Na⁺/H⁺ exchanger 1 inhibition reverses manifestation of peripheral diabetic neuropathy in type 1 diabetic rats

Abstract

Evidence for an important role for Na⁺/H⁺ exchangers in diabetic complications is emerging. The aim of this study was to evaluate whether Na⁺/H⁺ exchanger 1 inhibition reverses experimental peripheral diabetic neuropathy. Control and streptozotocin-diabetic rats were treated with the specific Na⁺/H⁺ exchanger 1 inhibitor cariporide for 4 wk after 12 wk without treatment. Neuropathy end points included sciatic motor and sensory nerve conduction velocities, endoneurial nutritive blood flow, vascular reactivity of epineurial arterioles, thermal nociception, tactile allodynia, and intraepidermal nerve fiber density. Advanced glycation end product and markers of oxidative stress, including nitrated protein levels in sciatic nerve, were evaluated by Western blot. Rats with 12-wk duration of diabetes developed motor and sensory nerve conduction deficits, thermal hypoalgesia, tactile allodynia, and intraepidermal nerve fiber loss. All these changes, including impairment of nerve blood flow and vascular reactivity of epineurial arterioles, were partially reversed by 4 wk of cariporide treatment. Na⁺/H⁺ exchanger 1 inhibition was also associated with reduction of diabetes-induced accumulation of advanced glycation endproduct, oxidative stress, and nitrated proteins in sciatic nerve. In conclusion, these findings support an important role for Na⁺/H⁺ exchanger 1 in functional, structural, and biochemical manifestations of peripheral diabetic neuropathy and provide the rationale for development of Na⁺/H⁺ exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

diabetic peripheral neuropathy is a complication of diabetes that has a complex etiology and even with good glycemic control can have severe consequences. To date, there is no effective treatment for diabetic peripheral neuropathy (54). There have been many mechanisms proposed to contribute to diabetes complications, and there is now widespread evidence for an important role for oxidative-nitrosative stress and its downstream effectors in the development of peripheral diabetic neuropathy (1, 16, 26, 40, 51). Hyperglycemia is thought to be an important contributing factor to the pathology of diabetic complications, but the intracellular metabolic pathways linking glucose and oxidative injury are not completely understood (9, 20, 56). Diabetes-associated inhibition or insufficient activation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase underlies diversion of excessive glycolytic flux toward the formation of methylglyoxal and α-glycerophosphate (5–7, 28). This in turn has been shown to lead to 1) a decrease in free cytosolic NAD⁺/NADH ratio and resulting activation of the superoxide-generating enzyme NAD(P)H oxidase and 2) a formation of advanced glycation end products (AGE) that generate free radicals during interaction with their receptors (3, 22, 62). The mechanisms underlying diabetes-associated activation of the upper part of glycolysis remain unidentified. One mechanism we have been investigating is the activation of Na⁺/H⁺ exchanger 1. Na⁺/H⁺ exchanger 1 is distributed ubiquitously in mammalian tissues and plays an important role in the regulation of intracellular pH by removing protons that are generated continuously in normal cells (8, 30, 32, 33, 37, 50, 65). Several reports suggest that upregulation of Na⁺/H⁺ exchanger-1 activity, demonstrated recently in several tissue sites for diabetes complications, leads to an increase in cytosolic pH and consequent activation of glucose transport and all enzymes in the upper part of glycolysis, especially phosphofructokinase (24, 53, 58). Given this information, we propose that diabetes-driven increased expression/activation of Na⁺/H⁺ exchanger 1 may cause an increase in the formation of advanced glycation end products that may then contribute to increased oxidative stress and development of peripheral diabetic neuropathy. We propose that inhibition of Na⁺/H⁺ exchanger 1 could be an effective treatment for peripheral diabetic neuropathy.

MATERIALS AND METHODS

Materials.

Unless stated otherwise, all chemicals used in these studies were obtained from Sigma Chemical (St. Louis, MO).

Animals.

The experiments were performed in accordance with regulations specified by the National Institutes of Health Principles of Laboratory Animal Care and the Pennington Biomedical Research Center and Iowa City Veterans Affairs Medical Center; both of these institutions approved the animal study protocols. Male Wistar rats (Charles River, Wilmington, MA) 10–11 wk of age were fed a standard rat chow (PMI Nutrition International, Brentwood, MO) and had access to water ad libitum. Type 1 diabetes was induced by injecting streptozotocin (50 mg/kg body wt ip). Hyperglycemia was verified (nonfasting blood glucose ≥13.8 mM via tail vein) 48 h after the streptozotocin injection. Control rats injected with vehicle and diabetic rats were monitored for 12 wk (weight and blood glucose). After 12 wk the rats were divided into four experimental groups: control and diabetic rats treated with or without cariporide, an Na⁺/H⁺ exchanger 1 inhibitor (10 mg·kg⁻¹·day⁻¹ in the drinking water), for 4 wk (37). Diabetic rats that lost >10% of their initial body weight were treated with 1–2 units of insulin every second day until their weight stabilized. Insulin treatments did not correct hyperglycemia.

