Etiology Of The Afferent Arteriolar Dilation Underlying Diabetic Hyperfiltration

Results from the Diabetes Control and Complications Trial (7,8) established that intensive insulin therapy aimed at near normalization of blood glucose levels reduces the occurrence of microalbuminuria and albuminuria by 39 and 54%, respectively, compared with conventional insulin therapy. These benefits were greatest when intensive therapy was initiated soon after onset of T1D, underscoring the importance of early hyperglycemia-induced processes in leading to development of DN. Whereas it seems likely that hyperglycemia triggers or promotes the development of diabetic hyperfiltration, the events linking hyperglycemia to impaired preglomerular microvascular function in T1D have not been definitively established. Indeed, intensive investigation of many different pathophysiological sequelae proposed to underlie diabetic hyperfiltration (renal prostaglandins, sorbitol, growth hormone, atrial natriuretic peptide, nitric oxide, glucagon, kallikrein-kinin system, the renin-angiotensin system (RAS), etc.) has failed to unveil the undisputed key insult. In this chapter, we consider the possibility that altered electromechanical regulation of vascular smooth muscle tone and renal oxidative stress act in concert to promote the afferent arteriolar dilation underlying diabetic hyperfiltration.

Renal Oxidative Stress in T1D

One widespread consequence of hyperglycemia in T1D is oxidative stress reflecting net accumulation of reactive oxygen species (ROS), which encompass a variety of partially reduced metabolites oxygen. At the forefront of the array of ROS lies superoxide anion (O2-), which is formed in biological systems as the result of a one-electron reduction

H2o2 Redutcion
Fig. 2. Cascade of endogenous ROS formation. O2'-, superoxide anion; NO, nitric oxide; ONOO-, peroxynitrite; H2O2, hydrogen peroxide; OH', hydroxyl radical; HOCl, hypochlorous acid; GSH-Px, glutathione peroxidase.

of molecular oxygen by oxidases and auto-oxidation processes. As summarized in Fig. 2, O2'- formation leads to production of several ROS. The initial fate of O2'- is either dismutation to form hydrogen peroxide (H2O2) or reaction with nitric oxide (NO) to form peroxynitrite (ONOO-). Spontaneous dismutation of O2'- is accelerated more than 30,000-fold by superoxide dismutase (SOD), a reaction that proceeds at approx 60% of the rate of ONOO- formation (9). Significant amounts of H2O2 are produced that, in turn, can be degraded to H2O and O2 by antioxidant enzymes. Alternatively, H2O2 can serve as a precursor of other damaging substances such as hypochlorous acid (HOCl) and hydroxyl radical (OH).

Whereas net accumulation of one or more ROS can result from excess formation and/or inadequate degradation mechanisms (enzymatic and nonenzymatic scavengers and so on), the initiating event in this cascade is O2'- production. The cellular sources of O2'- include the mitochondrial electron transport chain, NAD(P)H oxidase, NO synthase (NOS), xanthine oxidase, cyclooxygenase, and the oxygenated form of cytochrome P450 (9). O2'- production by the mitochondrial electron transport chain has been proposed to represent a common element that triggers glucose-induced activation of several processes implicated in the vascular complications of T1D (10,11); however, NAD(P)H oxidase and NOS have also been implicated as a sources of excess O2'- production in T1D (12,13). Activation of one or more of these oxidant pathways during T1D drives an increase in O2'- formation in the vasculature and in the renal cortex (14). Moreover, compensatory increases in renal antioxidant enzyme activities are evident (15-17). A consequence of accelerated O2'- production is the formation of ONOO- and/or H2O2. ONOO- can react with tyrosine residues on proteins, a process presumed to underlie the increase in renal cortical protein tyrosine nitration in T1D (14,18). The possibility of increased renal H2O2 production in T1D has received little attention, although this situation can be expected to result from the increases in both O2'- production and SOD activity (14). In recent preliminary studies, we found that STZ rats exhibit increased urinary H2O2 excretion, which could reflect

