Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin

HERG (human ether-à-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, IKr. This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37 °C of recombinant HERG channel current (IHERG), and of native IKr ‘tails’ from rabbit ventricular myocytes. Doxepin inhibited IHERG with an IC50 value of 6.5 ± 1.4 μM and native IKr with an IC50 of 4.4 ± 0.6 μM. The inhibitory effect on IHERG developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of IHERG. Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC50 for IHERG blockade by ∼4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.
1
Introduction
Diverse cardiac and non-cardiac drugs are associated with prolongation of the rate-corrected QT (QTc) interval of the electrocardiogram and with a risk of the potentially fatal arrhythmia Torsade de Pointes (TdP) [1–4]. The majority of such agents exert a common action of inhibiting the cardiac rapidly activating delayed rectifier K+ current (IKr). IKr is a major determinant of ventricular action potential repolarization and, thereby, of the QTc interval [2,4,5]. The pore-forming subunit of IKr channels is encoded by HERG (human ether-à-go-go-related gene [6,7]). HERG channels appear to have a larger pore cavity than other (Kv) six transmembrane domain K+ channels and possess particular aromatic amino-acid residues in the S6 region of the channel [5,8–10]. These features combine to confer a high susceptibility to pharmacological blockade upon the HERG channel. Indeed, the association between drug-induced QTc interval prolongation and pharmacological blockade of HERG channels is sufficiently strong that drug-screening against recombinant HERG channels is now an important component of cardiac safety-pharmacology during drug development [11–13].
Doxepin is a tricyclic antidepressant (TCA) structurally related to amitriptyline and imipramine that combines antidepressant and sedative actions [14]. Initially doxepin was suggested to have better cardiac safety than other TCA drugs [15]. However, a subsequent review of the clinical and animal data [16] and a study in depressed patients [17] found no evidence that doxepin had fewer cardiovascular effects than other TCAs. Moreover, similar to other TCAs, doxepin can be cardiotoxic in overdose [14]. Adverse cardiac effects associated with doxepin include premature ventricular complexes (PVCs) and wide QRS complexes on the electrocardiogram. Furthermore, there are a number of documented cases of QT interval prolongation with doxepin [18–20]. For example, in overdose doxepin has been associated with a greatly prolonged QTc interval (reaching 580 ms) and TdP [19], whilst QT interval prolongation and syncope have been reported for doxepin in combination with methadone and β blocker use [20]. At present there is no information on the basis for QTc prolongation with doxepin. However, other TCAs including imipramine and amitryptiline have been demonstrated to inhibit recombinant HERG channels [21–23] and we hypothesised that doxepin is also likely to act as an inhibitor of HERG channel current (IHERG). The present study was conducted to test this hypothesis and to characterise the nature of any observed IHERG blockade.
2
Methods
2.1
Maintenance of mammalian cell lines stably expressing wild-type and mutant HERG channels
Experiments on wild-type HERG were performed on a cell line (Human Embryonic Kidney; HEK 293) stably expressing HERG (donated by Dr Craig January, University of Wisconsin [24]), except for those in Fig. 8; these utilised a cell line stably expressing lower levels of HERG developed in this laboratory, for use with high external [K+] (for comparison with the F656A mutant, see Section 2.2 below). Cell lines stably expressing HERG and its mutants, F656A and Y652A were maintained as described previously [25]. Cell lines stably expressing the S631A [26] and N588K [27] mutants were made from appropriately mutated HERG sequences using previously described methods [25]. Cells were passed using a non-enzymatic dissociating agent (Splitase, AutogenBioclear) and plated out onto small sterilised glass coverslips in 30 mm petri dishes containing a modification of Dulbecco's modified Eagle's medium with Glutamax-1 (DMEM; Gibco, Gibco/Invitrogen, Paisley, UK), supplemented with 10% fetal bovine serum (Gibco), 400 μg ml−1 gentamycin (Gibco) and 400 μg ml−1 geneticin (G418; Gibco). Treatment of the mutant cell lines was identical to treatment of the wild-type cell line except that cultures were maintained with 800 μg ml−1 of hygromycin. The cells were incubated at 37 °C for a minimum of two days prior to any electrophysiological study.
2.2
Experimental solutions
Whole-cell patch-clamp measurements of wild-type (WT) and mutant IHERG were made at 37 ± 1 °C. Once in the experimental chamber cells were superfused with a standard extracellular Tyrode's solution containing (in mM): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, (titrated to pH 7.45 with NaOH). Similar to other studies from our laboratory (e.g. [28,29]), for experiments employing the S6 mutant F656A (which shows comparatively low levels of channel expression [8,25]) and its WT control, the external solution contained 94 mM KCl (the NaCl concentration was correspondingly reduced). Experimental solutions were applied using a home-built, warmed, solution delivery system that exchanged the solution surrounding a cell in <1 s. Doxepin powder (Sequoia Research Products and Sigma-Aldrich) was dissolved in Tyrode's solution to produce initial stock solutions of either 10 or 50 mM, which were serially diluted to produce working solutions ranging from 0.1 μM to 1 mM.
The pipette dialysis solution for IHERG measurement contained (in mM): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (titrated to pH 7.2 with KOH) [28,29]. Patch-pipettes were heat-polished to 2.5–4 MΩ. No correction was made for the ‘pipette-to-bath’ liquid junction potential, which was measured to be −3.2 mV.
2.3
Experiments on rabbit isolated ventricular myocytes
One series of experiments was performed to investigate blockade by doxepin of native IKr from adult ventricular myocytes (Results, Fig. 2). For these, male New Zealand white rabbits (2–3 kg) were killed humanely in accordance with UK Home Office legislation. Ventricular myocytes were then isolated by a combination of mechanical and enzymatic dispersion, using previously described methods [30,31]. Pipette and external solutions for IKr measurement were identical to those described above for IHERG measurement.
2.4
Electrophysiological recording and analysis
Whole-cell patch-clamp recordings were made using Axopatch 200 or 200B amplifiers (Axon Instruments) and a CV201 head-stage. Between 75 and 80% of the pipette series resistance was compensated. Voltage-clamp commands were generated using ‘WinWCP’ (John Dempster, Strathclyde University), Clampex 8 (Axon Instruments), or ‘Pulse’ software (HEKA Electronik). Data were recorded either via a Digidata 1200B interface (Axon Instruments) or an Instrutech VR-10B interface and stored on the hard-disk of a personal computer. The voltage-protocols employed for specific experiments are described either in the relevant ‘results’ text, or are shown diagrammatically on the relevant figures; unless otherwise stated in the text, the holding membrane potential between experimental sweeps was −80 mV. Data are presented as mean ± S.E.M. Statistical comparisons were made using, as appropriate, paired and unpaired t-tests or one-way analysis of variance (Anova) (Prism 3 or Instat, Graphpad Inc.). P values of less than 0.05 were taken as significant; ns = no statistically significant difference.
The following equations were used for numerical analysis and graphical fits to data:
The extent of IHERG inhibition by differing concentrations of doxepin was determined using the equation:where ‘Fractional block’ refers to the degree of inhibition of IHERG by a given concentration of doxepin; IHERG–DOXEPIN and IHERG–CONTROL represent current amplitudes in the presence and absence of doxepin.
Concentration–response data were fitted by a standard Hill equation of the form:where IC50 is [DOXEPIN] producing half-maximal inhibition of the IHERG tail and h is the Hill coefficient for the fit.
Half-maximal voltage values for IHERG activation were obtained by fitting IHERG tail-voltage (I–V) relations with a Boltzmann distribution equation of the form:where I is the IHERG tail amplitude following test potential Vm, Imax the maximal IHERG tail observed during the protocol, V0.5 the potential at which IHERG was maximally activated, and k is the slope factor describing IHERG activation. Data from each individual experiment were fitted by this equation to derive V0.5 and k values in ‘control’ and with doxepin. The resultant mean V0.5 and k values obtained from pooling values from each experiment were then used to calculate the mean activation relations plotted in Fig. 3.