Behavioral tests.

The paw withdrawal latency in response to radiant heat was recorded at a 15% intensity (heating rate of ∼1.3°C/s) with a cutoff time of 30 s, using the IITC model 336 TG combination tail-flick and paw algesia meter (IITC Life Sciences, Woodland Hills, CA) (42). Tactile responses were evaluated by quantifying the withdrawal threshold of the hindpaw in response to stimulation with flexible von Frey filaments, as described previously (23). The data were reported in seconds and grams.

Physiological tests.

On the day of terminal studies, rats were weighed and anesthetized with Nembutal (50 mg/kg ip; Abbott Laboratories, North Chicago, IL). Nonfasting blood glucose was determined. Sciatic motor nerve conduction velocity and digital sensory nerve conduction velocity were measured as described previously (51). The motor and sensory nerve conduction velocities were reported in meters per second. Sciatic nerve endoneurial blood flow was determined as described previously, using the hydrogen clearance method (45). The hydrogen clearance data were fitted to a mono- or biexponential curve using commercial software (Prism; Graphpad, San Diego, CA). Nutritive blood flow (ml·min⁻¹·100 g⁻¹) was calculated using the equation described by Young (64), and vascular conductance (ml·min·⁻¹100 g⁻¹·mmHg⁻¹) was determined by dividing the nutritive blood flow by the average mean arterial blood pressure.

Intraepidermal nerve fiber density.

Footpads were fixed in ice-cold Zamboni's fixative for 3 h, washed in 100 mM phosphate-buffered saline (PBS) overnight, and then washed in PBS containing increasing amounts of sucrose, i.e., 10, 15, and 20%, for 3 h in each solution. After washing, the samples were snap-frozen in optimum cutting temperature (OCT) and stored at −80°C. Three longitudinal 50-μm-thick footpad sections were cut using a Leica CM1950 cryostat (Leica Microsystems, Nussloch, Germany). Nonspecific binding was blocked by 3% goat serum containing 0.5% porcine gelatin and 0.5% Triton X-100 in SuperBlock blocking buffer (Thermo Scientific, Rockford, IL) at room temperature for 2 h. The sections were then incubated overnight with PGP 9.5 antiserum (UltraClone, Isle of Wight, UK) in 1:400 dilution at 4°C, after which secondary Alexa Fluor 488 antibody (Molecular Probes, Life Technologies, Grand Island, NY) in 1:1,000 dilution was applied at room temperature for 1 h. Sections were then coverslipped with VectaShield mounting medium (Vector Laboratories, Burlingame, CA). Intraepidermal nerve fiber profiles were counted blindly by three independent investigators using an Axioplan 2 microscope (Carl Zeiss Microscopy, Thornwood, NY) at ×400 magnification, and the average values were reported. The length of epidermis was assessed on the microphotographs of stained sections taken at ×50 magnification with a 3I Everest imaging system (Intelligent Imaging Innovations, Denver, CO) operated with an Axioplan 2 microscope, using the National Institutes of Health (Bethesda, MD) Image J software. An average of 2.8 ± 0.3 mm of the sample length was investigated to calculate a number of nerve fiber profiles per millimeter of epidermis. Representative images of intraepidermal nerve fibers were obtained by confocal laser scanning microscopy at ×400 magnification, using Leica TCS SP5 confocal system (Leica Microsystems, Nussloch, Germany) (51).

Vascular reactivity in epineurial arterioles.

Videomicroscopy was used to investigate in vitro vasodilatory responsiveness of epineurial arterioles vascularizing the region of the sciatic nerve, as described previously (55). The desired vessels were isolated by exposing the common iliac, and the branch points of the internal pudendal and superior gluteal arteries were identified. The vessels were then clamped, and tissues containing these vessels and the branches of the internal pudendal and superior gluteal arteries were dissected en bloc. The block of tissue was immediately submerged in a cooled (4°C), oxygenated (20% O₂, 5% CO₂, and 75% N₂) Krebs-Henseleit physiological saline solution (PSS) of the following composition (in mmol/l): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH₂PO₄, 1.2 MgSO₄, 20 NaHCO₃, 0.026 Na₂EDTA, and 5.5 glucose. Branches of the superior gluteal and internal pudendal arteries (50- to 150-μm internal diameter and 1–2 mm in length) were carefully dissected and trimmed of fat and connective tissue. Both ends of the isolated vessel segment were cannulated with glass micropipettes filled with PSS (4°C) and secured with 10-0 nylon Ethilon monofilament sutures (Ethicon, Cornelia, GA). The pipettes were attached to a single pressure reservoir (initially set at 0 mmHg) under conditions of no flow. The organ chamber containing the cannulated vessels was then transferred to the stage of an inverted microscope (CK2; Olympus, Lake Success, NY). Attached to the microscope were a closed-circuit television camera (WV-BL200; Panasonic, Secaucus, NJ), a video monitor (Panasonic), and a video caliper (VIA-100K; Boeckeler Instruments, Tucson, AZ). The organ chamber was connected to a rotary pump (Masterflex; Cole Parmer Instrument, Vernon Hills, IL), which continuously circulated 37°C oxygenated PSS at 30 ml/min. The pressure within the vessel was then slowly increased to 40 mmHg. At this pressure, we found that KCl gave the maximal constrictor response. Therefore, all of the studies were conducted at 40 mmHg. Internal vessel diameter (resolution of 2 μm) was measured by manually adjusting the video micrometer. After a 30-min equilibration, KCl was added to the bath to test vessel viability. Vessels failing to constrict by ≥30% were discarded. After they were washed with PSS, vessels were incubated for 30 min in PSS and then constricted with U-46619 (10⁻⁸ to 10⁻⁷ mol/l; Cayman Chemical, Ann Arbor, MI) to 30–50% of passive diameter. Afterwards, a cumulative concentration-response relationship was evaluated for acetylcholine (10⁻⁸ to 10⁻⁴ M). At the end of the dose response determination, a maximal dose of sodium nitroprusside (10⁻⁴ M) was added. Afterwards, papaverine (10⁻⁵ M) was added to determine maximal vasodilation.

