The effect of aliskiren on urinary cytokine/chemokine responses to clamped hyperglycaemia in type 1 diabetes
- First Online:
- 427 Downloads
Acute clamped hyperglycaemia activates the renin–angiotensin–aldosterone system (RAAS) and increases the urinary excretion of inflammatory cytokines/chemokines in patients with uncomplicated type 1 diabetes mellitus. Our objective was to determine whether blockade of the RAAS would blunt the effect of acute hyperglycaemia on urinary cytokine/chemokine excretion, thereby giving insights into potentially protective effects of these agents prior to the onset of clinical nephropathy.
Blood pressure, renal haemodynamic function (inulin and para-aminohippurate clearances) and urinary cytokines/chemokines were measured after 6 h of clamped euglycaemia (4–6 mmol/l) and hyperglycaemia (9–11 mmol/l) on two consecutive days in patients with type 1 diabetes mellitus (n = 27) without overt nephropathy. Measurements were repeated after treatment with aliskiren (300 mg daily) for 30 days.
Before aliskiren, clamped hyperglycaemia increased filtration fraction (from 0.188 ± 0.007 to 0.206 ± 0.007, p = 0.003) and urinary fibroblast growth factor-2 (FGF2), IFN-α2 and macrophage-derived chemokine (MDC) (p < 0.005). After aliskiren, the filtration fraction response to hyperglycaemia was abolished, resulting in a lower filtration fraction after aliskiren under clamped hyperglycaemic conditions (p = 0.004), and none of the biomarkers increased in response to hyperglycaemia. Aliskiren therapy also reduced levels of urinary eotaxin, FGF2, IFN-α2, IL-2 and MDC during clamped hyperglycaemia (p < 0.005).
The increased urinary excretion of inflammatory cytokines/chemokines in response to acute hyperglycaemia is blunted by RAAS blockade in humans with uncomplicated type 1 diabetes mellitus.
KeywordsClamped hyperglycaemia Type 1 diabetes mellitus Urine cytokines/chemokines Urine proteomic analysis
Direct renin inhibition
Effective renal plasma flow
Fibroblast growth factor-2
Granulocyte–monocyte colony stimulating factor
Monocyte chemoattractant protein
Macrophage inflammatory protein-1α
Platelet-derived growth factor-AB/BB
Glycaemic excursions may contribute to microvascular end-organ injury in patients with diabetes mellitus, independent of long-term glycaemic control . From a haemodynamic perspective in the kidney, the influence of hyperglycaemia manifests as a hyperfiltration response characterised by a rise in filtration fraction [2, 3, 4, 5, 6], which may cause renal injury through shear-stress effects [7, 8, 9]. The haemodynamic effects of hyperglycaemia are attributed, in part, to activation of the renin–angiotensin–aldosterone system (RAAS) .
In addition to haemodynamic effects, hyperglycaemia activates inflammatory pathways in the kidney, leading to further injury. We have recently demonstrated that acute clamped hyperglycaemia in a separate cohort of young patients with type 1 diabetes mellitus increases the urinary excretion of factors implicated in the initiation and progression of diabetic nephropathy, including eotaxin, fibroblast growth factor-2 (FGF-2), granulocyte–monocyte colony stimulating factor (GM-CSF), IFN-α2, IL-2, IL-12, monocyte chemoattractant protein (MCP)-3, macrophage-derived chemokine (MDC), macrophage inflammatory protein-1α (MIP-1α), platelet-derived growth factor-AB/BB (PDGF-AB/BB), sCD40ligand (sCD40K) and TNF-β . We have further demonstrated that urinary cytokine/chemokine levels are associated with protein excretion within the normal range in children, suggesting a role for renal inflammation in the early pathogenesis of diabetic nephropathy .
As inflammation and immune-mediated injury induced by intrarenal cytokine/chemokine expression are implicated in both the initiation and progression of diabetic nephropathy it is important to determine the relationship between existing pharmacotherapeutic agents and measures of renal inflammatory factors [12, 13, 14]. One of the pleiotropic protective effects of RAAS blockade on the kidney in patients with overt diabetic nephropathy is anti-inflammatory activity . The ability of clinicians to suppress renal inflammation may have particularly important implications for patients with uncomplicated type 1 diabetes mellitus, in whom urinary inflammatory markers may be the only sign of kidney disease prior to the onset of microalbuminuria or declining kidney function . New non-invasive biomarkers could therefore be used to target high-risk patients with otherwise clinically silent disease for earlier therapies .
