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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2020 Jan 6;318(2):F509–F517. doi: 10.1152/ajprenal.00518.2019

Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy

Michael D Wetzel 1, Ting Gao 2, Kristen Stanley 1, Timothy K Cooper 3, Sidney M Morris Jr 4, Alaa S Awad 1,2,✉
PMCID: PMC7052661  PMID: 31904280

Abstract

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

Keywords: asymmetric dimethylarginine, diabetic nephropathy, dimethylarginine dimethylaminohydrolase, nitric oxide

INTRODUCTION

Diabetes mellitus is a serious medical condition that leads to a variety of microvascular and macrovascular complications in addition to hyperglycemia. One of the most serious complications is diabetic nephropathy (DN) leading to end-stage renal disease (ESRD), affecting 20–40% of patients with type 1 and 2 diabetes (3, 14, 44, 48). Early histological changes in DN include glomerular hyperfiltration and hypertrophy, followed by glomerular basement membrane thickening, fibrosis, type I collagen deposition, macrophage infiltration, endothelial dysfunction, and mesangial matrix accumulation (5, 6, 9, 20, 70), leading to glomerular sclerosis and ESRD (5, 48). Current therapies, including blood pressure and glucose control and other lifestyle changes, have been only modestly successful in delaying the progression of renal failure in diabetes (2).

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide (NO) and increased oxidative stress, is a hallmark characteristic of diabetes (21) and DN (23). Under conditions of low arginine levels or hyperglycemia, endothelial NO synthase (eNOS; NOS3) is uncoupled, producing reactive oxygen species and oxidative stress in lieu of NO (16, 65). Low or absent NOS3 has been shown to exacerbate DN (63, 72). Thus, elucidating the basis for vascular dysfunction in DN is critical (11). Asymmetric dimethylarginine (ADMA) is a strong endogenous inhibitor of NO synthesis (19, 39, 58, 61). High levels of ADMA are observed in hypertension (24, 38), hypercholesterolemia (13, 34), diabetes (32), and chronic kidney disease (CKD) (61), and are a strong prognostic marker of cardiovascular events in patients with diabetes (1, 49, 54) and ESRD (61, 75). ADMA is highly elevated in adults with type 1 diabetes, leading to increased risk of ESRD and cardiac dysfunction (19). Clinical studies have shown that elevated ADMA is associated with increased mortality in patients with type 2 diabetes (74) and is an accurate indicator of albuminuria, impaired glomerular filtration rate, and hypertension (57). Elevated ADMA is also associated with insulin resistance in patients with type 1 diabetes (18), and vitamin E treatment decreased circulating ADMA levels while increasing NO levels (45). However, the precise mechanism underlying ADMA elevation in these diseases is not fully understood.

ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) into dimethylamine and citrulline (28, 40, 59), which are not inhibitors of NOS. Two isoforms of DDAH (DDAH-1 and DDAH-2) are encoded by two different genes, with distinct tissue distribution (29, 41). Although both are expressed in the kidney, mainly in glomerular endothelial cells, macula densa, and tubular cells (55, 56), DDAH-1 is the critical isozyme to degrade ADMA (25, 42). Low DDAH-1 levels have been associated with CKD, high systolic blood pressure, and increased urinary and plasma ADMA levels (36, 46). Introduction of DDAH in animal models with high ADMA reduced tubular fibrosis and improved systolic blood pressure (36, 37, 73). DDAH-1-deficient mice had increased plasma and kidney ADMA levels, reduced urine and plasma NO levels, and decreased endothelial cell proliferation (25), an effect completely reversed by DDAH-1 overexpression (71). However, a direct effect of DDAH on diabetic kidney disease had not previously been determined.

In the present study, we showed that diabetes is associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but has no effect on kidney DDAH-2 expression. Intrarenal DDAH-1 overexpression using an adenoviral vector significantly reduced kidney injury, as evidenced by reduced albuminuria, glomerular histology score, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid-reactive substances (TBARS) excretion, along with a significant increase in kidney DDAH activity and kidney NOS3 mRNA expression. Taken together, our data suggest that enhancing renal DDAH-1 activity might be a promising therapeutic modality for DN.

MATERIALS AND METHODS

Diabetic mouse model.