Vascular superoxide and nitrotyrosine staining.

Hydroethidine (Molecular Probes), an oxidative fluorescent dye, was used to evaluate in situ levels of superoxide (O₂⁻) in epineurial vessels (14, 15). Hydroethidine is permeable to cell membranes, and in the presence of O₂⁻ it is oxidized to fluorescent ethidium bromide, where it is trapped by intercalating with DNA. Unfixed frozen vessel segments imbedded in Tissue-Tek OCT were cut in 10-μm sections and placed on glass slides. Hydroethidine (2 μM) was topically applied to each tissue section and coverslipped. Slides were incubated in a light-protected humidified chamber at 37°C for 30 min. Images were obtained with a Zeiss LSM710 confocal microscope. Vessels from control and diabetic untreated and treated rats were processed and imaged in parallel. Laser settings were identical for acquisition of all images from control and diabetic rats. Superoxide anion can interact with nitric oxide to form peroxynitrite (60). This reaction reduces the efficacy of nitric oxide to act as a signal transduction agent. Peroxynitrite is a highly reactive intermediate known to nitrate protein tyrosine residues, and it is also known to cause cellular oxidative damage (4, 47). To determine formation of peroxynitrite, we measured 3-nitrotyrosine (a stable biomarker of tissue peroxynitrite formation) as described previously (19). Briefly, frozen tissue segments of arterioles were cut into 10-μm sections as described above and then incubated in phosphate-buffered saline solution containing 1% Triton X-100 and 0.1% bovine serum albumin for 30 min at room temperature. Afterwards, the samples were incubated in this buffer solution containing mouse anti-nitrotyrosine antibody (Upstate, Lake Placid, NY) overnight at 4°C. After washing, the sections were incubated for 2 h with Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes). Sections were then rinsed and mounted with VectaShield (Vector Laboratories). The labeled vessels derived from these studies were visualized with a Zeiss LSM710 confocal microscope. Images for superoxide and nitrotyrosine were quantified using the ZEN image analysis software. The amount of immunostaining was determined by dividing the total intensity of the stained regions by their area. This analysis excluded the area of the unstained lumen.

Western blot analysis of methylglyoxal-derived AGE and nitrated protein in rat sciatic nerve.

To assess methylglyoxal-derived AGE and nitrated protein expressions by Western blot analysis, sciatic nerve segments (∼20 mg) were placed on ice in 200 μl of radioimmunoprecipitation assay buffer containing 50 mmol/l Tris·HCl, pH 7.2, 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate, 1% NP-40, 5 mmol/l EDTA, 1 mmol/l EGTA, 1% sodium deoxycholate, and the protease/phosphatase inhibitors leupeptin (10 μg/ml), pepstatin (1 μg/ml), aprotinin (20 μg/ml), benzamidine (10 mM), phenylmethylsulfonyl fluoride (1 mM), and sodium orthovanadate (1 mmol/l) and homogenized on ice. The homogenates were sonicated and centrifuged at 14,000 g for 20 min. All the aforementioned steps were performed at 4°C. The lysates (40 μg of protein) were mixed with equal volumes of 2× sample-loading buffer containing 62.5 mmol/l Tris·HCl, pH 6.8, 2% sodium dodecyl sulfate, 5% β-mercaptoethanol, 10% glycerol, and 0.025% bromophenol blue and fractionated in 5–20% SDS-PAGE in an electrophoresis cell (Mini-Protean III; Bio-Rad Laboratories, Richmond, CA). Electrophoresis was conducted at 15 mA constant current for stacking and at 35 mA for protein separation. Gel contents were electrotransferred (80 V, 2 h) to nitrocellulose membranes using Mini Trans-Blot cell (Bio-Rad Laboratories) and Western transfer buffer [25 mmol/l Tris·HCl, pH 8.3, 192 mmol/l glycine, and 20% (vol/vol) methanol]. Free binding sites were blocked in 5% (wt/vol) BSA (methylglyoxal-derived AGE) or 3% (wt/vol) BSA (nitrated proteins), all diluted in 20 mmol/l Tris·HCl buffer, pH 7.5, containing 150 mmol/l NaCl and 0.1% Tween 20, for 1 h. After blocking free binding sites, primary antibodies to methylglyoxal-derived AGE (Trans Genic, Kumamoto, Japan) or nitrated proteins (Millipore, Billerica, MA) were applied overnight at 4°C. Then the anti-mouse secondary antibody was applied at room temperature for 1 h. Protein bands detected by the antibodies were visualized with Amersham ECL Western Blotting Detection Reagent (Little Chalfont, Buckinghamshire, UK). After incubation with secondary antibody, the membranes were stripped and reprobed with β-actin antibody to confirm equal protein loading. Stripping was conducted in 25 mmol/l glycine-HCl, pH 2.5 buffer containing 2% SDS. The data were quantified by densitometry (Quantity One 4.5.0 Software; Bio-Rad Laboratories).