Fig. 3. Suppressed influence of NOS inhibition (100 pM Nitro-L-arginine) on juxtamedullary afferent and efferent arteriolar diameter in STZ kidneys is restored by exogenous SOD (150 U/mL of blood perfusate). p < 0.05 vs Sham; n = 7-10 arterioles. (Data from Ohishi and Carmines [27].)

net increases in systemic and/or renal H2O2 production. H2O2 is relatively stable and easily crosses cell membranes; hence, any accumulation of H2O2 in the kidney could influence the function of a variety of cell types (19). Alternatively, H2O2 could be degraded by catalase or glutathione peroxidase, or it could serve as a substrate for formation of OH and/or HOCl. Winiarska et al. (20) have reported accelerated OH generation by renal cortical tubules from rabbits with alloxan-induced T1D. Because OH is highly reactive, it is very short-lived and thus assumed to exert its effects locally. An unexplored possibility is that OH is produced by (and subsequently influences the function of) the renal microvasculature in T1D. It is also possible that infiltration and activation of leukocytes (the only cells that express myeloperoxidase) leads to HOCl production in the kidney during the early stage of T1D. Indeed, although glomerular infiltration by leukocytes and monocytes/macrophages is evident within 1 wk of STZ injection in rats (21), no attempts have been made to determine if renal HOCl production occurs in diabetes.

Microvascular Impact of Renal Oxidative Stress in T1D

The best-documented vascular consequence of oxidative stress in T1D is a decreased half-life of NO (accelerated degradation as a result of ONOO- formation). The resulting impairment of agonist-induced NO-dependent relaxation in aorta and other vascular beds is normalized by antioxidant maneuvers (22-24). Agonist-induced endothelium-dependent relaxation is impaired in small intrarenal arteries from STZ rats, apparently as a result of O2'-, OH , and prostaglandin endoperoxide opposition of the effects of NO (25). O2'--dependent processes are also implicated in impaired endothelium-dependent relaxation of afferent arterioles isolated from diabetic rabbits (26). As illustrated in Fig. 3, renal arteriolar constrictor responses to NOS inhibition are attenuated in kidneys from STZ rats, but restored by acute exposure to SOD (27). This observation indicates that O2- production diminishes the tonic dilator influence of endogenous NO on the renal microvasculature. The O2--dependent decrease in NO bioavailability is evident in both afferent and efferent arterioles; thus, it is difficult to envision this phenomenon as contributing to diabetic hyperfiltration. In fact, the decrease in NO bioavailability in early T1D actually increases pre- and postglomerular vascular resistance, rather than producing the selective decline in preglomerular resistance that engenders diabetic hyperfiltration.

O2- may also alter renal vascular function via NO-independent mechanisms. In nonrenal vascular beds, O2- generally promotes vasoconstriction via effects on intracellular Ca2+ homeostasis and signaling events (28-33), although a few studies indicate attenuated contractile responsiveness (34,35). O2- has also been variably reported to influence K+ channels and l-type voltage-gated Ca2+ channels (VGCCs) (31,36), which are particularly critical to regulation of renal preglomerular microvascular function (37-39). However, no studies have examined the possibility that O2- exerts an NO-independent influence on the renal vasculature.

Systemic infusion of ONOO- evokes renal, hindquarter and mesenteric vasodilation possibly via a mechanism involving ATP-sensitive K+ channels (KATP channels) (40,41). ONOO- also dilates cerebral arterioles by activating KATP channels (35). In contrast, ONOO- contracts rat cerebral artery (42,43) and diminishes currents through voltage-sensitive K+ channels (Kv channels), as well as reducing the dilator influence of these channels in rat small coronary arteries (44). This phenomenon may result from nitration of tyrosine residues in the pore-forming a-subunit of the Kv channel (44). As ONOO- appears to activate KATP channels and inhibit Kv channels in vascular smooth muscle, the resulting change in vascular tone may reflect the relative prominence of these channel subtypes in a particular vascular bed. It is intriguing note that KATP channels have increased functional impact on afferent arteriolar function in T1D (see pp. 30-31), thus raising the possibility that this phenomenon might arise via a ONOO--dependent mechanism.