Parameters describing voltage-dependent inactivation of IHERG were derived from fits to voltage-dependent availability plots with the equation:where ‘inactivation parameter’ at any test potential, Vm, occurs within the range 1–0, V0.5 is the voltage at which IHERG was half-maximally inactivated and k describes the slope factor for the relationship.
3
Results
3.1
Doxepin produces concentration-dependent inhibition of WT IHERG
IHERG was elicited by the protocol shown in the inset of Fig. 1A, which is a standard protocol used to study IHERG pharmacology in this laboratory (e.g. [28,29,32]). Membrane potential was stepped from −80 to +20 mV for 2 s followed by a 4-s step to −40 mV to elicit IHERG tails. A brief (50 ms) pulse to −40 mV preceded the step to +20 mV in order to monitor the instantaneous current without activation of IHERG (peak outward IHERG tails on repolarization to –40 mV were compared with the instantaneous current during the 50 ms pulse to −40 mV, in order to measure IHERG tail amplitude). This voltage protocol was applied repeatedly (at 20 s intervals) prior to and during the application of doxepin. Fig. 1A shows a representative recording of IHERG in control, 5 min after the addition of 10 μM doxepin and 5 min following wash-out of the drug. Doxepin produced a substantial inhibition of IHERG; this was largely reversible upon washout (to 73 ± 9% of control in six cells to which 10 μM doxepin was applied). Fig. 1B shows records for the same cell before and at increasing times during doxepin exposure, indicating that blockade was maximal within 4–5 min of drug application. Fig. 1C shows a plot of mean ± S.E.M. fractional block of IHERG tails by four different concentrations of doxepin, fitted by Eq. (2). The IC50 for doxepin inhibition of IHERG with this protocol was 6.5 ± 1.4 μM and the Hill coefficient for the fit was 1.0 ± 0.2. IHERG blockade by doxepin was not accompanied by statistically significant changes to the time-course of deactivation of the time-dependent component of IHERG tails on repolarization to −40 mV: bi-exponential fitting of the time-dependent tail current decline yielded fast time-constants of deactivation (τfast) of 203 ± 15 and 273 ± 21 ms in control solution and 10 μM doxepin, respectively (n = 6; p > 0.05, paired t-test), and slow time-constants of deactivation (τslow) of 1164 ± 141 and 1499 ± 131 ms (p > 0.1). Doxepin did not influence significantly the relative proportions of deactivating current described by the fast and slow time-constants of deactivation (the proportion of deactivating current fitted by the τfast was ∼0.6 in both control and doxepin).
It has been suggested that pharmacological inhibition of IHERG by some drugs may vary between different stimulus protocols [33]. Therefore, we also investigated IHERG blockade by doxepin using an action potential (AP) voltage waveform [32,34]. The voltage-command used (shown in the lower panel of Fig. 2A) was a previously acquired, digitised AP from a rabbit ventricular myocyte. This was applied repeatedly (at 4 s intervals) from a holding potential of −80 mV [32]. The upper panel of Fig. 2A shows traces in the absence and presence of doxepin. Peak outward IHERG during the repolarizing phase of the AP was inhibited 60 ± 12% (n = 5) by 10 μM doxepin, which does not differ significantly from the extent of IHERG tail current blockade by this concentration obtained using the protocol shown in Fig. 1 (62 ± 4%; n = 6; p > 0.8; unpaired t-test).
In order to determine effects of doxepin on native IKr, three drug concentrations (1, 10 and 100 μM) were applied to ventricular myocytes under whole-cell patch clamp. The command protocol for these experiments (similar to [32]) is shown in Fig. 2B and IKr tails were monitored on repolarization from +20 to −40 mV. Under our conditions, the outward tail currents observed on repolarization to −40 mV were almost completely abolished by 1 μM dofetilide (91 ± 3% blockade; n = 7), verifying that these were carried by IKr with little or no contamination from overlapping currents. Fig. 2C shows representative IKr tails on repolarization from +20 to −40 mV in the absence and presence of 100 μM doxepin; at this concentration the current was largely abolished. Fig. 2D contains mean ± S.E.M fractional block data for the tested doxepin concentrations, fitted by Eq. (2). The derived IC50 for doxepin inhibition of native IKr tails was 4.4 ± 0.6 μM, in good agreement with the observed inhibitory potency of doxepin on IHERG.
3.2
Voltage dependence of IHERG blockade by doxepin
Voltage dependence of IHERG blockade by doxepin was determined by the application, in control and doxepin-containing solutions, of a series of 2 s duration depolarizing commands to a range of test potentials up to +40 mV [32]. Successive command pulses were applied at 20 s intervals. Representative control currents at selected command voltages are shown in Fig. 3Ai; currents from the same cell following equilibration in 10 μM doxepin are shown in Fig. 3Aii (upper traces; lower traces in each panel show corresponding voltage commands). At all test potentials, IHERG was inhibited by doxepin. For each of five similar experiments, fractional inhibition of IHERG tails following each voltage command was calculated using Eq. (1); the mean ± S.E.M. fractional block of IHERG tails is plotted against command voltage in Fig. 3B. Also shown in Fig. 3B are voltage-dependent activation relations for IHERG in control solution and in the presence of doxepin (see Section 2). The derived mean V0.5 and k values were: control V0.5 = −21.0 ± 3.1; doxepin V0.5 = −24.0 ± 2.2, p > 0.1; control k = 5.9 ± 0.2; doxepin k = 7.5 ± 1.7, p > 0.3, with the IHERG activation relations in control and doxepin closely overlying one another (Fig. 3B). Fractional block of IHERG showed no statistically significant dependence on voltage over the range from −40 to + 40 mV (p > 0.2; Anova).
The voltage dependence of IHERG availability/inactivation was assessed using a 3-step protocol similar to those used in previous IHERG investigations from our laboratory ([32,35], and shown schematically as an inset to Fig. 3C). Mean data from five experiments were corrected for deactivation [35] and the resulting values were plotted to give availability plots in the absence and presence of doxepin (Fig. 3C); the data-sets were fitted with Eq. (4) (see Section 2). In control and doxepin the inactivation V0.5 values were, respectively, −37.9 ± 2.4 and −43.5 ± 4.1 mV (ns, paired t-test), with corresponding k values of −16.2 ± 2.4 and −17.3 ± 8.8 mV (ns, paired t-test). Thus, doxepin did not alter the voltage-dependence of IHERG inactivation. Although there was a trend towards an acceleration in the time-course of IHERG inactivation, this did not attain statistical significance (inactivation time–constant of 3.3 ± 1.2 ms in control versus 1.4 ± 0.6 ms in doxepin, p > 0.1; obtained from exponential fits to the inactivating phase of the current elicited during the third step of the protocol, following a brief hyperpolarizing step to −80 mV).
3.3
Time dependence of IHERG inhibition by doxepin
Gating-dependence of IHERG inhibition by doxepin was investigated further by the use of two protocols to examine the time-dependence of development of IHERG blockade. The first protocol used a sustained (10 s) depolarizing step to 0 mV from a holding potential of −80 mV (shown in Fig. 4A). This protocol was applied first in the absence of doxepin to elicit control IHERG. It was then discontinued whilst cells were equilibrated in 10 μM doxepin (for 7 min), after which it was re-applied. The first current trace recorded on resumption of stimulation was used to determine development of fractional blockade throughout the applied depolarization. Fig. 4A shows representative traces in control and doxepin; these traces diverged rapidly following depolarization, suggesting that IHERG blockade developed rapidly with time. Fig. 4B shows plots of mean ± S.E.M. fractional block of IHERG at various time intervals throughout the applied depolarization (main panel; n = 8) and on an expanded time-scale to show development of blockade over the first 0.5 s (inset). These plots show clearly that IHERG blockade developed rapidly on depolarization, with little change in blockade after 200–300 ms following the onset of the voltage-command. The time-course of development of blockade was well-described by a mono-exponential fit to the data, with a rate constant (K) for the fit of 16.33 s−1 (equivalent to a time-constant (1/K) of 61 ms).