Although it is known that RAAS blockade is more effective under conditions of dietary sodium depletion, RAAS inhibitors are still effective in both diabetic and non-diabetic sodium-replete individuals [17, 18, 19, 20]. These observations suggest persistent intrarenal RAAS activation despite high dietary sodium intake. As a consequence, in sodium-replete individuals RAAS inhibitors exert important renal haemodynamic and blood-pressure effects, and in patients with established diabetic nephropathy these agents suppress urinary levels of inflammatory mediators [17, 21, 22, 23, 24, 25]. In patients with uncomplicated type 1 diabetes mellitus who would not otherwise be candidates for RAAS inhibition, the effect of these agents on urinary biomarker excretion or biomarker responses to acute clamped hyperglycaemia remain unknown. It is important to establish the effect of RAAS inhibitors on urinary markers of inflammation under sodium-replete conditions, as this would most closely mimic most typical clinical settings.
Accordingly, our goal was to examine whether RAAS blockade would mitigate the effect of acute hyperglycaemia on renal inflammatory pathways. We hypothesised that RAAS inhibition would reduce urinary cytokine/chemokine excretion and would also blunt the increased urinary excretion of these factors in response to clamped hyperglycaemia. We included young patients with uncomplicated type 1 diabetes mellitus who would not otherwise be treated with RAAS inhibitors, and studied participants in a sodium-replete state to replicate typical North American dietary conditions. Our goal was to better understand the effect of these agents on renal inflammatory pathways early in the natural history of the disease when clinical risk factors such as albuminuria may not identify those at elevated risk of renal disease progression.
Patients with uncomplicated type 1 diabetes mellitus (n = 27) were recruited. Inclusion criteria were: duration of type 1 diabetes ≥5 years; age ≥18 years; blood pressure <140/90 mmHg; no history of renal disease or macrovascular disease; and not taking any regular medications other than insulin, thyroid replacement therapy or inhalers to treat asthma. Patients who were normoalbuminuric were recruited based on a spot urine albumin-to-creatinine ratio <2.8 mg/mmol for women and <2.1 mg/mmol for men. All participants were subsequently shown to be normoalbuminuric on a 24 h urine collection on the day prior to all physiological assessments (<30 mg per 24 h).
We aimed to study women during the early follicular phase of the menstrual cycle, determined by cycle day and measurement of 17β-oestradiol levels. None was using oral contraceptive medication. The local Research Ethics Board at the University Health Network (Toronto, ON, Canada) approved the protocol and all participants gave informed consent.
In all phases of the experiment, blood glucose was maintained by a modified glucose-clamp technique, as described previously . In brief, a 16-gauge peripheral venous cannula was inserted into the left antecubital vein for infusion of glucose and insulin and a second cannula was inserted for blood sampling more distally. Blood glucose was measured approximately every 10 min and the insulin infusion was adjusted to maintain euglycaemia. All experiments were performed in the same warm (25°C), temperature-controlled room and in a dark, quiet environment in the supine position. The following day, identical sets of experiments were performed during clamped hyperglycaemia (blood glucose 9–11 mmol/l).
Renal and urinary biomarker studies were performed at baseline and then repeated in an identical fashion after 4 weeks of aliskiren therapy (300 mg orally per day).
Assessment of renal variables
Following maintenance of the euglycaemic clamp, a third intravenous line was inserted into the right arm and was connected to a syringe infusion pump for administration of inulin and para-aminohippurate (PAH). After collecting blood for inulin and blank, a priming infusion containing 25% inulin (60 mg/kg) and 20% PAH (8 mg/kg) was administered. Thereafter, inulin and PAH were infused continuously at a rate calculated to maintain their respective plasma concentrations constant at 2.0 and 0.15 mg/l, respectively. After a 90 min equilibration period, blood was collected for inulin, PAH and haematocrit. Two blood samples were further collected at 30 min intervals over 60 min for inulin and PAH. GFR and effective renal plasma flow (ERPF) were estimated from steady-state infusion of inulin and PAH, respectively. Filtration fraction was derived as GFR/ERPF .