All animal experiments were performed in 6-wk-old male DBA/2J background mice (strain no. 000671, The Jackson Laboratory, Bar Harbor, ME). Diabetes was induced by intraperitoneal injection of 50 mg/kg streptozotocin (STZ) for 5 days as recommended by the Animal Models of Diabetes Complications Consortium as an optimal model of DN (15, 17). Adenovirus expressing DDAH-1 (Ad-h-DDAH-1; 1 × 109 infectious units) or control vector [adenovirus expressing green fluorescent protein (Ad-GFP)] were directly and slowly injected into the superficial cortex in six locations (20 µL each) bilaterally as previously described (30, 31). Adenovirus was injected only into mice that had been confirmed to be diabetic at 2 wk following the final STZ injection. After 6 wk, plasma and 24-h urine samples were collected, mice were euthanized, and kidneys were removed for further experiments. All animal experiments were approved by the University of Texas Health Science Center at San Antonio and Penn State Hershey Institutional Animal Care and Use Committees.

Adenovirus vectors.

Adenovirus vectors with expression of human DDAH-1 (Ad-h-DDAH-1; catalog no. ADV-206691) or green fluorescent protein (Ad-GFP; catalog no. 1060) driven by the cytomegalovirus promoter were obtained from Vector BioLabs (Malvern, PA).

Analytical methodology.

Urine albumin excretion was measured by ELISA using an Albuwell M kit (Exocell, Philadelphia, PA) as previously described (5, 6, 64). Urine creatinine was determined using a Creatinine Liquid Reagents Assay Kit (Diazyme Laboratories, Poway, CA). Urinary TBARS Assay was performed according to the instruction provided by Animal Models of Diabetes Complications Consortium as previously described (68, 69). Blood glucose was measured using Contour Next glucose meter (Ascensia Diabetes Care, Parsippany, NJ) as previously described (5, 6, 64). Systolic blood pressure was determined using CODA Non-Invasive Blood Pressure System (Kent Scientific, Torrington, CT) as previously described (5, 6, 64).

Western blot analysis.

As previously described (69), kidney lysates were prepared by homogenization in lysis buffer (0.1% Triton X-100) supplemented with protease inhibitor cocktail tablets (Roche, Cambridge, MA). The BCA protein assay (ThermoFisher, Waltham, MA) was used to determine protein concentration. Thirty micrograms of kidney lysates were separated on a NuPAGE 4~12% bis-Tris Gel (Invitrogen, Carlsbad, CA), and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Invitrogen). After being blocked with 5% nonfat dry milk, membranes were incubated with DDAH-1 (Abcam, Cambridge, MA, 1:3,000 dilution), DDAH-2 (Abcam, 1:2,000 dilution), or GAPDH (Cell Signaling Technology, Danvers, MA, 1:1,000 dilution) primary antibody overnight at 4°C followed by incubation with anti-rabbit IgG-horseradish peroxidase secondary antibody (Cell Signaling Technology, 1:3,000 dilution) for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence solutions (ThermoFisher Scientific) after a wash followed by exposure to X-ray film. Densitometry was performed using ImageJ (NIH; https://imagej.nih.gov/ij/index.html).

Immunohistochemistry.

Mouse kidney tissues were fixed in 10% formalin and embedded in paraffin, and 3-μm sections were cut. Immunohistochemistry was performed on paraffin-embedded sections with anti-mouse Mac-2 antibody (clone M3/38, Cedarlane, Burlington, NC) as previously described (64, 69). Forty glomeruli per mouse were examined with a ×40 objective in a blinded manner, and the number of macrophages in the section were divided by 40 glomeruli to obtain the average macrophage/glomerulus value for each mouse. Images were taken with an Olympus BX51 microscope and DP71 digital camera using Microsuite Basic 2.6 image software. Images were obtained with a ×100 (oil) objective with a total magnification of ×1,000.

Renal histopathology.

Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm sections were cut. Sections were stained with periodic acid-Schiff and examined at ×400 in a blinded manner, and scores were averaged. Semiquantitative scores (0–4+) were assigned based on the masked reading, as previously described (72). In brief, 12 sections/mouse were graded from 0 to 4+, where 0 represents no lesion and 1, 2, 3, and 4+ represent mesangial matrix expansion or sclerosis, involving ≤25, 25–50, 50–75, or >75% of the glomerular tuft area, respectively. Images were taken with an Olympus BX51 microscope and DP71 digital camera using Microsuite Basic 2.6 image software. Images were taken at ×40 objective magnification. For ×100 images, pictures were taken on a Nikon Eclipse E600 scope using a Nikon Digital Camera DYM1200.

RT-PCR.

RNA was extracted from kidneys using TRIzol (Invitrogen, Carlsbad, CA), and cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR was performed on a CFX384 real-time system (Bio-Rad, Hercules, CA) using Taqman primers for mouse NOS3 (Mm00435217_m1), IL-1β (Mm00434228_m1), fibronectin (Mm01256744_m1), arginase-2 (Mm00477592_m1), and 18S rRNA (Mm03928990_g1) (all from ThermoFisher). The reaction was performed using a protocol of 50°C for 2 min, 95°C for 10 min, and then 39 cycles of 95°C for 15 s and 60°C for 1 min. Threshold cycle (Ct) values were normalized to 18S rRNA, and ΔCt values were compared with normal samples.

DDAH activity assay.

DDAH activity was determined using the procedure described by Tain and Baylis (52). Briefly, kidneys were homogenized in lysis buffer containing 0.1% Triton X-100, incubated with urease (1:10 ratio), mixed with 1 mM ADMA, incubated at 37°C for 45 min, and treated with 4% sulfosalicylic acid before centrifugation at 3,000 g for 10 min. The supernatant (300 µl) was mixed with an equal amount of color mixture (1 part of oxime reagent:2 parts of antipyrine/H2SO4) and incubated for 110 min at 60°C in the dark. The reaction was stopped by cooling samples at −20°C for 10 min, and 200-µl samples were then loaded into a 96-well plate. Citrulline was measured at 466 nm.

ADMA quantification.

ADMA levels were determined using a Mouse Asymmetrical Dimethylarginine ELISA kit (MBS1601018, MyBiosource, San Diego, CA) using the manufacturer’s instructions. Briefly, 20–45 mg of kidney tissue were homogenized in 300 µL of 1× PBS and then measured for protein concentration using Pierce BCA protein assay kit (ThermoFisher). Lysate (50 µL) was loaded in duplicate into strips and incubated with 10 µL of anti-ADMA antibody and 50 µL of streptavidin-horseradish peroxidase for 60 min at 37°C. Wells were then washed and developed for 10 min, and optical density was read at 450 nm. Data are presented as nanomoles per grams of tissue used.

Statistical analysis.

Comparisons between groups were conducted using GraphPad Prism software (version 7.04, San Diego, CA). Results are expressed as means ± SE. An unpaired t test was used for comparison between two groups. One-way ANOVA using Fisher’s least significant difference test as a post hoc test was used to compare significance between more than two groups. P < 0.05 represented a significant difference.

RESULTS

Diabetes regulates kidney ADMA and DDAH-1 levels.

Diabetic mice had elevated kidney ADMA levels (Fig. 1A), reduced kidney DDAH activity (Fig. 1B), and reduced DDAH-1 expression (Fig. 1C), but kidney DDAH-2 expression was unaffected (Fig. 1D). Since only DDAH-1 was significantly reduced in DN, we decided to use an adenovirus vector to overexpress DDAH-1 specifically in the kidney in all subsequent experiments.

Fig. 1.

Fig. 1.

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Diabetes increases kidney asymmetric dimethylarginine (ADMA) and reduces kidney dimethylarginine dimethylaminohydrolase (DDAH) activity and DDAH-1 expression. A: kidney ADMA levels were determined by ELISA. B: DDAH activity was determined as described in materials and methods. C and D: levels of DDAH-1 and DDAH-2 protein in normal and diabetic mouse kidneys were determined by Western blot analysis and normalized to GAPDH. Western blots are representative of 5 mice/group. Data are presented as means ± SE. *P < 0.05 compared with normal.

Kidney DDAH-1 delivery reduced albuminuria in diabetic mice.