Although the protocol used in Fig. 4 is well suited for examining the development of IHERG inhibition over a period of seconds following membrane depolarization, it is less well suited for accurate assessment of blockade of IHERG immediately following membrane depolarization than are protocols based on tail current measurements [29,32]. Therefore, a second protocol was also used [29]. Membrane potential was held at –100 mV and, from this, 10 and 200 ms duration depolarizations to +40 mV were applied, each followed by a period at −40 mV to monitor IHERG tails [29]. Fig. 5A shows representative currents from the same cell activated by 10 ms (Fig. 5Ai) and 200 ms (Fig. 5Aii) commands in the absence and presence of doxepin. Some IHERG tail inhibition was evident following the 10 ms duration command, with further blockade evident after the 200 ms command. Fig. 5B shows mean fractional block of IHERG tails for five similar experiments. Whilst blockade for 200 ms commands was significantly greater than that for 10 ms commands (p < 0.02), the occurrence of some blockade with only a very brief duration depolarizing command is concordant with either a very rapidly developing gating-dependent blockade on depolarization or with a contribution of closed-channel block to the overall effect of doxepin [25,29,36].
3.4
Effect of inactivation-attenuating mutants on IHERG inhibition by doxepin
In order to investigate further the gating-dependence of IHERG blockade by doxepin, experiments were performed using two inactivation-attenuating HERG mutants: S631A and N588K. Residue S631 is located towards the outer mouth of the HERG channel pore, and the S631A (serine → alanine) mutation has been reported to shift IHERG inactivation by ∼+100 mV [26]. Residue N588 is located in the external S5-Pore linker of the channel, and the N588K (asparagine → lysine) mutation, which is responsible for one form of the recently identified genetic short QT syndrome [27], has been reported to shift IHERG inactivation by ∼+60 to +100 mV [35,37]. The effect of each mutation on the potency of IHERG blockade by doxepin was determined using the same protocol as was used to establish concentration-dependence of WT IHERG blockade (Fig. 1). Fig. 6Ai shows representative currents in control and 10 μM doxepin for WT–HERG, whilst Fig. 6Aii and Aiii show similar records for S631A–HERG and N588K–HERG, respectively. In contrast to WT–HERG, for both S631A–HERG and N588K–HERG the IHERG tail magnitude was substantially smaller than the maximal current during the voltage-command (Fig. 6Aii and Aiii, respectively) reflecting the greatly attenuated IHERG inactivation of these HERG mutants [26,35,37]. Both mutant channels retained the ability to be inhibited by doxepin. Exponential fits to currents activated on depolarization to +20 mV for both mutants showed similar time-courses of current activation in control solution and following equilibration with 10 μM doxepin (S631A control: 60.6 ± 5.5 ms; doxepin: 64.5 ± 8.0 ms, p > 0.5; N588K control: 49.8 ± 6.2; doxepin: 41.7 ± 9.9 ms, p > 0.5; n = 5 for both). Three doxepin concentrations (1, 10 and 100 μM) were tested to obtain concentration-response data. Fig. 6B shows concentration-response relations (determined using Eq. (2)) for IHERG tail inhibition for both S631A–HERG and N588K–HERG, with corresponding data for WT–HERG plotted for comparison. Although the S631A and N588K mutations produced small and modest increases, respectively, in the IC50 derived from the fits to the concentration-response relations (from 6.6 μM for WT to 8.6 μM for S631A–HERG and 12.6 μM for N588K–HERG), these differences did not attain statistical significance (p > 0.05; Anova). These observations indicate the IHERG blockade by doxepin was not highly sensitive to attenuation of HERG channel inactivation.
3.5
Sensitivity of IHERG inhibition by doxepin to the S6 mutations Y652A and F656A
Two aromatic amino-acid residues, Y652 and F656, have been shown to be important components of the drug-binding site for a variety of HERG channel blockers [5,9,38]. Accordingly, we investigated whether or not doxepin inhibition of IHERG was sensitive to mutation of either residue, adopting a similar approach and protocols to those used in other recent IHERG pharmacology studies [29,32,39–41]. Fig. 7 shows the effects on doxepin inhibition of IHERG of the mutation Y652A (tyrosine → alanine). The experimental voltage protocol used (Fig. 7Aiii) was identical to that used for WT IHERG in Fig. 1 and to investigate the S631A and N588K mutants in Fig. 6. Representative traces showing the effect of 10 μM doxepin on WT IHERG and Y652A IHERG are shown in Fig. 7Ai and Aii. WT IHERG was inhibited substantially by this doxepin concentration (Fig. 7Ai). In contrast, inhibition of Y652A IHERG was noticeably reduced (Fig. 7Aii; 28 ± 3% peak tail current inhibition compared to 62 ± 4% for WT IHERG; p < 0.001). Four other doxepin concentrations (30, 100, 300μM and 1 mM) were also tested in order to construct a concentration response relation for inhibition of Y652A IHERG tails (Fig. 7B). A fit to the data with Eq. (2) (dashed line) yielded an IC50 for inhibition of Y652A IHERG of 27.8 ± 8.8 μM (with a Hill coefficient of 0.9 ± 0.3), which represented a modest, though significant (p < 0.05), ∼4.2 fold increase in IC50 over that for WT IHERG.
IHERG carried by F656A–HERG channels was studied using the protocol shown in the inset of Fig. 8Aiii and inward tails were measured at −120 mV [25,28,29]. Initial experiments employed a doxepin concentration of 100 μM (shown in Fig. 1 to produce extensive inhibition of WT IHERG with a standard [K+]e). Representative traces showing effects of this concentration on WT and F656A IHERG are shown in Fig. 8Ai and Aii. The F656A mutation produced a modest attenuation of IHERG inhibition by this concentration of doxepin. However, as observed previously for some other drugs (e.g. [28]), the extent of inhibition of WT IHERG by 100 μM doxepin under conditions of high [K+]e (see Section 2) was significantly smaller (56 ± 5%) than that observed with the same concentration in the experiments with a standard [K+]e in Fig. 1 (93 ± 4%; p < 0.001). Therefore, the effects of two further concentrations of doxepin (500 μM and 1 mM) were also examined. Fig. 8B shows bar-chart plots of the mean levels of inhibition of WT and F656A IHERG by the three doxepin concentrations. These concentrations resulted in progressive increases in the level of blockade of WT IHERG. In contrast, although Anova comparison of the data with the three drug concentrations confirmed that doxepin inhibition of F656A IHERG was concentration-dependent (p < 0.001), the observed concentration-dependence was unusual: there was no statistically significant difference between the observed level of blockade of F656A IHERG between 100 and 500 μM, whilst at 1 mM the observed level of inhibition of F656A IHERG was markedly increased (p < 0.001 compared to each of 100 and 500 μM, Bonferroni post-test) and approached that of WT IHERG. Taken together, the data with the three doxepin concentrations suggest that the F656A mutation exerted some influence on the ability of doxepin to inhibit IHERG, though the F656A data did not appear to follow a conventional monotonic concentration dependence.
4
Discussion
Despite a strong association between TCA use and QT interval lengthening [42], and although doxepin itself has been linked with both QT interval prolongation and TdP [18–20], the effects of this drug on HERG K+ channels have not hitherto been reported. Moreover, whilst IHERG blockade has been investigated previously for the TCAs imipramine and amitriptyline [21–23], to our best knowledge the present study is the first in which molecular determinants of IHERG inhibition have been investigated for any member of the TCA family.
