help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0602
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/5/2016    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yuan, G.
Right arrow Articles by Wang, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yuan, G.
Right arrow Articles by Wang, D. W.
Endocrinology Vol. 148, No. 5 2016-2026
Copyright © 2007 by The Endocrine Society

Tissue Kallikrein Reverses Insulin Resistance and Attenuates Nephropathy in Diabetic Rats by Activation of Phosphatidylinositol 3-Kinase/Protein Kinase B and Adenosine 5'-Monophosphate-Activated Protein Kinase Signaling Pathways

Gang Yuan1, Juanjuan Deng1, Tao Wang, Chunxia Zhao, Xizheng Xu, Peihua Wang, James W. Voltz, Matthew L. Edin, Xiao Xiao, Lee Chao, Julie Chao, Xin A. Zhang, Darryl C. Zeldin and Dao Wen Wang

Department of Internal Medicine and Gene Therapy Center (G.Y., J.D., T.W., C.Z., X.Xu., P.W., X.Xi., D.W.W.), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China; Division of Intramural Research (J.W.V., M.L.E., D.C.Z.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Departments of Molecular Genetics and Biochemistry and Gene Therapy Center (X.Xi.), University of Pittsburgh, Pittsburgh, Pennsylvania 15260; Department of Biochemistry and Molecular Biology (L.C., J.C.), Medical University of South Carolina, Charleston, South Carolina 29425; and Vascular Biology Center and Departments of Medicine and Molecular Science (X.A.Z.), University of Tennessee Health Science Center, Memphis, Tennessee 38163

Address all correspondence and requests for reprints to: Dao Wen Wang, M.D., Ph.D., Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, People’s Republic of China. E-mail: dwwang{at}tjh.tjmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that iv delivery of the human tissue kallikrein (HK) gene reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with insulin resistance. In the current study, we evaluated the potential of a recombinant adeno-associated viral vector expressing the HK cDNA (rAAV-HK) as a sole, long-term therapy to correct insulin resistance and prevent renal damage in streptozotocin-induced type-2 diabetic rats. Administration of streptozotocin in conjunction with a high-fat diet induced systemic hypertension, diabetes, and renal damage in rats. Delivery of rAAV-HK resulted in a long-term reduction in blood pressure, and fasting plasma insulin was significantly lower in the rAAV-HK group than in the control group. The expression of phosphatidylinositol 3-kinase p110 catalytic subunit and the levels of phosphorylation at residue Thr-308 of Akt, insulin receptor B, and AMP-activated protein kinases were significantly decreased in organs from diabetic animals. These changes were significantly attenuated after rAAV-mediated HK gene therapy. Moreover, rAAV-HK significantly decreased urinary microalbumin excretion, improved creatinine clearance, and increased urinary osmolarity. HK gene therapy also attenuated diabetic renal damage as assessed by histology. Together, these findings demonstrate that rAAV-HK delivery can efficiently attenuate hypertension, insulin resistance, and diabetic nephropathy in streptozotocin-induced diabetic rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES IS a worldwide epidemic associated with significant morbidity and mortality. People with diabetes have higher all-cause mortality rates than those without diabetes, mainly attributable to cardiovascular causes (1, 2). The pathogenesis of type 2 diabetes includes reductions in insulin sensitivity and ß-cell function. Insulin resistance may play a major role in type 2 diabetes, especially when glucose tolerance is still normal (3).

Insulin resistance in animal and humans is characterized by decreased rates of insulin-mediated glucose uptake and glycogen synthesis in peripheral tissues including skeletal muscle, liver, and fat. It has been previously demonstrated that bradykinin increases insulin sensitivity and stimulates glucose uptake both in vivo and in vitro (4). Isami et al. (5) reported that bradykinin increased insulin-induced glucose uptake, stimulated insulin-induced translocation of glucose transporter (GLUT4), and potentiated insulin-induced phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1) in isolated dog adipocytes. Tissue kallikrein is a serine protease that converts kininogen to the peptide hormone bradykinin. A reduction of renal kallikrein has been found in non-insulin-treated diabetic individuals, suggesting that an impaired kallikrein-kinin system may be involved in the development of diabetes (6).