Sample collection and analytical methods
Blood samples collected for inulin and PAH determinations were immediately centrifuged at 96 g for 10 min at 4°C. Plasma was separated, placed on ice and then stored at −70°C before the assay. Inulin and PAH were measured in serum by colorimetric assays using anthrone and N-(1-naphthyl)ethylenediamine, respectively [27, 28, 29]. The mean of two baseline clearance periods represent GFR and ERPF, expressed per 1.73 m2 body surface area [27, 29].
On arrival at the renal physiology laboratory, participants were asked to void and this final volume completed the 24 h urine collection. Participants then voided again mid-morning at approximately 10:00 hours to empty the bladder of urine that was produced while the euglycaemic clamp was being stabilised. The subsequent spot urine sample that was used for the analysis was collected prior to the first set of renal haemodynamic variables. This 50 ml mid-stream sterile urine specimen was used to measure levels of cytokines/chemokines using a Millipore Cytokine/Chemokine Panel Luminex Assay (Eve Technologies, Calgary, AB, Canada) corrected for urine creatinine concentration [10, 11]. Immediately after collection, urine was centrifuged at 1,500 g for 15 min to remove cells, then separated into 1 ml aliquots and frozen at −80°C. Urine was then thawed at 4°C 1 day prior to use. As the protocol for handling urine specimens was designed to avoid protein degradation, this analysis included spot urines and not the timed collections that were carried out before the study visit. Furthermore, a priori we only included analytes that we have observed either to increase in response to hyperglycaemia or that were associated with the highest tertile of normoalbuminuria in our previous work [10, 11]. Our analysis therefore included eotaxin, FGF-2, GM-CSF, IFN-α2, IL-2, IL-12, MCP-1, MCP-3, MDC, MIP-1α, TNF-β, sCD40K and PDGF-AB/BB. We limited our analysis to these factors to maintain statistical power, to minimise false-positive results and to further elucidate mechanisms that may link high intraglomerular pressure with factors that may promote the initiation of renal disease. The accuracy and precision of the urinary cytokine/chemokine assay is available from the manufacturer’s website (www.millipore.com/userguides/tech1/proto_mpxhcyto-60k). The detection limits of our assays have also been published previously . The investigator performing data analysis was blinded to all study variables.
Urinary albumin excretion rate was determined from a 24 h urine collection by immunoturbidimetry. HbA1c was measured by high-performance liquid chromatography and plasma renin concentration and plasma insulin were measured using previously described methods [8, 30].
Descriptive statistics were used to compare baseline clinical and demographic characteristics. Based on our previous work with MCPs, to be able to detect a 16 unit or greater change in response to RAAS blockade (80% power, α 0.05, standard deviation 20 units), a minimum of 24 patients were required . Within-subject responses to hyperglycaemia and RAAS inhibition were determined by repeated measures analysis of variance (ANOVA), followed by Bonferroni’s correction for multiple comparisons using a two-tailed test and p ≤ 0.005 to indicate statistical significance (SPSS 20.0 software package, IBM, Armonk, NY, USA).