4.1
Characteristics of IHERG blockade by doxepin
Previously, imipramine has been reported to inhibit IHERG recorded in experiments using a mammalian expression system with an IC50 of 3.4 μM [23] whilst an IC50 of 10 μM was reported for amitriptyline [21]. A different study of amitriptyline, using the Xenopus oocyte expression system, reported IC50's of ∼3.3–4.8 μM (depending on [K+]e) [22]. Thus, the potency of doxepin as an IHERG inhibitor found here (IC50 of 6.5 μM) is broadly comparable to that seen previously for these other two TCAs. However, in terms of the characteristics of observed IHERG blockade, doxepin appears to be closer to imipramine than to amitriptyline: amitriptyline block of IHERG has been reported to show significant voltage-dependence [22] whereas imipramine showed only weak voltage-dependence [23]. Moreover, no significant effects of doxepin on the voltage-dependence of activation or inactivation were seen in this study. Unfortunately, comparative data for the TCAs imipramine and amitriptyline are lacking [21–23] and, therefore, a direct comparison between doxepin and these agents cannot be made in this regard. However, the atypical tetracyclic antidepressant maprotiline has recently been reported to show no alteration to the voltage-dependence of activation and inactivation [43], though another study contradicted this with regard to activation [44]. The lack of a significant leftward shift in the voltage-dependence of inactivation with doxepin in our study suggests that this agent does not act to stabilise IHERG inactivation, and the time-course of WT IHERG inactivation was not significantly accelerated by the drug. Moreover, the results with inactivation-attenuating mutations to two residues from distinct parts of the channel (S631A: outer mouth of the channel pore; N588K: in the S5-Pore linker) provide evidence that IHERG inactivation does not play an obligatory role in doxepin's binding to the channel. This is further supported by unaltered levels of WT IHERG blockade with progressive depolarization over voltages at which IHERG was maximally activated, but over which inactivation increased (Fig. 3C and D). Doxepin is quite distinct in this regard from a number of other drugs, including the archetypal high affinity HERG blocking methanesulphonanilide drugs E-4031 and dofetilide, for which IHERG inactivation exerts a strong influence on blocking potency – either as a direct consequence of inactivation-state dependent block, or due to conformational changes during inactivation facilitating optimal orientation of S6 helical residues to which drugs bind [10,45–48].
Blockade of IHERG by imipramine has been reported to develop rapidly during a sustained depolarization, with a component of inhibition visible even for comparatively brief depolarizing voltage commands [23]. These features of imipramine's action correspond well with those seen for doxepin in the present study. They are also similar to those reported for the serotonin-selective reuptake inhibitors (SSRIs) fluvoxamine and citalopram [25,49], but differ significantly from the methanesulphonanilides, for which little blockade is observed immediately upon depolarization, with blockade then increasing progressively during the maintained depolarization [24,49,50]. The lack of a strong dependence of HERG channel blockade by doxepin on IHERG inactivation suggests that gating-dependent blockade by this drug is likely to arise predominantly to activated/open channels. Accordingly, the observed time-dependence of IHERG inhibition by doxepin in this study is consistent with either a mixed state-dependence of blockade (with components of both closed and open channel blockade) or with the presence of a very rapidly developing component of activation-dependent inhibition immediately on depolarization.
For the majority of drugs that have been studied, one or both of the Y652 and F656 aromatic amino acid residues in the S6 helices of the HERG channel comprise key elements of the drug binding site [5,9,38,51]. For example, the Y652A and F656A mutations increased the IC50 for HERG blockade by the methanesulphonanilide MK-499 by 94-fold and 650-fold, respectively, and that for terfenadine by ∼100-fold [8]. In comparison, the ∼4-fold increase in IC50 for doxepin produced by the Y652A mutation in this study, is rather modest, suggesting that this residue is less influential for binding of doxepin than for either of these high affinity blockers. The unusual concentration dependence seen with the F656A mutation indicated that at a high doxepin concentration of 1 mM IHERG blockade was little affected by mutation of this residue, whilst at concentrations of 100 and 500 μM significant attenuation of blockade occurred with no significant increase in block at 500 μM compared to 100 μM. It was not possible to obtain an adequate fit of these data with Eq. (2). A cautious interpretation of the lack of a conventional concentration-dependence of F656A–HERG inhibition by doxepin is that blockade may depend partly, but incompletely, on this residue. Neither the Y652A nor F656A mutations attenuated blockade by doxepin concentrations producing very high levels (>90%) of blockade of WT IHERG, suggesting that neither residue is absolutely obligatory for doxepin binding to HERG channels to occur. Whilst unusual, this is not unprecedented; IHERG block by both the SSRI fluvoxamine and the antiarrhythmic agent dronedarone has been reported to be only partially attenuated by the Y652A and F656A mutations [25,28]. Comparative data for other TCAs are lacking, though recently these residues have been implicated in IHERG inhibition by the tetracyclic drug maprotiline [43,44]; one of these two studies obtained IC50 values for the Y652A and F656T mutants, with respective (modest) 3-fold and 7-fold increases in IC50
[43]. Whilst obligatory molecular determinants of doxepin-binding to HERG remain to be found, the fact that IHERG inhibition by doxepin developed progressively over several minutes following rapid external solution exchange is consistent with the drug crossing the cell membrane to reach its site of action. The reduced IHERG inhibition by doxepin of WT IHERG in the presence of raised [K+]e may also be of significance. Since IHERG inhibition by doxepin appears not to be critically dependent on channel inactivation, decreased blockade with high [K+]e is unlikely to result from any effect of [K+]e on IHERG inactivation. Rather, reduced inhibition in high [K+]e may be accounted for by an interference with drug-binding due to an electrostatic repulsion or “knock-off” process [28,52], consistent with drug binding to the channel at a site close to the ion conduction pathway. It remains to be determined whether this site would need to reside within the channel pore. However, in a limited series of experiments using the D540K mutant [53], we did not find evidence that doxepin can readily unbind on hyperpolarization-induced channel opening (data not shown); though not all drugs that bind within the pore exhibit marked ‘untrapping’ [54]. Nevertheless, given the presence of a significant component of IHERG blockade with brief depolarization and incomplete attenuation of inhibition by the Y652A and F656A mutations, we cannot exclude the possibilities that a proportion of the observed blockade with doxepin involves binding outside of the channel pore, or binding to closed HERG channels.
4.2
Clinical significance of IHERG blockade by doxepin
TCAs are associated with QTc interval lengthening in clinical use [42], and IHERG inhibition by doxepin observed in the present study is consistent both with documented cases of QT interval prolongation and TdP with doxepin [18–20] and with QT interval increases seen in anaesthetised guinea-pigs receiving doxepin infusion [15]. Our study was conducted at a physiologically relevant temperature and the extent of IHERG blockade by doxepin was similar between ventricular action potential and conventional voltage step protocols. A lack of relief of IHERG blockade on membrane hyperpolarization for the D540K mutant (mentioned in Section 4.1, above) is suggestive of relatively poor drug unbinding at negative voltages in the maintained presence of drug, whilst the data shown in Fig. 5 indicate that at 10 μM (the same concentration as used for the AP clamp experiment in Fig. 2) doxepin was able to bind and inhibit IHERG rapidly on membrane depolarization to a positive voltage. These factors may combine to account for the similar levels of IHERG inhibition by doxepin observed with step and AP clamp protocols. Although we did not co-express HERG with MiRP1, a putative β subunit suggested to be necessary to recapitulate native IKr
[55], it has recently been suggested that MiRP1 is unlikely to interact with HERG outside of the cardiac conduction system [51] and, additionally, the pharmacological sensitivity of HERG channels expressed in mammalian cells without MiRP1 co-expression has been found to be similar to that of native IKr
[56]. This notion is reinforced by the close concordance of inhibitory potency of doxepin on IHERG and native IKr in our experiments (IC50 values of 6.5 and 4.4 μM, respectively). A question therefore arises as to the relationship between the potency of IHERG/IKr blockade seen in this study and plasma concentrations of doxepin in patients. As a class, the TCAs are lipophilic and are known to become concentrated in some tissues, including the myocardium [57]. In the case of doxepin, one experimental study has reported doxepin concentration in cardiac muscle to be 41-fold higher than plasma levels [58]. This makes it difficult to extrapolate with accuracy from known plasma concentrations to likely levels of IHERG/IKr blockade by doxepin in vivo. The therapeutic plasma level of doxepin is thought to be between 50 and 250 ng/ml (0.16–0.8 μM), although a wide variety of recommendations from university psychiatric departments and laboratories (up to 1000 ng/ml; 3.2 μM) have been reported [59]. Whilst IHERG (or IKr) blockade at the lower end of this range might be anticipated to be modest, inhibition at higher concentrations would be significant and, taking into account also potential cardiac accumulation, the observed potency of IHERG inhibition by doxepin in this study is likely to be clinically relevant, particularly in overdose. Such an effect may be exacerbated in individuals exhibiting pre-existing QT interval prolongation (congenital or acquired), electrolyte abnormalities or with impaired drug metabolism. Thus, as for other IHERG blocking medications, caution is warranted in its use in patients with pre-existing QT interval prolongation or with risk factors likely to exacerbate the effects of IHERG blocking medications.
