusion, GFR is partially regulated by A2a receptor signaling, which dilates efferent arterioles and thereby maintains normal FF. This mechanism for maintaining GFR within the normal range is abolished in diabetes, which provides a novel mechanistic explanation for the development of diabetes-induced glomerular hyperfiltration, a parameter considered to be an early risk factor for disease progression. Diabetes-induced glomerular hyperfiltration is generally considered to predict progressive development of diabetic nephropathy. Current therapies to inhibit angiotensin II signaling via AT1 receptors, using either angiotensin-converting enzyme inhibition or direct AT1 receptor blockade, are used in order to reduce proteinuria by inducing efferent arteriole dilation, which reduces filtration pressure and FF. However, this study demonstrates that reduced A2a receptor signaling in the efferent arteriole, probably because of reduced ligand availability, causes vasoconstriction, increased FF, and diabetes-induced glomerular hyperfiltration. We also demonstrate that direct A2a receptor stimulation reduces diabetes-induced glomerular hyperfiltration, which may provide a novel target to treat and prevent the development of diabetic nephropathy. Age-matched male Sprague-Dawley rats (250g body weight) were purchased from Charles River (Sulzfeld, Germany). Animals had free access to pelleted standard rat chow and tap water. All experiments were performed in accordance with national guidelines and approved by the local animal care and use committee of Uppsala University. Diabetes was induced by a single intravenous injection of streptozotocin (55mg/kg body wt; Sigma-Aldrich, St Louis, MO) in the tail vein. Animals were considered diabetic if blood glucose increased to >15mmol/l within 48h after injection. Blood glucose was determined with test reagent strips (FreeStyle Lite, Abbott, Alameda, CA) on blood sampled from the cut tip of the tail in all animals. Experimental groups consisted of untreated control and diabetic rats with diabetes duration of 14±4 days. Eight normoglycemic and 10 diabetic rats were used for the study of kidney function and micropuncture after administration of the selective adenosine A2a antagonist ZM241385.33Klotz K.N. Adenosine receptors and their ligands.Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 382-391Crossref PubMed Scopus (297) Google Scholar Eight normoglycemic controls and 11 diabetic animals were used to study kidney function after administration of the selective adenosine A2a agonist CGS21680,33Klotz K.N. Adenosine receptors and their ligands.Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 382-391Crossref PubMed Scopus (297) Google Scholar with an additional five normoglycemic controls and 8 diabetics allocated to micropuncture experiments. Six normoglycemic controls and 10 diabetics were used to study the kidney function after administration of unselective NOS inhibitor L-NAME, alone or in combination with CGS21680. On the day of acute experiments, rats were anesthetized with thiobutabarbital (Inactin, 120mg/kg body wt for controls and 80mg/kg body wt for diabetics, intraperitoneally) and placed on a table with a servo-controlled heating pad to maintain body temperature at 37.5°C. Animals were tracheostomized and catheters were placed in the femoral artery and vein for monitoring of blood pressure, withdrawal of blood samples, and infusion of Ringer solution (5ml/kg body wt/h for controls and 10ml/kg body wt/h for diabetics). The bladder was catheterized for drainage. The left kidney was exposed through a subcostal flank incision and immobilized in a plastic cup surrounded by saline-soaked cotton wool. The left ureter was catheterized for collection of urine for subsequent analysis. A lumbar artery close to the renal artery was catheterized and the catheter tip was advanced into the renal artery. The Ringer solution containing Lissamine green was infused to assure proper position of the catheter. In studies using the adenosine A2a antagonist ZM241385, only the bladder was catheterized for urine collection and the lumbar artery was not catheterized as this substance was administered systemically. GFR was estimated by the measurement of clearance of 3H-inulin. 3H-inulin was initially given as bolus dose 185kBq (American Radiolabeled Chemical, St Louis, MO) followed by a continuous infusion in Ringer solution (185kBq/kg body wt/h). 3H-activity in urine and plasma samples was determined using standard liquid scintillation techniques. GFR was calculated according to GFR=U × V/P, where U and P denote activity in urine and plasma, respectively, and V denotes urine flow. Total RBF was measured using an ultrasound flow probe (Transonic Systems, Itacha, NY) placed around the left renal artery. RVR was calculated as RVR=MAP/RBF. Segmental vascular resistance of afferent (RA=(MAP-PGC)/RBF) and efferent (RE=(PGC-PPC)/(GFR-RBF)) arterioles was calculated using the whole-kidney RBF combined with micropuncture data estimating PGC as well as peritubular capillary pressure (PPC) taken from the literature.47Oken D.E. Wolfert A.I. Laveri L.A. et al.Effects of intra-animal nephron heterogeneity on studies of glomerular dynamics.Kidney Int. 1985; 27: 871-878Abstract Full Text PDF PubMed Scopus (8) Google Scholar Animals were allowed a 45-minute recovery period after surgery before the experiment was commenced. All parameters were followed during baseline (30min for ZM241385 and 20min for CGS21680, and L-NAME). Thereafter, either intravenous injection of a bolus dose of the adenosine A2a antagonist ZM241385 (0.5mg/kg, intravenous; Tocris Biosciences, Bristol, UK) or continuous lumbar artery infusion of the selective adenosine A2a agonist CGS21680 (final concentration of 3nmol/l and 6nmol/l in the renal artery for first and second period, respectively; Tocris Biosciences), or L-NAME (1mg/kg/min), was started and all parameters were followed for one experimental period with ZM241385 and for two periods with CGS21680 and L-NAME. CGS21680 and ZM241385 were dissolved in dimethyl sulfoxide and Tween80 and diluted in Ringer solution to a final dimethyl sulfoxide concentration <0.25% and Tween80 <5%. L-NAME was dissolved in Ringer solution. Proximal tubular free flow (PFF) and stop flow pressure (PSF) were determined in randomly chosen proximal tubular segments on the kidney surface using a servo-controlled micropressure device (World Precision Instruments, New Haven, CT) during baseline and after intravenous administration of ZM241385 (0.5mg/kg) or after lumbar artery infusion of CGS21680 (6nmol/l). PFF was determined in the unobstructed free-flowing nephron and PSF after nephron flow was interrupted by injecting mineral oil distal to the pressure pipette. Net filtration pressure (PNET) was calculated according to PNET=PSF-PFF. Urine flow was determined gravimetrically and plasma protein concentration using DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Kidney weights were determined after the kidney had been removed at the end of the experiments. All statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA). All data were analyzed by two-sided two-way analysis of variance, followed by Bonferroni's multiple comparison test analyzing for differences between baseline and treatment within each group as well as analyzing between-group differences for each intervention. Descriptive statistics are presented as means±s.e.m. For all comparisons P<0.05 was considered significant. This study was supported by grants from the Swedish Research Council, the Swedish Diabetes Foundation, and the Åke Wiberg Foundation.