Demographic characteristics at baseline and after aliskiren therapy in patients with uncomplicated type 1 diabetes
25.6 ± 2.1
Diabetes duration (years)
17.2 ± 1.2
8.8 ± 1.0
73 ± 3
24.4 ± 0.6
Albumin excretion rate (mg/day)
12 ± 3
9 ± 2
24 h Na+ intake (mmol/day)
171 ± 6
174 ± 6
Estimated protein intake (g kg−1 day−1)
1.03 ± 0.01
0.92 ± 0.01
Oestrogen in women (pmol/l)
188 ± 42
176 ± 78
Systolic blood pressure (mmHg)
113 ± 2
108 ± 3***
Diastolic blood pressure (mmHg)
64 ± 2
59 ± 2***
Pulse (beats per minute)
57 ± 6
63 ± 4
Plasma insulin (pmol/l)
188 ± 53
201 ± 98
175 ± 68
232 ± 136
Plasma glucose (mmol/l)
4.79 ± 0.10
4.44 ± 0.21
11.33 ± 0.15
11.00 ± 0.22
Fractional Na+ excretion
1.21 ± 0.12
1.19 ± 0.12
1.17 ± 0.11
1.20 ± 0.10
Renal haemodynamic function
GFR (ml min−1 1.73 m−2)
136 ± 6
140 ± 5
143 ± 5†
146 ± 7†
Effective renal plasma flow (ml min−1 1.73 m−2)
718 ± 26
763 ± 37
710 ± 30
816 ± 40‡
0.188 ± 0.007
0.193 ± 0.011
0.206 ± 0.007§
0.183 ± 0.008¶
Baseline response to clamped hyperglycaemia
Effects of RAAS blockade on haemodynamics and urinary cytokines/chemokines
Compared with levels during baseline clamped hyperglycaemia, RAAS blockade similarly reduced the urinary levels of eotaxin, FGF-2, IFN-α2, IL-2, MCP-3, MDC, PDGF-AB/BB and MCP-1 under clamped hyperglycaemic conditions (Fig. 3). Differences in each of these mediators except for MCP-3 (p = 0.015), PDGF-AB/BB (p = 0.022) and MCP-1 (p = 0.006) were significant after correcting for multiple comparisons (Fig. 3). In addition, RAAS blockade reversed cytokine/chemokine responses to clamped hyperglycaemia, such that none of the ten biomarkers increased in response to clamped hyperglycaemia.
Renal hyperfiltration, RAAS blockade and urinary cytokines/chemokines
The goal of this study was to determine whether the effect of acute clamped hyperglycaemia on urinary cytokines/chemokines could be reversed with RAAS inhibition in patients with uncomplicated type 1 diabetes mellitus. Previous work in animals and humans has demonstrated that pathways that are upregulated by hyperglycaemia, including RAAS and oxidative stress pathways, produce increased systemic and renal levels of cytokines/chemokines [21, 33]. These deleterious effects have been attributed to chronic hyperglycaemia-induced angiotensin II and aldosterone activity [34, 35, 36], RAAS blockade is thought to be renal protective in part by abrogating these inflammatory responses. We have recently demonstrated that inflammatory urine biomarkers also increase in response to acute clamped hyperglycaemia; however, the role of the RAAS in mediating this response and the reversibility of this phenomenon was unknown. We hypothesised that blockade of the RAAS would mitigate the effect of clamped hyperglycaemia on urinary cytokines/chemokines [37, 38, 39, 40].
Our first major finding was that in patients with uncomplicated type 1 diabetes mellitus, the effect of clamped hyperglycaemia on urinary cytokines/chemokines was inhibited by RAAS blockade. Similar to our previous observations in an independent cohort, factors that are involved in either chemotaxis or inflammatory kidney disease initially increased in response to clamped hyperglycaemia, prior to any RAAS-modulating therapy. We have previously hypothesised that the increase in urinary cytokine/chemokine excretion occurred because of increased tubular secretion, although it is also possible that the urinary excretion of these factors could reflect systemic ‘spillover’ into the urine. Regardless of the responsible mechanism, previous studies have demonstrated that renoprotective medications, including protein kinase C-β inhibitors [13, 31, 41] and ACE inhibitors and angiotensin-receptor blockers , reduce urinary cytokines/chemokines in diabetes mellitus patients with established nephropathy. Less was known about the urinary excretion of inflammatory biomarkers in response to RAAS inhibition in humans with uncomplicated type 1 diabetes mellitus, or the effect of these agents on urinary biomarker responses to clamped hyperglycaemia . To our knowledge this is the first study to demonstrate potentially positive effects of RAAS inhibition on urinary biomarkers in patients with uncomplicated type 1 diabetes mellitus under different conditions of clamped euglycaemia and hyperglycaemia.