ELISA measurements of 4-hydroxynonenal adducts in rat sciatic nerve.

For 4-hydroxynonenal adduct measurements, the samples were homogenized in 20 mM PBS, pH 7.4 (1:10, wt/vol), on ice. Homogenates were centrifuged at 14,000 g (4°C, 20 min). Supernatants were used for measurements of 4-hydroxynonenal adducts with the OxiSelect HNE-His Adduct ELISA kit (Cell BioLabs, San Diego, CA). 4-Hydroxynonenal adducts were normalized per milligram of protein. Protein was measured with the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL).

Fluorescent immunohistochemistry in dorsal root ganglia.

Dorsal root ganglia were dissected and fixed in normal buffered 4% formaldehyde for 24 h at 4°C, dehydrated, and embedded in paraffin. Sections were cut at 5-μm thickness, dewaxed in xylene, hydrated in decreasing concentrations of ethanol, washed in distilled water, and subjected to heat-induced epitope retrieval in 10 mM citrate buffer (pH 6.0) with 0.05% Tween 20. To create positive control for anti-nitrotyrosine antibody, several deparaffinized sections from random rats were incubated with 1 mM peroxynitrite in 100 mM sodium acetate buffer, pH 5.0, for 30 min, preceding the antigen retrieval step. Then the sections were subsequently incubated with Image-iT FX signal enhancer for 30 min and blocking solution containing 2% BSA, 5% normal goat serum, and 0.3% Triton X-100 in 50 mM Tris-buffered saline (pH 8.4) for 1 h, with thorough washes between the steps. The incubation with anti-nitrotyrosine (1:100) and anti-Na⁺/H⁺ exchanger 1 (1:50) primary antibodies was performed overnight at 4°C. The secondary Alexa Fluor 594-conjugated goat anti-rabbit antibody (Molecular Probes) was used in a working dilution of 1:400. Sections were mounted in VectaShield mounting medium. All sections were processed by a single investigator and evaluated blindly. Color images were captured at ×400 magnification with a 3I Everest imaging system (Intelligent Imaging Innovations) equipped with an Axioplan 2 microscope (Zeiss). Nitrotyrosine and Na⁺/H⁺ exchanger 1 fluorescence intensity of individual dorsal root ganglion neurons was quantified using the Image J software (National Institutes of Health) and normalized per neuronal area. For nitrotyrosine immunofluorescence analysis, nuclei of individual cells were excluded from the regions of interest. Neurons (15–20/rat) were counted, and the average values for each animal were used to calculate group means. Fluorescence intensity was expressed as means ± SE for each experimental group.

Statistical analysis.

The results are presented as means ± SE. Data were subjected to equality of variance F test and then to log transformation if necessary before one-way analysis of variance. Where overall significance (P < 0.05) was attained, individual between-group comparisons for multiple groups were made using the Student-Newman-Keuls multiple-range test. When between-group variance differences could not be normalized by log transformation (data sets for body weights and plasma glucose), the data were analyzed by the nonparametric Kruskal-Wallis one-way analysis of variance, followed by the Bonferroni-Dunn test for multiple comparisons. Individual pairwise comparisons in Table 1 and Fig. 1 were made using the unpaired two-tailed Student t-test. Concentration-response curves for acetylcholine were compared using a two-way repeated-measures analysis of variance with autoregressive covariance structure using the proc mixed program of SAS (14, 15). Significance was defined at P < 0.05.

Table 1.

Effect of diabetes on thermal algesia, IENF density, tactile response, MNCV, and SNCV

Determination	Control	Diabetic
Thermal response latency, s	9.6 ± 0.5	15.5 ± 0.8*
IENF, profiles/mm	21.1 ± 2.6	14.1 ± 3.0*
Tactile response threshold, g	14.3 ± 1.5	6.3 ± 0.5*
MNCV, m/s	54.9 ± 1.0	48.3 ± 1.3*
SNCV, m/s	42.4 ± 0.4	35.8 ± 0.3*

Data are presented as means ± SE; n = 8 experimental animals. IENF, intraepidermal nerve fiber; MNCV, motor nerve conduction velocity; SNCV, sensory nerve conduction velocity. Control rats and rats with 12-wk duration of diabetes.