Our previous study demonstrated that iv delivery of the pcDNA3.1 vector carrying the human tissue kallikrein (HK) cDNA reduced blood pressure and plasma insulin levels in fructose-induced hypertensive rats with diabetes (7). Recently, Montanari et al. (8) used an adenovirus vector to deliver the HK gene to diabetic rats to further support our findings (7). Together, these data suggest the tissue kallikrein may be an effective gene for the treatment of insulin resistance in type 2 diabetes. Plasmid or adenovirus vectors mediate only transient gene expression and produce short-term therapeutic effects (~3–4 wk), and the latter is toxic and immunogenic. Because of these complications, neither is suitable for gene therapy of a chronic disease such as insulin-resistant diabetes. Comparatively, recombinant adeno-associated virus (rAAV) vectors possess numerous advantages, including stable and long-term transgene expression, the ability to infect both dividing and nondividing cells (such as muscle and liver), nonpathogenecity, and low vector toxicity. Recently, the establishment of adenovirus-independent package and replication systems and the improvement of carrying capacity (9, 10, 11) make rAAV the most attractive vector for long-term gene therapy.

The objectives in the current study were 1) to confirm that tissue kallikrein gene delivery attenuates insulin resistance in streptozotocin-induced diabetes, 2) to determine whether HK gene delivery attenuates the kidney complications associated with diabetes, and 3) to determine the impact of rAAV-HK gene therapy on the signaling mechanisms involved in diabetic pathology. To address these issues, we have evaluated the effectiveness of HK gene-expressing rAAV as a sole therapy to correct insulin resistance and to prevent renal complications in type 2 diabetic rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Horseradish-peroxidase-conjugated goat antirabbit or antimouse IgG were obtained from Jackson ImmunoResearch Laboratories (Soham, UK). Enhanced chemiluminescence reagent was purchased from Pierce Chemical (Rockford, IL), prestained molecular weight standards from Bio-Rad (Hercules, CA), and polyvinylidene difluoride membranes from Schleicher and Schuell (Dassel, Germany). Akt (p-Thr-308) phosphorylation-specific polyclonal antibody was purchased from AnaSpec, Inc. (San Jose, CA). Polyclonal antibodies against phosphatidylinositol 3-kinase (PI3-kinase), Akt, p42/p44 MAPK, and phosphorylated MAPK (p-MAPK), and monoclonal antibodies against Bax and Bcl-2 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). AMP-activated protein kinases (AMPK), p-AMPK, insulin receptor ß (IR-ß), and p-IR-ß were from Cell Signaling Technology Inc. (Danvers, MA). All other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Construction and preparation of rAAV
The rAAV vector plasmid pXXUF1 and rAAV plasmid containing the reporter gene pdxII-lacZ were from Dr. Xiao Xiao. The pXXUF1 contains two inverted terminal repeats, a cytomegalovirus promoter and a poly-A tail. The rAAV packaging plasmids were constructed based on plasmid pAAV/Ad as we reported previously (9, 12). Packaging plasmid pXX2 (for serotype 2 of adeno-associated virus) was constructed from pACG2 by inserting a promoter p5 element downstream of the capsid gene. The adenovirus helper plasmid pXX6 was constructed by inserting the large ClaI/SalI fragment of pXX5 into plasmid pBluescript KS. An 860-bp HK cDNA fragment (NotI/NotI) containing the open reading frame (NM 002257) was subcloned into pXXUF1 downstream of the cytomegalovirus promoter to create the plasmid pUF1-HK. rAAVs containing either LacZ or HK genes were prepared and purified as described previously (13, 14), and the rAAV titers were determined by dot-blot hybridization. The resultant rAAV vectors were designated rAAV-LacZ and rAAV-HK, respectively.