A survey of literature concerning the vasoactive effects of H2O2 yields a mixed bag of constrictor (31,45) and dilator effects (46-48). In some cases, contraction is evident at low concentrations with higher concentrations provoking a transient contractile response followed by a sustained dilation (49,50). Most studies indicating vasodilator responses to H2O2 have implicated increased K+ channel activity in the response (49,51-53). Renal medullary interstitial infusion of H2O2 decreases medullary blood flow (54); however, our preliminary data unveiled a concentration-dependent, NOS-independent afferent arteriolar dilator response to H2O2 (55). Although we do not know if this phenomenon is endothelium dependent or if it reflects vasoactive effects of H2O2 metabolism products (OH and HOCl), this observation suggests that increased H2O2 production in the renal cortex during T1D might contribute to the afferent arteriolar dilation and hyperfiltration.

Little information exists concerning the potential renal vascular effects of OH, except for one report indicating that OH contributes to impaired endothelium-dependent dilation of small intrarenal arteries from STZ rats (25). A similar effect of OH has been described in the basilar artery of STZ rats (56). Thus, it appears likely that OH impairs NO-dependent dilator events, possibly by oxidizing NO (57).

However, conflicting reports regarding the effects of OH' on vascular tone include a vasodilator impact on cerebral arterioles (58), but an endothelium-independent constrictor influence on rat aorta (59).

Among the ROS, the vasoactive effects of HOCl are the most poorly characterized. HOCl increases vascular resistance in the perfused rat liver (60). In isolated pulmonary arteries from sheep, HOCl produces vasoconstriction under resting force and vasodilation when the pulmonary arteries are precontracted (61). HOCl also appears capable of impairing NO-dependent vasodilation (62,63). Moreover, a recent report indicates that exposure of rat aortic rings to 20 mM glucose for 5 h sensitizes the vessel to myeloperoxidase-induced endothelial dysfunction (64), an observation that underscores the potential for a vascular effect of HOCl if even only modest renal leukocyte infiltration/ activation occurs during the early stage of T1D. However, nothing is known about the effects of HOCl on renal hemodynamics, even in inflammatory states in which increased renal myeloperoxidase activity and HOCl-oxidized protein have been documented (65-67). It is interesting to note that immunosuppressant/anti-inflammatory therapy is beneficial in preventing glomerular injury in uninephrectomized STZ rats (which display accelerated development of diabetic nephropathy), although this phenomenon appeared to arise independent of the hemodynamic changes associated with diabetic hyperfiltration (68).

In addition to direct effects on the renal vasculature, ROS production in T1D may elicit tubuloglomerular feedback-dependent alterations in afferent arteriolar tone. A complex interaction exists between O2'- and NO in determining tubuloglomerular feedback regulation of afferent arteriolar tone (69). Moreover, ROS can influence Na+ transport by various segments of the nephron. For example, in the medullary thick ascending limb, O2'- increases net NaCl reabsorption, ONOO- inhibits Na+-K+-ATPase activity, and H2O2 has no effect on Na+ transport (70). The possibility that HOCl and OH' might influence renal Na+ transport has not been explored. The pluripo-tential effects of the various ROS on Na+ transport might change solute delivery to the macula densa, thereby indirectly altering afferent arteriolar resistance. Obviously, this phenomenon could represent a mechanism through which ROS influence the magnitude of diabetic hyperfiltration.