Previous studies have routinely demonstrated that diabetic mice have elevated 24-h urine albumin excretion and the urine albumin-to-creatinine ratio compared with normal mice (67, 69). Similarly, Ad-GFP-treated diabetic mice showed a significantly higher urine albumin-to-creatinine ratio (Fig. 2) compared with normal mice after 6 wk of diabetes. Intrarenal Ad-h-DDAH-1-treated diabetic mice had a significantly reduced urine albumin-to-creatinine ratio compared with Ad-GFP-treated diabetic mice. Importantly, there were no significant differences between Ad-GFP-treated and Ad-h-DDAH-1-treated diabetic mice in blood glucose levels (499 ± 1 vs. 485 ± 7 mg/dL) or systolic blood pressure (118 ± 3 vs. 123 ± 4 mmHg), respectively.

Fig. 2.

Fig. 2.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces albuminuria in diabetic mice. Urine was collected from mice and analyzed for the urine albumin-to-creatinine ratio. Data are presented as means ± SE; n = 10–13 mice/group. *P < 0.005 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Kidney DDAH-1 delivery restored kidney DDAH-1 activity and ADMA levels in diabetic mice.

Since diabetes was associated with reduced kidney DDAH activity along with increased kidney ADMA levels (Fig. 1), we next determined whether intrarenal Ad-h-DDAH-1 delivery could restore these parameters. As shown in Fig. 3, Ad-GFP-treated diabetic mice showed significantly lower kidney DDAH activity (Fig. 3A) and increased kidney ADMA levels (Fig. 3B) compared with normal mice after 6 wk of diabetes. Intrarenal Ad-h-DDAH-1-treated diabetic mice restored kidney DDAH activity and kidney ADMA levels to their normal levels.

Fig. 3.

Fig. 3.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) improves mouse kidney DDAH activity and reduces renal asymmetric dimethylarginine (ADMA) in diabetic mice. A: kidneys were analyzed for DDAH activity as described in materials and methods. B: ADMA levels were measured in mouse kidney samples by ELISA. Data are presented as means ± SE; n = 8–14 mice/group. *P < 0.05 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Kidney DDAH-1 delivery restored kidney NOS3 mRNA expression and urinary TBARS levels in diabetic mice.

Since ADMA is a strong endogenous inhibitor of NO synthesis (19, 39, 58, 61) and plays a role in NOS uncoupling in endothelial cells, resulting in the production of reactive oxygen species and oxidative stress in lieu of NO (4, 50), we examined the effect of intrarenal Ad-h-DDAH-1 delivery on NOS3 expression and oxidative stress as measured by TBARS. Whereas kidney NOS3 mRNA expression was reduced (Fig. 4A) and levels of urinary TBARS greatly increased (Fig. 4B) in Ad-GFP-treated diabetic mice compared with normal mice, kidney NOS3 mRNA expression and urinary TBARS excretion were restored to normal levels in Ad-h-DDAH-1-treated diabetic mice.

Fig. 4.

Fig. 4.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) increases kidney endothelial nitric oxide synthase (NOS3) mRNA and reduces urinary thiobarbituric acid-reactive substances (TBARS) in diabetic mice. A: total RNA was isolated from mouse kidneys, and RT-PCR was performed to determine NOS3 mRNA levels normalized to 18S rRNA. B: mouse 24-h urine was collected for the measurement of TBARS. Data are presented as means ± SE; n = 5–10 mice/group. *P < 0.05 and **P < 0.001 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Kidney DDAH-1 delivery decreased renal histological score in diabetic mice.

Periodic acid-Schiff staining showed significantly increased glomerular cellularity and mesangial expansion after 6 wk of diabetes in Ad-GFP-treated diabetic mice compared with normal mice (Fig. 5). In contrast, Ad-h-DDAH-1-treated diabetic mice had significantly reduced glomerular scores compared with Ad-GFP-treated diabetic mice (Fig. 5).

Fig. 5.

Fig. 5.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces kidney pathology in diabetic mice. Paraffin-embedded mouse kidney sections were subjected to periodic acid-Schiff staining. Scale bar = 50 µm (×40) and 20 µm (×100). Data are presented as means ± SE; n = 8–11 mice/group. *P < 0.005 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Kidney DDAH-1 delivery decreased glomerular macrophage infiltration in diabetic mice.