The findings of this study have further clinical relevance in a second, perhaps less expected, respect. The attenuated-inactivation N588K–HERG mutant used in this study has been shown recently to underlie the SQT1 familial form of the recently identified genetic ‘Short QT syndrome’, which carries a risk of cardiac arrhythmia and sudden death [27,60]. Pharmacological approaches to correcting the QT-interval of SQT1 patients are currently very limited. These patients are comparatively insensitive to Class III IKr/HERG blocking drugs [27,61] and the N588K–HERG blocking potencies of the IKr/HERG blockers E-4031and D-sotalol are reduced ∼12–20-fold compared to their effects on WT–HERG [62,63], presumably due to a role (direct or otherwise) of channel inactivation in facilitating drug binding to the HERG channel. To date, only the Class Ia antiarrhythmic drug quinidine has been found both to inhibit N588K–HERG effectively and to correct the QT interval in such patients [27,62,63]; however, very recently, another Class Ia antiarrhythmic, disopyramide, has been shown to be effective against N588K–HERG in vitro [63]. The present study identifies doxepin as both an IHERG-blocker for which channel inactivation does not play a major role in drug binding and as an additional drug that is an effective inhibitor of N588K–HERG. Whilst the sedative effects of doxepin may make it unsuitable as a corrective treatment for SQT1 patients, our findings prompt the question as to whether chemical structures related to doxepin might feasibly offer viable IHERG blocking agents in SQT1.

herg
potassium channel
antidepressant
doxepin
rapid delayed rectifier
qt interval
arrhythmia
short qt syndrome
torsade de pointes
i kr
long qt syndrome
qt-prolongation


What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium?

Microalbuminuria is an important risk factor for cardiovascular disease and progressive renal impairment. This holds true in the general population and particularly in those with diabetes, in whom it is common and marks out those likely to develop macrovascular disease and progressive renal impairment. Understanding the pathophysiological mechanisms through which microalbuminuria occurs holds the key to designing therapies to arrest its development and prevent these later manifestations.
Introduction
The associations between microalbuminuria, cardiovascular disease and progressive renal impairment are well described, but how these are linked mechanistically is something of a conundrum [1]. Here we focus on the pathogenesis of microalbuminuria in patients with diabetes, in whom it occurs commonly and has particular significance. In type 1 diabetes the prevalence gradually increases from onset of disease (6% after 1–3 years), reaching over 50% after 20 years [2]. In type 2 diabetes the prevalence is 20–25% in both newly diagnosed and established diabetes [3]. However, it is also instructive to review the general epidemiology of microalbuminuria, including those conditions with which it is associated and those for which it is a risk factor. Such an analysis reveals generalised endothelial dysfunction as a common denominator in microalbuminuria in both the general and diabetic populations. In 1989, this observation led to the hypothesis that a common process underlies both microalbuminuria and generalised endothelial dysfunction in diabetes. This process was suggested to be the dysregulation of enzymes involved in metabolism of extracellular matrix, the ‘Steno hypothesis’ [4].
Nearly 20 years on from the Steno hypothesis, the determinants of selective glomerular permeability to proteins at the cellular and molecular level are much better understood. In particular, the importance of podocyte-specific proteins in the regulation of selective permeability has been recognised. Similarly, much is now known about the biochemical derangements important in the pathogenesis of diabetic complications. We draw together these elements to consider the pathophysiological mechanisms through which diabetes exerts its effects on glomerular permeability in the initiating stages of diabetic nephropathy, i.e. at or before the appearance of microalbuminuria. These early changes establish the milieu in which the more advanced changes of overt diabetic nephropathy develop. Defining the mechanistic links from biochemical derangements to the appearance of increased urinary albumin highlights key elements in the pathophysiological pathway of the development of both diabetic nephropathy and micro- and macrovascular disease elsewhere.
We hold with the established view that increased transglomerular passage of albumin is the major source of microalbuminuria [5]. While other hypotheses have been advanced, for example, failure of tubular reuptake of albumin, none are sufficiently robust to seriously challenge this position.
In both general and diabetic populations, conditions associated with endothelial damage predispose to microalbuminuria
In the general (non-diabetic) population, hypertension is the major risk factor for microalbuminuria, and the prevalence of microalbuminuria in essential hypertension is around 25%. Individuals with essential hypertension who develop microalbuminuria have a higher incidence of biochemical disturbances, implying that hypertension per se may not be the cause of microalbuminuria, but, rather, these additional derangements [6]. Microalbuminuria is strongly associated with vascular disease in hypertensive patients, suggesting that it is a marker of vascular and/or endothelial damage in this condition [7].
The insulin resistance syndrome describes a clustering of disorders the underlying pathology of which is thought to be related to insulin resistance and/or endothelial dysfunction [8]. Microalbuminuria is associated with several of the disturbances found in the insulin resistance syndrome, including endothelial dysfunction and obesity, in addition to type 2 diabetes. Proinflammatory cytokines produced by visceral adipocytes (adipokines) have recently emerged as important mediators of the increased cardiovascular risk associated with the insulin resistance syndrome. These adipokines represent a possible link from insulin resistance and obesity to microalbuminuria in the non-diabetic population.
Microalbuminuria can be detected in patients undergoing major surgery, particularly when complicated by sepsis [9], and is associated with other inflammatory states, including rheumatoid arthritis and inflammatory bowel disease [10]. Microalbuminuria can also be detected in a significant proportion of the normal non-diabetic, normotensive population (6.6% in one study [11]), where it also associates with cardiovascular disease. Male sex [11] and hormone replacement therapy in women [12] seem to increase susceptibility to microalbuminuria, and although the basis for this is not clear, the fact that men have a higher incidence of vascular disease in general implies a common aetiology.
Hypertension is approximately twice as frequent in individuals with diabetes as in those without, and hypertensive individuals are predisposed to the development of diabetes [13]. Hypertension is certainly a major determinant of microangiopathy in diabetes, but the relationship between hypertension and microalbuminuria in diabetes is complex. Hypertension and microalbuminuria often coexist in diabetic patients, and reducing blood pressure reduces microalbuminuria in type 1 diabetes [14]. However, it is unclear whether hypertension contributes to the development of microalbuminuria in diabetes. At least in type 1 diabetes, hypertension and microalbuminuria appear to develop together: in longitudinal studies there is no evidence that hypertension develops before microalbuminuria [15]. This is more difficult to demonstrate in type 2 diabetes, perhaps because of the heterogeneity of the disease.
One third of patients with type 1 diabetes develop advanced nephropathy, and the renal status of probands of diabetic patients makes a difference of nearly 50% in risk [16]. A family history of hypertension also predisposes to microalbuminuria in diabetes [17]. These observations suggest a genetic predisposition in individuals with type 1 diabetes who develop diabetic nephropathy, but as yet no gene has been identified. Perhaps most significant is that this and other evidence suggests that the susceptibility of type 1 diabetic patients to diabetic nephropathy is increased in response to increased genetic risk of insulin resistance [18]. Consequently, it is the combination of this genetic risk with hyperglycaemia and its sequelae that leads to microalbuminuria and, eventually, diabetic nephropathy.
Thus, in the diabetic as well as in the general population the risk factors for the development of microalbuminuria can be grouped into those associated with vascular disease, including endothelial dysfunction, inflammation and insulin resistance. This implies that microalbuminuria may also, at least in these situations, result from endothelial dysfunction. This does not preclude significant damage to other components of the vessel wall, including basement membrane, pericytes, matrix components (collagen, elastin) and vascular smooth muscle cells. However, as the principal regulators of vascular permeability, endothelial cells are likely to be involved in systemic increases in permeability.