Summary of the Potential Role of Oxidative Stress in Diabetic Hyperfiltration

Oxidative stress in T1D is implicated in multiple complications of the disease, including diabetic nephropathy, which has its origin in dysregulation of renal pre-glomerular microvascular tone. Intense investigative effort has established several pathways through which ROS production results from hyperglycemia. However, it is not clear which renal cells produce excess ROS in T1D, nor is it clear which ROS predominates in the kidney (and in the renal microvasculature) under these conditions. Even less well understood are the functional effects of the various ROS—information that is critical for understanding the mechanisms through which these substances contribute to the deleterious effects of T1D. Each ROS has the potential to influence renal microvascular tone via direct effects on the resident vascular smooth muscle cells, through effects on NO bioavailability (as documented for O2-), or indirectly via effects on tubular transport that evoke tubuloglomerular feedback-mediated vascular responses. Any or all of these events can be expected to influence the magnitude of diabetic hyperfiltration.

Disrupted Electromechanical Regulation of Preglomerular Microvascular Resistance in STZ-Induced T1D

Basal tone and contractile responsiveness of the preglomerular microvasculature are highly dependent on Ca2+ influx through voltage-gated channels, whereas efferent arteriolar function seems much less reliant on this process (37). Accordingly, alterations in electromechanical coupling events would have a functional impact on a variety of processes in the preglomerular microvasculature, with little impact on the efferent arte-riole (71). In light of the fact that multiple aspects of afferent arteriolar function are impaired in T1D, with relative preservation of efferent arteriolar function, we have proposed that T1D provokes a functional defect in electromechanical regulation of afferent arteriolar function.

Functional Impairment of Afferent Arteriolar Voltage-Gated Ca2+ Channels in T1D

Bank and colleagues (72) first suggested that insulinopenia in T1D might impair Ca2+ movement through VGCCs in the renal vasculature. Subsequently, Williams and Schrier (73) found that cultured rat aortic vascular smooth muscle cells exposed to high extracellular glucose concentrations for 12 h exhibited suppressed voltage-sensitive and agonist-induced [45Ca2+] uptake responses. In recent preliminary studies, we confirmed that culture of renal preglomerular microvascular smooth muscle cells for 3-5 d in high-glucose media significantly impaired [Ca2+]i responses to depolarization (74). These observations link hyperglycemia with depressed VGCC activation. We have reported attenuation of afferent arteriolar contractile responses to BAY K 8644 (a dihy-dropyridine activator of l-type VGCCs) in kidneys from STZ rats. Moreover, the EC50 for KCl-induced afferent arteriolar contraction was 40% greater in STZ kidneys than in kidneys from Sham rats (75). We also found that afferent arterioles from STZ rats isolated and studied in media containing 20 mM glucose exhibited blunted [Ca2+]i responses to K+-induced depolarization (Fig. 4) (75). Although afferent arterioles from Sham rats were unaffected by bath glucose concentration, [Ca2+]i responsiveness to depolarization in arterioles from STZ rats was restored within 10 min of exposure to normal bath glucose values. The rapid reversibility of this phenomenon is reminiscent of the ability of acute restoration of euglycemia to normalize GFR in STZ rats, as noted earlier (4), and also makes it unlikely that a decrease in VGCC expression in afferent arteriolar smooth muscle cells is responsible. Rather, these observations suggest that hyperglycemia in T1D provokes altered regulation of afferent arteriolar VGCCs, probably representing a diminished sensitivity to membrane potential. This phenomenon can be expected to result in reduced responses to a variety of stimuli that normally rely on depolarization-dependent Ca2+ influx to evoke increases in preglomerular vascular resistance—including autoregulatory and tubuloglomerular feedback responses, as well as contractile responses to AngII, vasopressin, norepi-nephrine, and other agonists (37,76-78). Accordingly, the impaired afferent arteriolar responsiveness to depolarization in T1D should promote afferent arteriolar dilation and diabetic hyperfiltration (71).