To determine whether kidney DDAH is critical for kidney macrophage infiltration in DN, we examined the distribution and number of Mac-2-positive macrophages in the glomeruli by immunohistochemistry (Fig. 6). The number of glomerular macrophages in normal mice was low but increased significantly in Ad-GFP-treated diabetic mice after 6 wk of diabetes (Fig. 6). Intrarenal Ad-h-DDAH-1-treated diabetic mice had significantly reduced glomerular macrophage recruitment compared with Ad-GFP-treated diabetic mice.

Fig. 6.

Fig. 6.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces glomerular macrophage infiltration in diabetic mice. A: glomerular macrophage recruitment was visualized by immunohistochemical staining using Mac-2 antibody, and pictures were taken at ×100 magnification. Red arrows indicate macrophages. Scale bar = 20 µm. B: macrophage numbers per glomerulus were counted in a blinded manner. The number of glomerular macrophages was counted in 40 glomeruli/section (number of macrophages in glomeruli divided by the number of glomeruli) under ×40 magnification and averaged. Data are presented as means ± SE; n = 8–12 mice/group. *P < 0.001 compared with normal; #P < 0.005 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

Kidney DDAH-1 delivery decreased inflammatory cytokines and fibrotic markers in diabetic mice.

Increased inflammatory cytokines and fibrotic markers are major features of and important predictors of DN (6, 9, 67). Therefore, we further assessed the anti-inflammatory and antifibrotic effects of intrarenal DDAH-1 delivery in diabetic mice (Fig. 7). Kidney arginase-2, fibronectin, and IL-1β mRNA expression was significantly increased in Ad-GFP-treated diabetic mice compared with normal mice. Intrarenal Ad-h-DDAH-1-treated diabetic mice resulted in significantly reduced kidney arginase-2, fibronectin, and IL-1β mRNA expression after 6 wk of diabetes.

Fig. 7.

Fig. 7.

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Administration of adenovirus expressing dimethylarginine dimethylaminohydrolase-1 (Ad-h-DDAH-1) reduces inflammatory cytokines and fibrotic markers in diabetic mice. RT-PCR was performed on RNA extracted from kidney and analyzed for the indicated genes using indicated Taqman primers. Threshold cycle (Ct) values were normalized to 18S rRNA values and then compared with normal mice. Data are presented as means ± SE; n = 6–7 mice/group. *P < 0.01 and **P < 0.001 compared with normal; #P < 0.05 compared with adenovirus expressing green fluorescent protein (Ad-GFP)-treated diabetic mice.

DISCUSSION

DDAH-1 plays a pivotal role to maintain endothelial function under physiological and pathological conditions, yet its role in diabetic kidney injury had not previously been determined. This study shows that intrarenal DDAH-1 delivery mediates renal tissue protection as proven by a reduction in albuminuria, histopathological changes, kidney macrophage recruitment, and inflammatory cytokine and fibrotic markers during diabetes. The renal tissue-protective effect of DDAH-1 is likely mediated by a reduction of ADMA and restoration of kidney NOS3 expression and activity, thus reducing oxidative stress. These findings reveal an important role for DDAH-1 in the pathogenesis of DN and provide evidence for enhancing renal DDAH-1 activity as a potential therapeutic modality for treating patients with diabetes.

Although both DDAH-1 and DDAH-2 are expressed in the kidney (29), albeit in different renal cell types, we show that expression DDAH-1, but not of DDAH-2, is altered in diabetes. Zhu et al. (73) showed that treatment of diabetic rats with DDAH-2 improved myocardial function and lower blood glucose levels. Unlike DDAH-2, DDAH-1 exerts its effects mainly in the kidneys (41, 51). DDAH-1 is a critical enzyme to degrade ADMA (25, 42). Low DDAH-1 levels have been associated with CKD and hypertension and increased cardiovascular risk (36, 46, 60). DDAH-1-deficient mice had increased ADMA levels, reduced NO production, and decreased cell proliferation; an effect completely reversed by DDAH-1 overexpression (71). Although previous reports have shown that diabetes is associated with reduced renal DDAH activity and increased renal and urinary ADMA (27, 33, 47), a direct effect of DDAH on diabetic kidney disease remained unknown.