While the factors responsible for the effect of RAAS blockade on urinary cytokines/chemokines cannot be determined from this study, it is perhaps important that aliskiren also blocked the effect of hyperglycaemia on filtration fraction, which is often used as a surrogate for intraglomerular pressure in humans . The decline in filtration fraction occurred because of a predominant increase in ERPF without affecting GFR responses, suggesting similar afferent compared with efferent vasodilatory effects. As reductions in glomerular stretch and shear-stress mechanical forces suppress renal inflammation in animals, it is tempting to speculate that the effect of RAAS blockade on urine cytokines/chemokines was caused by a decline in intraglomerular pressure . Interestingly, the effect of DRI on MCP-1 was more pronounced in patients with renal hyperfiltration under clamped hyperglycaemic conditions. As hyperfiltration is associated with high intraglomerular pressure, the exaggerated response in hyperfiltering individuals may highlight a possible physiological benefit of earlier RAAS blockade in this group . Alternatively, changes in urinary factors may have been on the basis of known ‘non-haemodynamic’ effects of RAAS blockade on critical pro-inflammatory pathways, including nuclear factor-κβ . Ultimately, the clinical importance of our observations will have to be clarified in long-term prospective trials in humans to assess whether RAAS blockade exerts protective effects on early markers of renal injury, including albuminuria and declining renal function, and to determine if certain patient subgroups such as hyperfiltering individuals derive additional benefit.
Hyperglycaemia is necessary but not sufficient for the initiation and progression of diabetic nephropathy. It is instead the individual response to hyperglycaemia that is the critical determinant of diabetic complications. The acute effect of glucose on renal markers of inflammation and fibrosis is therefore important because our work may give insight into pathways that permit the initiation of nephropathy in some patients and not others. Moreover, in light of the observations in the present study, these hyperglycaemia-inducible pathways are sensitive to existing pharmacological agents. As ‘untargeted’ primary-prevention trials in type 1 diabetes mellitus have failed, it is now more important than ever to translate observations from experimental animal models and from pre-clinical patient studies into the clinical trial arena. A better understanding of the clinical significance of urinary cytokines/chemokines and their ability to reflect biological effects of experimental and therapeutic interventions in patients with type 1 diabetes mellitus may lead to a more targeted, personalised approach to primary-prevention strategies. Our observations suggest that non-invasive early biomarkers that may contribute to progressive kidney injury over time are dynamic and responsive to RAAS blockade. Future work should define the time course of this response, and should also correlate these factors with the development of proteinuria and declining kidney function. Finally, as the effects of RAAS blockade on urinary cytokines/chemokines was greatest under conditions of clamped hyperglycaemia, future studies should consider taking into account the level of ambient glycaemia at the time biomarkers are measured.
It is also important to recognise that our observations were made under conditions of a relatively high-sodium diet that are common in the North American clinical context. As we studied patients under these conditions of high dietary sodium, the magnitude of our results may be less than would be expected under conditions of salt depletion, when the RAAS is activated and the response to RAAS blockers is enhanced . The effect of sodium intake may be of particular importance in light of recent clinical trial data from the Reduction in End points in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study and the Irbesartan Diabetic Nephropathy Trial (IDNT) that demonstrated enhanced renal protective effects of RAAS inhibition in participants with the two lowest tertiles of dietary sodium intake . Whether the protective effect observed with low-sodium diet and RAAS inhibition in this post hoc analysis was due to greater haemodynamic effects, enhanced anti-inflammatory effects or both, is unknown and requires further study. Nevertheless, as previously reported by our group [17, 18, 19] and by other investigators , RAAS inhibitors still exert prominent haemodynamic effects on the intrarenal RAAS in the presence of relative RAAS suppression induced by a high-salt diet in diabetic patients. Our results extend this previous haemodynamic-focused body of work by suggesting that RAAS inhibition during salt repletion also reduces inflammatory urinary cytokines/chemokines.