Exaggerated Impact of ATP-Sensitive K+ Channels on Afferent Arteriolar Tone in T1D

Studies from our laboratory have also explored the possibility that K+ channel regulation of afferent arteriolar function is altered in T1D. In particular, we have focused on

Fig. 4. Typical [Ca2+]; responses to depolarization (40 mM K+ bath) of isolated afferent arterioles from Sham and STZ rats. (Data from Carmines et al. [75].)

the Katp channel (79), the activation of which may reflect the cellular metabolic state. Several studies have provided functional evidence of KATP channel expression in the renal microcirculation (80-83). We confirmed that potassium channel openers (pinacidil or PCO-400; both specific for KATP) evoke concentration-dependent afferent arteriolar dilation in kidneys from nondiabetic rats (Fig. 5), indicating the functional expression of Katp channels; however, the normal afferent arteriole is minimally responsive to gliben-clamide (a sulfonylurea KATP inhibitor). Thus, the normal afferent arteriole possesses a recruitable pool of KATP channels having a low open probability in the absence of pharma-gological activators and in the presence of physiologic intracellular ATP levels. In contrast, afferent arterioles from STZ rats contracted significantly in response to glibenclamide (Fig. 5), indicating the emergence of a KATP channel-dependent component of afferent arteriolar tone in T1D. Indeed, as the afferent arteriole is typically vasodilated in the hyperfiltering kidney of the STZ rat, glibenclamide-induced contraction of these vessels restored arteriolar diameter to values that did not differ significantly from arterioles in Sham kidneys (79). Afferent arterioles from STZ rats also exhibited an exaggerated vasodilator response to pinacidil and PCO-400, consistent with an increased functional expression of KATP channels and/or an enhanced impact of these channels on membrane potential (and hence arteriolar tone). As opening of KATP channels promotes afferent arteriolar dilation, this phenomenon can be expected to contribute to the etiology of diabetic hyperfiltration (71).

Summary of the Potential Role of Impaired Afferent Arteriolar Electromechanical Coupling in Diabetic Hyperfiltration

Increased functional availability and basal activation of KATP channels in T1D should result in dysregulation of membrane potential (favoring hyperpolarization). Moreover, this situation should be exacerbated by impaired VGCC responsiveness

Fig. 5. Impact of KATp channels on juxtamedullary afferent arteriolar lumen diameter in kidneys from Sham and STZ rats. Responses to 100 pM glibenclamide (Katp blocker) and 100 pM pinacidil (Katp opener) are illustrated. p < 0.05 vs Sham; n = 7-9 arterioles. (Data from Ikenaga et al. [79].)

to membrane depolarization. These synergistic effects of T1D on KATp channels and VGCCs would tend to diminish afferent arteriolar responsiveness to myriad constrictor stimuli, thus promoting the characteristic afferent arteriolar dilation that evokes diabetic hyperfiltration. However, the events that underlie altered electromechanical coupling in the preglomerular microvasculature during T1D have not been elucidated.

Could Oxidative Stress in T1D Underlie the Alterations in Afferent Arteriolar

Electromechanical Coupling?

In considering the potential roles of renal oxidative stress and impaired afferent arteriolar electromechanical coupling in the etiology of diabetic hyperfiltration, it is intriguing to note a potential link between these phenomena. Indeed, many of the direct vasoactive effects of ROS arise via changes in K+ channels that regulate membrane potential or Ca2+ channels that respond to changes in membrane potential. These channels play prominent roles in the electromechanical regulation of afferent arteriolar tone (and are much less prominent in the efferent arteriole). Thus, as illustrated in Fig. 6, it is conceivable that renal oxidative stress in T1D might activate K+ channels (perhaps the KATp channel) in afferent arteriolar smooth muscle cells, resulting in hyperpolarization and diminished Ca2+ influx through VGCCs, thus engendering afferent arteriolar dilation and hyperfiltration. These events may occur in parallel with effects of ROS that reduce NO bioavailability (tempering hyperfiltration) and/or ROS-stimulated changes in tubular transport that evoke tubuloglomerular feedback-dependent responses (which could either favor or blunt hyperfiltration). This complex scenario of factors impacting the magnitude of diabetic hyperglycemia awaits validation through detailed experimental scrutiny.

Diabetic Hyperglycemia

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