DDAH-1 knockdown reduced proximal tubule fluid reabsorption and induced adverse kidney pathology without affecting systolic blood pressure (12). Our results show that increased renal expression of DDAH-1 did not affect systolic blood pressure in diabetic mice despite restoring ADMA levels and renal NOS3 mRNA expression.

DDAH-1 converts ADMA to dimethylamine and citrulline, and increased levels of ADMA are widely used as an indicator of diabetes complications (36, 46). Polymorphisms in both DDAH isoforms have been linked with type 1 and 2 diabetes as well as elevated serum ADMA (22, 35) but not albuminuria. Only one DDAH polymorphism (single-nucleotide polymorphism rs805304) is correlated with increased risk of glomerular filtration rate decline (53). Our data indicate that DDAH-1 activity is critical for regulating ADMA levels in diabetes.

Although the role of NO in DN is well established, the expression and regulation of NO and NOS in DN is controversial (26). We have previously shown that the production of NO is diminished in diabetes and restored to normal levels by insulin (8). Lai et al. (27) showed an association between elevated ADMA and the reduction in NO and DDAH activity in diabetic rats. ADMA, equipotent with NG-monomethyl-l-arginine, inhibits NOS3, which becomes uncoupled and generates superoxide in lieu of NO (43). Our data show increased NOS3 expression as a result of increased kidney DDAH-1, which could explain the protective effect of DDAH-1 in DN via reducing ADMA levels. These results are in agreement with a previous report showing that ADMA infusion resulted in a reduction of NOS3 expression (mRNA, protein, and phospho-eNOS) and NO levels, along with increased superoxide production, in the porcine coronary artery (66). The mechanism by which DDAH-1 overexpression increases NOS3 mRNA expression is not known. However, as DDAH-1 overexpression likely promotes ADMA conversion to citrulline and dimethylamine, decreases in ADMA levels result in reduced inhibition of NOS3 activity and expression, NOS3 uncoupling, and generation of superoxide in lieu of NO (43). Furthermore, reduced ADMA levels may promote NO synthesis because ADMA can competitively inhibit cellular uptake of arginine, the nitrogenous substrate for NOS3. Our study is only focused on ADMA/NOS3 interaction and therefore is limited for not examining other NOS isoforms and measuring NOS protein expression, NOS uncoupling, or activity, which also could play a role in the protective effect of DDAH-1 overexpression in DN. Additional studies are needed to elucidate the role of ADMA in regulating NOS3 expression.

DDAH-1 resulted in less mesangial expansion and glomerular hypercellularity in diabetes, indicating a possible contribution of DDAH-1 deficiency to the initiation and/or progression of diabetic renal fibrosis. This is also confirmed by reduced fibronectin in DDAH-1-overexpressing mice in our study. The renal protective effect of DDAH-1 also correlates with a significant reduction of kidney macrophage infiltration and inflammatory cytokines. Whether the reduction in macrophage recruitment is mediated directly by DDAH-1 or indirectly by reducing diabetic renal injury is not clear at this time. Additional studies are required to elucidate the role of DDAH-1 in kidney macrophage infiltration. However, we previously showed that arginase-2 mediates renal tissue injury and pharmacological blockade or genetic deficiency of arginase-2 protects both the development and progression of DN via a NOS3-dependent pathway (39a, 67a, 68). Whether DDAH-1 and arginase-2 interact directly or indirectly is not clear at this time.

In conclusion, we have shown that intrarenal adenoviral delivery of DDAH-1 reduces DN by preventing the increase in ADMA and restoring expression of NOS3. Thus, strategies to enhance renal DDAH-1 activity may represent promising therapeutic modalities for treatment of DN.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-094930 and DK-094930S1.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

S.M.M. and A.S.A. conceived and designed research; M.D.W., T.G., and K.S. performed experiments; M.D.W., K.S., T.K.C., and A.S.A. analyzed data; M.D.W., S.M.M., and A.S.A. interpreted results of experiments; M.D.W. and A.S.A. prepared figures; M.D.W. and A.S.A. drafted manuscript; M.D.W., S.M.M., and A.S.A. edited and revised manuscript; M.D.W., T.G., K.S., T.K.C., S.M.M., and A.S.A. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank R. Padmanabhan for technical assistance.

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