Microalbuminuria is a risk factor for macro- and microangiopathy, including advanced diabetic nephropathy
Microalbuminuria is a significant risk factor for cardiovascular mortality in type 1 [19] and 2 diabetes [20], as well as in the non-diabetic population [21]. In type 1 diabetes, microalbuminuria is closely associated with microangiopathy elsewhere (e.g. retinopathy). Although this association is also present in type 2 diabetes, in a proportion of these patients microalbuminuria does not appear to reflect generalised microvascular damage [22].
Microalbuminuria predicts the development of overt diabetic nephropathy in type 1 and 2 diabetes; however, the relationship in type 2 diabetes is less clear because of the greater heterogeneity of this condition and the presence of other risk factors for microalbuminuria in these, usually elderly, patients [23]. Microalbuminuria invariably precedes overt diabetic nephropathy, and although microalbuminuria may regress spontaneously in a proportion of cases, it remains the best documented predictor for high risk of development of diabetic nephropathy in both type 1 and type 2 diabetes [24].
The relationship between microalbuminuria and vascular disease suggests a common causality, as established risk factors explain at most a small part of these associations [25]. Unifying mechanisms, such as generalised endothelial dysfunction or inflammation, are therefore implicated [8]. Indeed, AER is correlated with endothelial dysfunction in type 1 and type 2 diabetes, and in the non-diabetic population [26]. Markers of chronic low-grade inflammation, including C-reactive protein (CRP), are also correlated with microalbuminuria in type 1 and type 2 diabetes [27], and increasing evidence from the non-diabetic population indicates the importance of inflammation in the pathogenesis of cardiovascular disease [28].
Generalised and glomerular endothelial dysfunction
Markers of endothelial dysfunction, including elevated serum von Willebrand factor (vWF) and increased transcapillary albumin escape rate, are present before the onset of microalbuminuria in type 1 diabetes and worsen in association with it [27, 29]. Type 2 diabetes is often complicated by the presence of other risk factors for vascular disease, and discerning the contribution of hyperglycaemia and its sequelae to endothelial dysfunction in this condition is more difficult. While in some type 2 diabetic patients microalbuminuria may occur in the absence of evidence of endothelial dysfunction, in others, vWF levels predict its development [30]. The close association between endothelial dysfunction and microalbuminuria in type 1 diabetes may underlie the predictability of development of diabetic nephropathy and the greater susceptibility to micro- and macrovascular disease in other organs.
As endothelial dysfunction is an important antecedent of microalbuminuria in both types of diabetes (albeit with a less predictable relationship in type 2), it provides an attractive explanation for the association between microalbuminuria and vascular disease in diabetes (Fig. 1). But to what extent can endothelial dysfunction be said to cause microalbuminuria? As the glomerular endothelium is exposed to the same diabetic milieu as other endothelia, it is highly likely that it is also dysfunctional. This begs the question of how glomerular endothelial dysfunction could lead to microalbuminuria. To address this we now turn to consider the structure and function of the glomerular filtration barrier (GFB).
Fig. 1The relationship between hyperglycaemia, insulin resistance, endothelial dysfunction, macrovascular disease and microalbuminuria in type 1 and type 2 diabetes. Proposed major pathways are represented by red arrows; those of less certain significance by black arrows. The diagram illustrates, for the example, a possible mechanism for the increased risk of microalbuminuria in patients with type 1 diabetes and a susceptibility to insulin resistance. Particularly in type 2 diabetes, other pathways, not directly involving endothelial dysfunction, are likely in the pathogenesis of macrovascular disease and may also contribute to microalbuminuria (broken arrows)
The glomerular filtration barrier is a complex biological sieve
Unlike other capillaries, glomerular capillaries have a high permeability to water (hydraulic conductivity) yet, like other capillaries, are relatively impermeable to macromolecules. These fundamental permeability properties depend on the unique, three-layer structure of the GFB: the endothelium with its glycocalyx, the glomerular basement membrane (GBM) and podocytes (glomerular epithelial cells; Fig. 2) [31].
Fig. 2Representation of a cross-section through the GFB showing the three-layer structure consisting of glomerular endothelium and glycocalyx, glomerular basement membrane (GBM) and podocyte foot processes. Albumin, represented by orange ellipses, does not pass through the normal GFB in significant amounts
Glomerular endothelial cells Glomerular endothelial cells are highly specialised cells with regions of attenuated cytoplasm punctuated by numerous fenestrae, circular transcellular pores 60–80 nm in diameter [31, 32]. Initially these fenestrations were thought of as empty and to therefore provide little barrier to the passage of proteins. Standard fixation protocols for electron microscopy do not preserve the glycocalyx, but newer fixation techniques have allowed the demonstration of a glomerular endothelial glycocalyx of 200–400 nm in thickness [33, 34]. This glycocalyx covers both fenestral and inter-fenestral domains of the glomerular endothelial cell luminal surface.Studies of the systemic endothelial glycocalyx are instructive here. The glycocalyx is a dynamic, hydrated layer largely composed of glycoproteins and proteoglycans with adsorbed plasma proteins. Heparan sulphate proteoglycans (HSPGs) are largely responsible for the negative charge characteristics of the glycocalyx. Removal of the glycocalyx increases vascular protein permeability, providing evidence that it hinders the passage of macromolecules [35, 36]. Compared with those without, capillaries with fenestrations are much more permeable to water and small solutes, but not to proteins. These characteristics can only be explained by the glycocalyx [37].Therefore, the presence of a significant glomerular endothelial glycocalyx implies that the glomerular endothelium significantly contributes to the barrier to macromolecules [31, 32, 38]. Experimental data support this view: in mice treated with glycocalyx-degrading enzymes the distance between luminal lipid droplets and the glomerular endothelium was decreased, and this was accompanied by an increase in AER [39]. In rats, under normal perfusion conditions albumin is confined to the glomerular capillary lumen and endothelial fenestrae [40]. Endothelial glycocalyx has the correct anatomical distribution (on the surface of endothelial cells, including in fenestral openings) to explain this distribution. Reactive oxygen species (ROS), which are known to disrupt the glycocalyx [41], cause torrential proteinuria without any identifiable structural changes in the GFB using standard electron microscopy techniques [42]. Furthermore, the ability of human glomerular endothelial cell glycocalyx to form a permeability barrier to macromolecules can be directly demonstrated in vitro [43].
The GBM The GBM is a basal lamina specialised for the structural requirements of the GBM and its filtration function. It is a hydrated meshwork of collagens and laminins to which negatively charged HSPGs are attached. Traditional concepts have therefore characterised the GBM as a charge-selective barrier. However, more recent analyses indicate that it only makes a small direct contribution to the barrier to protein passage [31].
Podocytes Podocytes, or, more specifically, their interdigitating foot processes, form the outer layer of the GFB (Fig. 2). The gaps between adjacent foot processes, the ‘filtration slits’ (25–60 nm), are spanned by the slit diaphragm. This is a molecular structure thought to form the most restrictive barrier to the passage of water and macromolecules. The effect of mutations in podocyte-specific proteins (e.g. nephrin mutations result in congenital nephrotic syndrome) indicate the importance of podocytes in resisting the passage of protein [44]. However, exactly how podocytes and their foot processes contribute to selective permeability is not yet clear. Electron microscopy studies have suggested that the slit diaphragm has a porous structure with a pore half-width of 2 nm. However, this figure is too small to account for the experimentally observed passage of solutes of various radii, emphasising that our understanding of slit diaphragm function is incomplete [45]. Podocyte biology has been reviewed in detail elsewhere [44].
Passage of albumin across the normal GFB
Filtration Water filters across the GFB via gaps through or between cells rather than through the cell cytoplasm. Therefore, the high hydraulic conductivity of the GFB depends on the presence of fenestrae and filtration slits. Fifty per cent of the hydraulic resistance of the GFB is afforded by the two cell layers (endothelium and podocyte foot processes), and 50% by the GBM [46].