^*P < 0.05 compared with control.

Fig. 1.

A: representative Western blot analysis of rat sciatic nerve from control (C) and diabetic (D) rats of expression of Na⁺/H⁺ exchanger 1. Equal protein loading was confirmed with β-actin antibody. Left 3 lanes, control rats; right 3 lanes, diabetic rats (12-wk duration). B: Na⁺/H⁺ exchanger 1 content in sciatic nerves of C and D rats. Na⁺/H⁺ exchanger 1 content in control rats was taken as 100%. Means ± SE; n = 3/group. *P < 0.05 vs. controls.

RESULTS

Baseline changes in diabetic rats prior to treatment.

Data in Table 1 demonstrate that after 12 wk of untreated diabetes thermal sensitivity, tactile response, motor and sensory nerve conduction velocity, and intraepidermal nerve fiber density were all significantly impaired compared with age-matched controls. Data in Fig. 1 demonstrate that expression of Na⁺/H⁺ exchanger 1 in the sciatic nerve is significantly increased after 12 wk of untreated diabetes.

Effect of treatment of diabetic rats with cariporide on weight and blood glucose.

Data in Table 2 demonstrate that treating diabetic rats for 4 wk with cariporide, an inhibitor of Na⁺/H⁺ exchanger 1, following 12 wk of no treatment did not correct hyperglycemia or weight gain. Treatment of nondiabetic rats with cariporide for 4 wk did not affect blood glucose levels or weight gain.

Table 2.

Change in body weight and blood glucose in streptozotocin-induced diabetic rats treated with or without cariporide

Determination (No. of Experimental Animals)	Control (n = 10)	Control + Cariporide (n = 10)	Diabetic (n = 16)	Diabetic + Cariporide (n = 17)
Initial weight, g	314 ± 10	323 ± 3	300 ± 10	316 ± 4
Final weight, g	570 ± 26	547 ± 9	318 ± 17*	301 ± 14*
Initial blood glucose, mM	3.9 ± 0.1	4.5 ± 0.1	26.2 ± 1.2*	23.8 ± 0.6*
Final blood glucose, mM	5.9 ± 0.2	5.9 ± 0.1	24.6 ± 1.1*	24.9 ± 1.1*

Data are presented as means ± SE. Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes.

^*P < 0.05 compared with control.

Effect of treatment of diabetic rats with cariporide on neural and vascular complications.

Data in Table 3 demonstrate that 4 wk of treatment with cariporide significantly improved thermal and tactile responses and motor and sensory nerve conduction velocity compared with untreated diabetic rats. However, each of these neural complications remained significantly impaired compared with control rats. This could be for several reasons, including suboptimal dosing, and a longer duration of treatment may be required to achieve full recovery, or multiple mechanisms may be involved. Data in Fig. 2 demonstrate that treating diabetic rats for 4 wk with cariporide stimulated the recovery of intraepidermal nerve fibers in the hindpaw. Treating diabetic rats for 4 wk with cariporide following 12 wk of no treatment significantly improved endoneurial blood flow (Fig. 3, A and B) and vascular relaxation to acetylcholine by epineurial arterioles of the sciatic nerve compared with untreated diabetic rats (Fig. 4A). In these studies, streptozotocin-induced diabetes did not impair blood pressure or relaxation to sodium nitroprusside (Fig. 4C and data not shown, respectively). Similarly to neural complications that were partially improved with 4 wk of treatment, cariporide vascular relaxation to acetylcholine by epineurial arterioles was not fully corrected. Previously, we had shown that acetylcholine-mediated relaxation by epineurial was significantly decreased as early as 1 wk after the induction of diabetes (55). Furthermore, we had shown previously that increased oxidative stress as determined by increased levels of superoxide and nitrotyrosine staining in epineurial arterioles derived from diabetic rats was responsible for vascular impairment (14, 15). In these studies, we demonstrated that treating diabetic rats with cariporide for 4 wk reduced superoxide and nitrotyrosine staining significantly in epineurial arterioles of diabetic rats (Fig. 4, B and C). Treatment of nondiabetic rats with cariporide for 4 wk did not affect neural or vascular end points.

Table 3.

Effect of treatment with cariporide of control and diabetic rats on thermal algesia, tactile response, MNCV, and SNCV

Determination (No. of Experimental Animals)	Control (n = 10)	Control + Cariporide (n = 10)	Diabetic (n = 16)	Diabetic + Cariporide (n = 17)
Thermal response latency, s	11.0 ± 0.7	11.8 ± 0.3	27.3 ± 0.7*	20.4 ± 0.8*+
Tactile response threshold, g	18.6 ± 1.8	17.0 ± 1.7	7.1 ± 0.3*	11.0 ± 0.8*+
MNCV, m/s	63.5 ± 1.6	65.2 ± 1.0	44.0 ± 0.9*	55.2 ± 1.6*+
SNCV, m/s	46.8 ± 1.0	44.7 ± 1.0	40.0 ± 0.5*	42.0 ± 0.5*+

Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes. Data are presented as means ± SE.