Animal treatment
Animal treatment has been described in detail elsewhere (15). Briefly, 2-month-old male Wistar rats weighing 180–200 g were supplied from the Experimental Animal Center in Shanghai, China. Experimental protocols complied with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by The Academy of Sciences of China. All animals were housed (four per cage) in a temperature (25 ± 3 C) and humidity (50 ± 20%) controlled room with 12-h light, 12-h dark cycles and were allowed free access to normal rat chow (9% fat, 20% protein, 53% starch, and 5% fiber) plus water for 1 wk to obtain baseline blood pressure and blood samples. The animals were then divided randomly into two groups. Normal rats (n = 16) were fed normal rat chow and remained untreated for the duration of the study. Experimental rats (n = 32) were fed a diet enriched in fat (25% fat, 15% protein, 51% starch, and 5% fiber) as previously described (16). After 1 month on their respective diets, experimental rats were injected iv with streptozotocin (25 mg/kg body weight), and normal rats were injected with vehicle (0.05 mol/liter citric acid, pH 4.5). Both the low dose of streptozotocin and the high-fat diet are essential elements of the model designed to induce type 2 diabetes with insulin resistance. After 1 month, rats were fasted overnight and given 20% glucose (3 g/kg body weight). Blood samples were taken from the tail vein to measure glucose and insulin levels at 2 and 12 h after glucose administration. Only rats with increased postprandial glucose (>200 mg/dl) were considered diabetic and were randomly divided into two groups: rAAV-HK treatment (HK group) and rAAV-lacZ treatment (LacZ group) (n = 16 rats in each group).

Gene delivery
Diabetic animals were first anesthetized with pentobarbital at a dose of 50 mg/kg body weight ip. A single iv injection of rAAV-HK or rAAV-LacZ (1 x 1011 viron particles in 1 ml saline solution) was then given via tail vein of diabetic rats. This dose has previously been shown to result in persistent HK gene expression for up to 19 wk (13). After the injection, the rats were kept warm under an infrared lamp until they returned to consciousness. All animals were killed 12 wk after rAAV gene delivery under pentobarbital anesthesia (50 mg/kg body weight), and heart, kidney, skeletal muscle, liver, lung, and fat were collected, frozen on liquid nitrogen, and stored at –80 C until additional analyses were done.

Systolic blood pressure measurements
After rAAV injections, systolic blood pressure was measured every 2 wk in conscious rats with a manometer-tachometer (Rat Tail NIBP system; ADInstruments, New South Wales, Australia) by means of the tail-cuff method (17). The rats were minimally warmed (usually 15 min) before blood pressure determination and after a brief period of restraint in a plastic cage. For each animal, systolic blood pressure was taken as the mean of five daily recordings. All measurements were made between 0900 and 1200 h.

Plasma and urine analysis
Blood samples were taken after an overnight fast from the tail vein immediately before injection of rAAV and at the end of the experiment. Plasma was prepared and stored at –80 C. Every 2 wk, 24-h urine samples were collected in metabolic cages into brown bottles containing 500 µl toluene to prevent decay. Plasma glucose, cholesterol, triglyceride, creatinine, and urine creatinine were measured in duplicate on an AEROSET Clinical Chemistry System (Abbott Laboratories, Chicago, IL). Plasma insulin was measured with the use of a magnetic solid-phase enzyme immunoassay kit from BioChem ImmunoSystem. Insulin resistance was calculated by means of the homeostasis model assessment for insulin resistance (HOMA-IR) method (18), which has been used previously in rodents (19, 20, 21). Urine osmolarity was measured on a model 3D3 osmometer (Advanced Instruments, Inc., Norwood, MA). Urinary microalbumin was measured to estimate albumin excretion rate (AER) by ELISA method with a kit from Shanghai Debo Biotechnology Inc. (Shanghai, China). Creatinine clearance was calculated according to the method of Gault and Cockcroft (22).

ELISA specific for HK
ELISA reagents specific for HK were a gift from Dr. Lee Chao (Medical University of South Carolina). ELISA for HK in animal urine samples was performed according to previously described methods (23, 24). The plates were read at 405 nm on Elx 800 ELISA reader (Bio-Tek Instruments, Inc., Winooski, VT).

RT-PCR analysis of HK mRNA
Total RNA was extracted from frozen rat lung, liver, heart, kidney, skeletal muscle, and fat using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA). RT-PCR analysis specific for HK mRNA used the following primers: 5' primer, 5'-CATTTCAGCACTTTCCA-3', and 3' primer, 5'-GCCACAAGGGACGTAGC-3'. RT-PCR analysis for ß-actin housekeeping gene used the following primers: 5' primer, 5'-GGAGAAGATGACCCAGATC-3', and 3' primer, 5'-GATCTTCATGAGGTAGTCAG-3'. The RT-PCR method was performed according to manufacturer’s instructions using an RT-PCR kit from Takara Biotechnology Co. Ltd. (Dalian, China). After incubation with Moloney murine leukemia virus reverse transcriptase, amplification was performed with reagents from Applied Biosystems (Foster City, CA) under the following conditions: denaturing phase of 1 min at 94 C, annealing phase of 40 sec at 65 C, and extension phase of 1 min at 72 C for 20 cycles. PCR products were electrophoresed on 1.5% agarose gels. The quantity of specific HK transcripts was normalized to the expression of ß-actin to control for RNA quality and amount.