Solute flux Solute flux (including albumin) from the glomerular capillary occurs by a combination of convection (i.e. being swept along by the filtration of water) and diffusion [31]. The rate of convection of a solute depends on the filtration rate and on its reflection coefficient. Reflection coefficients are related to solute size, larger solutes having higher reflection coefficients. A value of 1 indicates that a molecule is totally excluded. The rate of diffusion of a solute depends on the concentration gradient and its diffusivity (solute permeability). The diffusivity of a solute decreases with increasing size. A measure of overall flux of a solute is given by the sieving coefficient: the filtrate to plasma concentration ratio. The sieving coefficient for albumin across the normal GFB is <0.001 [47]. The ratio of the contribution of convective to diffusive flux (the Peclet number) increases with increasing molecular size. That is, for small molecules, diffusion dominates; for larger molecules, convection dominates. The Peclet number for albumin across the normal GFB is near to unity, so both diffusion and convection contribute [48]. Hence, an increase in GFR will result in an increase in the convective flux of albumin. However, despite this, the best estimates indicate that even a 50% increase in filtration rate would only increase urinary albumin in the sub-microalbuminuric range, as the majority of the excess albumin is reabsorbed by the tubules [47]. Therefore, for albumin flux to increase sufficiently to produce microalbuminuria (assuming normal tubular reuptake), the GFB must be physically altered in such a way as to increase the sieving coefficient of albumin across it.Models have been developed to correlate the macromolecular sieving properties of the GFB with the structure and properties of its components [31]. These indicate that the GFB functions as a whole, with each layer having an important contribution to selective permeability. Whereas hydraulic resistances are essentially additive, sieving coefficients are multiplicative. This relationship means that a change in one element significantly affects the overall protein permeability to the same degree, i.e. a 10% change in permeability of any one layer will produce a 10% change in overall GFB permeability. Importantly, this means that, regardless of which layer is most restrictive, a change in the permeability of any layer of the GFB could potentially account for microalbuminuria.
Structural alterations in the GFB associated with microalbuminuria in diabetes
Glomerular structural changes typical of diabetic nephropathy are commonly established by the time microalbuminuria becomes apparent [49, 50]. However, the changes seen are heterogeneous, and all may be found in normoalbuminuric diabetic patients [51]. Early changes described include an increase in glomerular size, GBM thickening, mesangial expansion and broadening of podocyte foot processes [49–51]. The increase in glomerular size is due both to mesangial expansion and to enlargement in glomerular capillaries. The latter occurs at least in part through angiogenesis, an endothelium-dependent process [52, 53]. Glomerular structural changes are less marked in type 2 diabetes, with only a third conforming to the classical pattern observed in type 1 diabetes [22].
Specific assessment of structural changes in glomerular endothelial cells and associated glycocalyx in diabetes has not been performed. Evidence is emerging, however, that total systemic glycocalyx volume is reduced by acute hyperglycaemia in humans [54]. Furthermore, type 1 diabetic patients have decreased systemic glycocalyx volume, and this correlates with the presence of microalbuminuria [55]. GBM thickening alone, without change in composition, does not significantly affect its protein permeability characteristics.
Broadening of podocyte foot processes (effacement) is an indicator of podocyte injury but generally correlates poorly with degree of proteinuria [56]. Indeed, proteinuria may occur in the complete absence of structural changes to podocytes [57]. This is the case with diabetic microalbuminuria, which can occur in the absence of such changes, at least in type 2 diabetes [58]. Some have suggested that podocyte loss occurs in early diabetes and that this would contribute to the filtration barrier defect [51]. Others have argued that significant early podocyte loss does not occur [59]. A likely reconciliation is that the lower proportion of podocytes seen in early disease is due to a relative increase in mesangial and endothelial cells, while podocyte loss occurs at a later stage.
Functional alterations in GFB selective permeability associated with microalbuminuria in diabetes
Increased flux of albumin across the GFB in diabetic microalbuminuria can be confirmed in experimental models using isolated glomeruli [60] and by inhibition of tubular reabsorption of albumin [61]. Analysis of the permeability of the GFB to molecules of varying size and charge can be used to estimate whether the increased flux of albumin is due to loss of size or charge selectivity of the GFB. In animal models of diabetes the defect is primarily in charge selectivity [61]. In clinically healthy non-diabetic individuals with microalbuminuria, loss of both size and charge selectivity of the GFB can be demonstrated [62]. However, in type 1 diabetes, defects in charge selectivity occur earlier than loss of size selectivity [63]. Similarly, in Pima Indians with type 2 diabetes, microalbuminuria is associated with loss of charge selectivity. Loss of size selectivity is seen only in those developing macroalbuminuria [58].
We have already noted that physical alteration of the GFB is necessary to increase its permeability to albumin. Therefore, this confirmation of changes in GFB selective permeability in diabetic microalbuminuria in the absence of clearly identified structural correlates suggests that the key changes are yet to be elucidated. These considerations and the predominance of defects in charge selectivity point to alterations in the negatively charged glomerular endothelial glycocalyx as the missing link. We now move to consider what aspects of the diabetic milieu are responsible for these GFB changes and how they might cause endothelial, including glycocalyx, dysfunction.
Metabolic pathways and effectors from hyperglycaemia to microalbuminuria
There is now overwhelming evidence that hyperglycaemia is the major initiating factor in the pathogenesis of diabetic complications, including microalbuminuria. However, most adverse effects of glucose are mediated indirectly through diverse metabolic pathways. Four major hypotheses have highlighted the roles of AGEs, increased activity of the polyol pathway, activation of protein kinase C and increased flux through the hexosamine pathway (Fig. 3). Activation of these pathways in turn causes dysregulation of a number of effector molecules which cause cellular damage and dysfunction. The roles of these pathways and effectors have been studied in detail in overt diabetic nephropathy, but the importance of these elements individually in its initiation and the appearance of microalbuminuria are less clear.
Fig. 3Pathways to microalbuminuria in diabetes. Hyperglycaemia, through increased mitochondrial superoxide production, dysregulates key intracellular metabolic pathways. These in turn lead to the production of effectors that directly cause glomerular endothelial cell (GEnC) dysfunction (particularly of the glycocalyx) and disturb podocyte–endothelial cell communication. This results in microalbuminuria. Progression of these lesions and development of other glomerular changes, including podocyte damage, lead to overt diabetic nephropathy
For example, TGFβ is an effector molecule with a clear role in the progression of established diabetic nephropathy, but there is no evidence that it is important in the mechanism of proteinuria per se. It is a key pro-fibrotic mediator, and in type 1 and type 2 diabetes, TGFβ is elevated in serum, urine and glomeruli from an early stage of disease. Its levels correlate with the degree of mesangial expansion, interstitial fibrosis and renal insufficiency, but not with microalbuminuria. Similarly, in animal models, blockade of TGFβ signalling inhibits the development of these pathological features, apart from the microalbuminuria [64, 65].
Here we focus on selected intermediaries and effectors that have an identifiable role in microalbuminuria. In large part, dissection of these pathways and elucidation of their role in glomerular disease has necessarily relied on tissue culture or animal models. Such are described where human studies are lacking or where they provide additional significant insights.