^*P < 0.05 compared with control; +P < 0.05 compared with untreated diabetic.

Fig. 2.

A: representative images of intraepidermal nerve fiber profiles; magnification ×40. B: intraepidermal nerve fiber densities in control rats and rats with 16-wk duration of diabetes maintained with or without cariporide treatment for 4 wk after 12 wk of untreated diabetes. C + CP, control + cariporide-treated rats; and D + CP, diabetic + cariporide-treated rats. Means ± SE; n = 10/group. *P < 0.05 vs. controls; +P < 0.05 vs. untreated diabetic group.

Fig. 3.

A: sciatic nerve nutritive endoneurial blood flow. B: sciatic nerve conductance endoneurial blood flow. C: mean systemic blood pressure in control and diabetic rats. Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes. Means ± SE; n = 8/group. *P < 0.05 vs. controls; +P < 0.05 vs. untreated diabetic group.

Fig. 4.

A: vascular reactivity of epineurial arterioles of the sciatic nerve in response to acetylcholine. Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes. Means ± SE; n = 8/group. B: representative images of epineurial arterioles stained for superoxide anion radicals. C: analysis of fluorescence as relative light units (RLU) for superoxide staining. D: representative images of epineurial arterioles stained for nitrotyrosine. E: analysis of fluorescence as RLU for nitrotyrosine staining. Means ± SE; n = 6. *P < 0.05 vs. controls; +P < 0.05 vs. untreated diabetic group.

Effect of treatment of diabetic rats with cariporide on AGE, nitrated proteins, and 4-hydroxynonenal in sciatic nerve.

Data in Fig. 5 demonstrate that methylglyoxal-derived AGE (Fig. 5A), nitrated proteins (Fig. 5B), and 4-hydroxynonenal (Fig. 5C) are all increased significantly in the sciatic nerve of diabetic rats (16-wk duration). Treating diabetic rats for 4 wk with cariporide significantly reduced these markers associated with advanced glycation end product accumulation and oxidative/nitrosative stress. Treatment of nondiabetic rats with cariporide for 4 wk did not affect the presence of these markers in the sciatic nerve.

Fig. 5.

A: Western blot analysis by densitometry of rat sciatic nerve of expression of methylglyoxal-derived advanced glycation end products (AGE). Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes. Control was arbitrarily assigned a value of 100%. B: Western blot analysis by densitometry of rat sciatic nerve of expression of sciatic nerve nitrated proteins. Control was arbitrarily assigned a value of 100%. C: ELISA analysis of sciatic nerve of 4-hydroxynonenal adduct. Value is expressed as μg/mg protein. Means ± SE; n = 6. *P < 0.05 vs. controls; +P < 0.05 vs. untreated diabetic group.

Effect of treatment of diabetic rats with cariporide on expression of Na⁺/H⁺ exchanger 1 and nitrated proteins in dorsal root ganglion neurons.

In this study, we determined whether Na⁺/H⁺ exchanger 1 overexpression occurs in dorsal root ganglion neurons and whether it is related to oxidative/nitrosative stress. As in the above studies/diabetic rats were treated with the Na⁺/H⁺ exchanger 1 inhibitor cariporide at a dose of 10 mg·kg⁻¹·day⁻¹ for 4 wk after 12 wk of untreated diabetes. Expression of Na⁺/H⁺ exchanger 1 is increased in dorsal root ganglion neurons from untreated diabetic rats compared with control rats (Fig. 6, A and B), and after 4 wk of treatment with cariporide the expression of Na⁺/H⁺ exchanger 1 remained significantly higher and was not significantly different compared with untreated diabetic rats (Fig. 6, A and B). Nitrotyrosine fluorescence was increased in dorsal root ganglion neurons of untreated diabetic rats, indicative of neuronal oxidative/nitrosative stress (Fig. 6, C and D). Treating diabetic rats with cariporide significantly decreased nitrotyrosine staining in dorsal root ganglion neurons (Fig. 6, C and D).

Fig. 6.

A: representative images of dorsal root ganglion neurons stained for Na⁺/H⁺ exchanger 1. B: analysis of fluorescence as RLU for Na⁺/H⁺ exchanger 1 staining. Means ± SE; n = 8–9 (10–20 neurons/rat were counted). C: representative images of dorsal root ganglion neurons stained for nitrotyrosine. D: analysis of fluorescence as RLU for nitrotyrosine staining. Control rats and rats with 16-wk duration of diabetes maintained with or without cariporide for 4 wk after 12 wk of untreated diabetes. Means ± SE; n = 8–9 (20–25 neurons/rat were counted). *P < 0.05 vs. controls; +P < 0.05 vs. untreated diabetic group.