Although we have focused on neurohormonal RAAS-related factors that have been implicated in the pathogenesis of renal haemodynamic dysfunction and renal inflammation, it is also important to recognise that tubuloglomerular feedback mechanisms have been implicated in the initiation and progression of diabetic nephropathy . In brief, increased proximal sodium–glucose cotransport via sodium–glucose cotransport-2 (SGLT2) reduces delivery of sodium to the macula densa, thereby leading to afferent vasodilatation through tubuloglomerular feedback . Perhaps as a result of upregulated SGLT2 activity in diabetes mellitus, animals and humans with type 1 diabetes mellitus exhibit a phenomenon call the ‘salt paradox of diabetes mellitus’, whereby sodium restriction leads to exaggerated renal haemodynamic responses (hyperfiltration and afferent vasodilatation) despite blunted levels of circulating RAAS mediators [48, 49]. This again suggests that under conditions of sodium restriction when the RAAS is activated and tubuloglomerular feedback mechanisms may play a more prominent role, pharmacological agents such as RAAS inhibitors or novel SGLT2 inhibitors (that may influence tubuloglomerular feedback), may exert more complete protective effects on renal haemodynamic dysfunction, intraglomerular pressure and renal inflammation . However, as fractional sodium excretion did not change in response to aliskiren, alterations in tubuloglomerular feedback may not have contributed significantly to the declines in urinary cytokines/chemokines in this cohort.
Our study has limitations. We attempted to minimise the effect of the small sample size by using homogeneous study groups and by using a careful pre-study preparation phase with a focus on known factors that influence neurohormonal activation, including dietary sodium intake. We also scheduled studies in women to coincide with the early follicular phase of the menstrual cycle to avoid the confounding effects of oestrogen on vascular function. In addition, we decreased variability and increased statistical power by using a study design that allowed each participant to act as his/her own control. Next, we are unable to comment about the relative effect of direct renin inhibitors compared with ACE inhibitors or angiotensin-receptor blockers. While this was not the goal of this study, based on comparable haemodynamic effects we would expect similar findings to be observed using these older classes of agents. Finally, it is important for future studies to determine whether changes in urinary cytokine/chemokine excretion are systemically derived (i.e. ‘spillover’) or due to renal production. In light of the exaggerated effects of DRI on MCP-1 in hyperfiltering patients in our post hoc analysis, future adequately powered trials should determine whether hyperfiltering patients exhibit exaggerated urinary cytokine/chemokine responses, as this may identify a group that could benefit from earlier primary-prevention strategies.
In conclusion, RAAS blockade for 30 days reduces the effect of acute clamped hyperglycaemia on filtration fraction and urinary cytokines/chemokines. Our results suggest that RAAS inhibitors may result in a beneficial urinary cytokine/chemokine physiological profile in patients with type 1 diabetes mellitus prior to the clinical onset of diabetic nephropathy.
The authors would like to thank Paul Yip and Jenny Cheung in the Special Testing Laboratory at the University Health Network (Toronto, ON, Canada) for their invaluable assistance with the biochemical assays included in this work. Finally, the authors are grateful to the study participants whose time and effort are critical to the success of our research program.
This work was supported by operating grants from the Canadian Institutes of Health Research, Heart Stroke Foundation (to DZIC), a University of Toronto Dean’s Fund Grant and by a grant from the JDRF to DD, FHM and EBS. DZIC was also supported by a Kidney Foundation of Canada Scholarship, a Canadian Diabetes Association-KRESCENT Program Joint New Investigator Award and a University of Toronto Dean’s Fund Award. RH is supported by a Banting and Best Graduate Award and an Institute of Medical Science Open Fellowship Award. JWS is the Canadian Institutes of Health Research (CIHR)/Amgen Canada Kidney Research Chair at the University Health Network, University of Toronto (ON, Canada).
Duality of interest
DZIC is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. The results presented in this paper have not been published previously in whole or in part. The authors declare that there is no duality of interest associated with this manuscript
DZIC and RH were involved in study design, and acquisition and interpretation of data. JWS, DD, FHM, EBS and HNR were involved in the interpretation of data. All authors revised the manuscript and approved the final version.
- 5.Gordin D, Forsblom C, Ronnback M et al (2008) Acute hyperglycaemia induces an inflammatory response in young patients with type 1 diabetes. Ann Med 1–7Google Scholar
- 44.Hernandez-Presa M, Bustos C, Ortego M et al (1997) Angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 95:1532–1541PubMedCrossRefGoogle Scholar
- 46.Hollenberg NK, Fisher ND, Nussberger J, Moukarbel GV, Barkoudah E, Danser AH (2011) Renal responses to three types of renin-angiotensin system blockers in patients with diabetes mellitus on a high-salt diet: a need for higher doses in diabetic patients? J Hypertens 29:2454–2461PubMedCrossRefGoogle Scholar