ROS Brownlee has proposed oxidative stress as a unifying mechanism whereby the above-mentioned four pathways are inter-linked in the pathogenesis of diabetic complications (Fig. 3) [66]. Hyperglycaemia increases oxidative stress through overproduction of superoxide and other ROS by the mitochondrial electron transport chain. ROS have direct cellular effects (e.g. they increase activity of nuclear factor κB [NFκB], a key inflammatory regulator), increase oxidative stress and interact with the above four pathways. Normalisation of mitochondrial superoxide production blocks all four of these pathways implicated in hyperglycaemic damage and corrects a variety of hyperglycaemia-induced phenotypes in target cells of diabetic complications. Endogenous superoxide dismutase normally neutralises excess superoxide but is overwhelmed in the diabetic state.As well as forming a nexus of metabolic pathways dysregulated by hyperglycaemia, ROS can also be considered as effectors through direct cellular actions. ROS decrease glomerular HSPG production [67], directly disrupt the endothelial glycocalyx [68], interfere with nitric oxide bioavailability and activate NFκB.Glomerular ROS production is increased in experimental diabetes [69] and transgenic overexpression of superoxide dismutase attenuates renal injury, including increases in AER [70]. A superoxide dismutase mimetic also ameliorates increases in glomerular permeability both in vivo and ex vivo in non-diabetic models [71]. Little work has yet been done on the use of antioxidants in human diabetic nephropathy, but in vitro work confirms the importance of ROS in cells of the GFB. Podocytes produce ROS in response to high glucose [72], and genetic overexpression of superoxide dismutase prevents inhibition of endothelial nitric oxide synthase (eNOS) activity by hyperglycaemia in endothelial cells [73].
Vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is a key regulator of vascular permeability and angiogenesis and is implicated in the pathogenesis of diabetic retinal neovascularisation. In the glomerulus it is produced in large amounts by podocytes and is thought to be important in maintaining glomerular endothelial cell fenestrations [74]. In rat models of type 1 diabetes, VEGF is upregulated in podocytes throughout the course of disease [75]. VEGF inhibition attenuates glomerular hypertrophy and albumin excretion and prevents upregulation of eNOS production in glomerular endothelial cells [76]. Similar effects are observed in some, but not all, models of type 2 diabetes [77].In human type 1 diabetes, serum VEGF concentrations vary according to glycaemic control, and higher levels are associated with microvascular complications, including microalbuminuria [78]. In type 2 diabetes, VEGF is upregulated early in the course of disease and urinary VEGF levels are correlated with microalbuminuria [79]. Further studies have confirmed initial upregulation of VEGF signalling in type 2 diabetes followed by a downregulation as podocyte loss and sclerosis develops [80].VEGF has the potential to induce the new vessel growth seen in early diabetic nephropathy [53] and to alter the permeability characteristics of the endothelium. Indeed, there is increasing evidence of the importance of precise control of glomerular VEGF for normal GFB function: both transgenic podocyte-specific under- or overexpression result in glomerular abnormalities, including glomerular endothelial changes and proteinuria [81]. Retardation of albuminuria in experimental diabetes by angiogenesis inhibitors further implies the importance of angiogenesis and/or endothelial-related processes in the development of diabetic microalbuminuria [52, 82].
The growth hormone/IGF system Dysregulation of the growth hormone/IGF system can be detected early in experimental diabetes and is associated with both glomerular hypertrophy and microalbuminuria [83]. All components of the growth hormone/IGF system can be detected at the mRNA level in the normal kidney. However, as detailed histochemical studies have not been performed, the exact location of system components with the glomerulus and the likely role of particular cell types cannot be defined.The rapid renal growth in experimental diabetes is preceded by a rise in the renal concentration of IGF-1 [84]. Somatostatin analogues ameliorate the increase in IGF-1 and renal hypertrophy and reduce AER [85]. In human type 1 diabetes, serum growth hormone levels and urinary IGF-1 levels are elevated and correlate with microalbuminuria, while an IGF-1 gene polymorphism modifies the risk of development of microalbuminuria [86]. The somatostatin analogue octreotide reduces macroalbuminuria and endothelial dysfunction in type 2 diabetes [87].Taken together, this evidence points to a role for the growth hormone/IGF system early in human diabetic nephropathy. However, there are few clues to its contribution to the pathogenesis of microalbuminuria at the structural level. IGF-1 activates intracellular intermediates, including NFκB, and an IGF-1 receptor inhibitor suppresses VEGF production, suggesting that VEGF is a downstream mediator of the effects of IGF-1 [88].
Proinflammatory cytokines and adipokines TNFα is a proinflammatory cytokine with diverse actions, including increased production of endothelial cell adhesion molecules and IL-6, which in turn regulates CRP. TNFα also directly increases endothelial permeability and disrupts the glycocalyx [41, 89]. In experimental diabetes, TNFα levels rise in urine and the renal interstitium prior to the onset of albuminuria and correlate with it [90], as they do in human type 2 diabetes [91]. IL-6 levels also correlate with albuminuria in type 1 and 2 diabetes [91, 92]. CRP, often thought of as a downstream marker of inflammation, is elevated in both type 1 and 2 diabetes [27, 93] and correlates strongly with cardiovascular disease.Adipokines, which include leptin and adiponectin (and, arguably, TNFα and IL-6), also have the potential to contribute to the development of microalbuminuria in both non-diabetic and diabetic populations. Enlargement of fat cells in obesity is associated with a generalised proinflammatory state, including increased levels of TNFα and IL-6, and hypersecretion of adipokines, with the exception of adiponectin, which is downregulated [94]. Leptin induces vascular permeability and synergistically stimulates angiogenesis with VEGF [95]. Its serum levels correlate strongly with nephropathy in type 2 diabetes [96]. Adiponectin, unlike other adipokines, appears to have protective effects in the vasculature in general by reducing endothelial cell activation and inflammation.What do these changes in regulation and expression of these mediators tell us about the mechanism of microalbuminuria? It is clear that ROS and oxidative stress have a central role, given their importance in various metabolic pathways and direct cellular effects, including in the disruption of endothelial glycocalyx, which may be relevant in pathogenesis of microalbuminuria. There is strong evidence for a role of VEGF early in the course of diabetic nephropathy, probably through upregulation of production by podocytes and actions on glomerular endothelial cells disrupting their contribution to the GFB. The importance of dysregulation of angiogenic factors is emphasised by the protective effects of angiogenesis inhibitors. The growth hormone/IGF system is also dysregulated early in diabetes, and it appears that IGF-1 is an important contributor to microalbuminuria through VEGF. Involvement of inflammatory mediators again points to a role for glomerular endothelial cells through endothelial activation, and hence, an increase in permeability through disruption of the glycocalyx.
Conclusions
In summary, the various avenues of study of diabetic microalbuminuria reviewed converge on the glomerular endothelium. Our analysis therefore leads to the conclusion that this is the site of the initial damage that leads to the development of microalbuminuria in diabetes. The most important aspect of this damage is disruption of the endothelial glycocalyx through actions of mediators dysregulated by the diabetic milieu. Key players include ROS, VEGF and proinflammatory cytokines (Fig. 4). Disturbance of endothelial cell–podocyte communication contributes to and amplifies the endothelial lesion. Progression of microalbuminuria to overt nephropathy is accompanied by predictable structural changes in the glomerulus, including podocyte damage and loss. This is the result of the ongoing diabetic milieu and disturbed cell–cell communication, but is also secondary to increased penetration of the GFB by serum proteins [97].
Fig. 4Proposed mechanism of glomerular filtration barrier damage leading to diabetic microalbuminuria. High glucose causes dysregulation of mediators including TNFα and enhanced production of ROS, which directly damage the glomerular endothelial glycocalyx leading to microalbuminuria. Increased levels of pro-angiogenic molecules, including VEGF and inflammatory mediators, induce an activated and more permeable glomerular endothelial cell phenotype
Thus recent evidence remains broadly supportive of the Steno hypothesis [4] but it points specifically to disturbance of the endothelial glycocalyx as the common process underlying both microalbuminuria and generalised endothelial dysfunction. While it would be overly simplistic, particularly in type 2 diabetes, to suggest that this is the only factor involved (Fig. 4), it follows nevertheless that microalbuminuria is an indicator of generalised endothelial dysfunction. Most interestingly, these conclusions imply that glycocalyx dysfunction is involved in the pathogenesis of other vascular disease, both microvascular and macrovascular. Therefore, therapies aimed at protecting or repairing endothelial cells and their glycocalyx would be expected to retard these diseases. Reduction or reversal of microalbuminuria implies resolution of generalised endothelial dysfunction and has potential to be a useful indicator of successful reduction of overall cardiovascular risk [98].

microalbuminuria
diabetes
podocyte
glomerular filtration barrier
glycocalyx
glomerular endothelial cell