DISCUSSION

Diabetic neuropathy is a progressive multifactorial complication with no effective treatment other than good glycemic control, and even with intense insulin therapy, diabetic neuropathy develops. Following years of research, many mechanisms have been proposed to contribute to diabetic neuropathy (29, 39). One of the more highly investigated theories has been increased oxidative stress. Oxidative stress has been documented in animal models of type 1 and type 2 diabetes (13, 15, 41, 43, 44, 52). It is clearly manifest in neurons, Schwann cells, axons, and endothelial cells of the peripheral nervous system (42). Accumulation of nitrotyrosine (a footprint of peroxynitrite-induced protein nitration) has also been documented in the peripheral nerve of diabetic rats, indicating that diabetes creates not just oxidative but nitrosative stress also in the peripheral nervous system (11, 38, 42).

In this study, we investigated the inhibition of Na⁺/H⁺ exchanger 1 as a potential new treatment aimed at reducing oxidative/nitrosative stress in diabetes. The 10 members of the Na⁺/H⁺ exchanger family described so far show a particular tissue distribution pattern (53). In this study, we were interested in the Na⁺/H⁺ exchanger 1 isoform, which is found in the plasma membrane of most mammalian cells and is normally described as the housekeeping isoform (53). Na⁺/H⁺ exchanger 1 plays a critical role in intracellular pH and cell volume homeostasis and regulates a number of cell behaviors, including adhesion, shape determination, migration, and proliferation (8, 48). Another important function of Na⁺/H⁺ exchanger 1 of interest for diabetes complications is regulation of glycolysis. Activation of Na⁺/H⁺ exchanger 1 causes cytosol alkalinization and resulting activation of glycolysis; furthermore, a Na⁺/H⁺ exchange-dependent increase in intracellular pH by ∼0.3 units was recently found to cause a one-order magnitude increase in the rate of glycolysis (25, 34, 46, 49). It is well known that upregulation of glycolysis contributes to the formation of by-products of glycolysis, i.e., methylglyoxal, α-glycerophosphate, and diacylglycerol, with concomitant activation of advanced glycation end product formation.

Hyperglycemia is associated with stimulation of Na⁺/H⁺ exchanger 1 (53, 57). In the diabetic kidney, it has been demonstrated that Na⁺/H⁺ exchanger 3 activity is increased (53). Both hyperglycemia and oxidative stress stimulate Na⁺/H⁺ exchanger 3 activity via angiotensin II receptor activation (53). Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers reduce progression of diabetic nephropathy, and we have shown that these drugs also improve diabetic neuropathy (13, 35, 36, 45, 63). It is not known whether angiotensin-converting enzyme inhibitors or angiotensin receptor blockers decrease Na⁺/H⁺ exchanger 1 activity in diabetes. These data imply that one possible mechanism for the beneficial effects of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers on diabetic complications, including neuropathy, may be through inhibiting the activation of Na⁺/H⁺ exchangers.

Hyperglycemia and increased metabolic rate could accentuate proton production and lead to increased proton efflux through Na⁺/H⁺ exchange, which would stimulate Na⁺/Ca²⁺ exchange and calcium overload (57). Imbalance of sodium and calcium with hyperglycemia is associated with endothelial dysfunction and apoptosis (57), proliferation of vascular smooth muscle cells (31), retinal microangiopathy and ischemic damage (17, 37), and myocardial damage (2, 10, 18). It has been shown that inhibition of Na⁺/H⁺ exchanger 1 yields beneficial effects on diabetes vascular, retinal, and renal complications, but no information is available on whether inhibiting Na⁺/H⁺ exchanger 1 improves diabetic peripheral neuropathy (37, 53).

In this study, we demonstrated that after 12 wk of untreated diabetes, expression of Na⁺/H⁺ exchanger 1 is increased in the sciatic nerve and dorsal root ganglion neurons and that cariporide treatment for 4 wk after 12 wk of untreated diabetes improved vascular and neural defects of diabetic neuropathy, including nerve conduction velocity, thermal and tactile sensitivity, endoneurial blood flow, and regeneration of intraepidermal nerve fibers. Improvement in diabetic neuropathy end points with cariporide treatment was associated with improvement in vascular reactivity of epineurial arterioles, reduction of oxidative stress in the sciatic nerve, dorsal root ganglion neurons, and epineurial arterioles, and reduction of a marker of advanced glycation end product in the sciatic nerve. It is unlikely that cariporide treatment reduced the expression of Na⁺/H⁺ exchanger 1 since expression of Na⁺/H⁺ exchanger 1 remained elevated in dorsal root ganglion neurons from diabetic rats treated with cariporide.

In this study, 12 wk of untreated diabetes resulted in hyperalgesia based upon the response to stimulation with flexible von Frey filaments applied to the hindpaw and hypoalgesia based upon the latent response to a thermal stimulus. These procedures test the response of different nerve fibers in the hindpaw, so it was not surprising that the behavioral response was different depending on whether a mechanical or heat stimulus was applied. The myelinated Aδ fibers are responsive to a mechanical stimulus, whereas unmyelinated C-fibers are responsive to a thermal stimulus. Our study suggests that, after 12 wk of untreated diabetes, a state of increased sensitivity exists in response to a mechanical stimulus, but decreased sensitivity when a thermal stimulus is applied. This could be due to the signaling mechanisms propagating these behavioral responses being affected differently by long-term diabetes or to the preferential loss of C-fibers in the skin of diabetic rats. What is interesting is that treatment with cariporide improved both outcomes as well as intraepidermal nerve fiber density in the hindpaw.

Previously, we have demonstrated that diabetes-induced vascular dysfunction of epineurial arterioles precedes deficits in nerve conduction velocity, suggesting that vascular impairment is a contributing factor to diabetic neuropathy (12, 44). We have also shown that diabetes-induced impairment of vascular relaxation to acetylcholine, which is endothelium dependent, is in part due to increased oxidative stress and that treating diabetic rats with an antioxidant improves both vascular and neural deficits (14, 15). In this study, treatment of diabetic rats with cariporide improved vascular relaxation to acetylcholine and reduced both superoxide levels and nitrotyrosine staining in epineurial arterioles. Improvement in vascular relaxation of blood vessels that provide circulation to peripheral nerves such as the epineurial arterioles would be expected to reduce ischemia and improve neural function. In aorta and coronary arteries, hyperglycemia was demonstrated to cause impairment in endothelium-dependent vasodilation, and this was prevented by inhibition of Na⁺/H⁺ exchanger 1 (57, 59). In mesentery vessels, diabetes-induced hypertrophy was prevented by inhibiting Na⁺/H⁺ exchanger 1 (21). In the former studies, the improvement in endothelial function was associated with maintenance of the cytosolic redox potential. Since prevention/reversal of oxidative stress in the endothelium improves vascular relaxation, the increase in oxidative stress in the vasculature may be an indicator of cytosolic redox imbalance.

This study demonstrated that inhibition of Na⁺/H⁺ exchanger 1 in diabetic rats reverses oxidative stress and accumulation of advanced glycation end products by the nerve, which has been shown to contribute to diabetic neuropathy. In nerves, calcium overload, a consequence of Na⁺/H⁺ exchanger 1 activation, has been shown to cause ishemic like injuries (27). In a model of spinal cord injury, an increase in markers of oxidative stress, nitrotyrosine staining and 4-hydroxynonenal, and calcium overload was shown to activate cysteine protease calpain and the degradation of cytoskeletal proteins (61). In diabetes we have shown that peripheral nerves accumulate the same markers, and with the activation of Na⁺/H⁺ exchanger 1 possibly leading to calcium overload, loss of neural function in part could be attributed to axonal degeneration. Interestingly, in the endothelium the activation of Na⁺/H⁺ exchanger 1 by hyperglycemia has been shown to activate calpain, which contributes to hyperglycemia-induced endothelial dysfunction through dissociation of heat shock protein 90 from endothelial nitric oxide synthetase (58). In that study, as in ours, treatment with cariporide attenuated the hyperglycemia-induced impairment of acetylcholine-induced relaxation in streptozotocin diabetic rats.

In summary, it is widely believed that oxidative stress plays an important role in the pathogenesis of peripheral diabetic neuropathy and that several pathways activated by hyperglycemia contribute to the generation of reactive oxygen/nitrogen species. We have shown that Na⁺/H⁺ exchanger 1 is overexpressed in peripheral nerve and contributes to the accumulation of markers for oxidative/nitrosative stress and advanced glycation end product. Inhibiting Na⁺/H⁺ exchanger 1 in diabetic rats reduced the levels of these markers in nerve and vascular tissue and improved diabetic neuropathy. These studies provide a rationale for further development of Na⁺/H⁺ exchanger 1 inhibitors for treatment of diabetic vascular and neural complications.

GRANTS

This material is based on work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, and Biomedical Laboratory Research and Development (BX001680-01; M. A. Yorek) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-073990 (MAY) and DK-077141 (I. G. Obrosova and M. A. Yorek).

DISCLOSURES

The authors have no conflicts of interest, financial or otherwise, to declare. The contents of this article are new and solely the responsibility of the authors and do not necessarily represent the official views of the granting agencies.

AUTHOR CONTRIBUTIONS

S.L., P.W., H.S., I.V., A.O., I.G.O., and M.A.Y. performed the experiments; S.L., P.W., H.S., I.V., A.O., I.G.O., and M.A.Y. analyzed the data; S.L., A.O., and M.A.Y. prepared the figures; S.L., H.S., A.O., and M.A.Y. edited and revised the manuscript; S.L., P.W., H.S., I.V., A.O., and M.A.Y. approved the final version of the manuscript; I.G.O. and M.A.Y. contributed to the conception and design of the research; I.G.O. and M.A.Y. interpreted the results of the experiments; M.A.Y. drafted the manuscript.