Analysis of renal morphology and collagen deposition
A portion of the kidney from each animal was preserved in 4% PBS-buffered formaldehyde solution and embedded in paraffin. Four 3-µm paraffin sections were randomly chosen from one kidney from each animal and stained with periodic acid Schiff (PAS) reagent or Masson’s trichrome to evaluate morphological changes and with Sirius red using the method described previously to evaluate collagen deposition (25). In each section, six fields of kidney cortex were randomly selected at a magnification of x40 using an Olympus BH-2 light microscope (Olympus, Tokyo, Japan). Extracellular matrix (ECM) production was quantified as the ratio of collagen to noncollagen area (percentage of ECM) with the use of HMIAS (High Medical Image Analysis System). All sections were evaluated by investigators who were blinded to treatment group assignment.

Apoptosis assays
Apoptotic cells were detected using a terminal dUTP nick-end labeling (TUNEL) assay on paraffin-embedded sections using a commercially available kit for detecting end-labeled DNA (R&D Systems, Inc., Minneapolis, MN) as described previously (26). All experiments were repeated at least three times for each animal kidney. Double-stranded DNA in nuclei was counterstained after TUNEL staining with methyl green. TUNEL-positive cells per high-power field were counted in 10 random high-power fields from each of five sections, and the counts were averaged as the apoptotic index.

Western blotting
Proteins for Western blotting were obtained from skeletal muscle and liver using TRIZOL reagent. Protein concentrations were estimated by the Bradford method (27). Equal amounts of protein sample were separated by electrophoresis on 10% SDS-PAGE gels. The resolved proteins were electrophoretically transferred to polyvinylidene difluoride membranes using a transfer buffer containing 192 mmol/liter glycine, 20% (vol/vol) methanol, and 0.02% SDS. The membranes were incubated in 10 mmol/liter Tris-Cl (pH 7.5), 100 mmol/liter NaCl, and 0.1% Tween 20 with 5% nonfat dried milk for 2 h at room temperature and then incubated overnight at 4 C with the indicated primary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence using horseradish-peroxidase-labeled antirabbit or antimouse IgG (Amersham Life Science, Arlington Heights, IL) and quantified by densitometric analysis using the Bio image system (Syngene, Cambridge, UK).

Statistical analysis
Results are expressed as mean ± SEM. Data were analyzed by either the unpaired Student’s t test or ANOVA, with the use of SYSTAT software (Systat Inc., Evanston, IL). Values were considered significantly different if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Streptozotocin and high-fat diet induce diabetes in rats
Table 1Go shows the biochemical and physiological parameters in streptozotocin-treated rats on a high-fat diet and normal rats. There were no significant differences in systolic blood pressure, body weight, glucose, insulin, triglycerides, cholesterol, and the HOMA-IR between the two groups at baseline. After streptozotocin injection and high-fat feeding, experimental rats displayed lower body weight (P < 0.05), higher systolic blood pressure (P < 0.01), higher serum postprandial glucose (P < 0.01), higher fasting insulin levels (P < 0.01), higher triglycerides, and higher cholesterol compared with nondiabetic control rats. HOMA-IR was significantly increased in experimental rats compared with nondiabetic controls. These data validate the use of streptozotocin and high-fat diet to induce the metabolic abnormalities characteristic of type 2 diabetes in rats.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Physiological parameters in normal and diabetic rats after induction of type 2 diabetes but preceding HK gene delivery

 
rAAV-HK induces in vivo expression of HK
We analyzed HK mRNA levels in rats after rAAV gene delivery by RT-PCR. Total RNA was prepared from lung, liver, heart, kidney, skeletal muscle, and fat 16 wk after iv injection of rAAV-HK. HK mRNA was detected in lung, liver, heart, kidney, and skeletal muscle in rAAV-HK-treated rats but not in rAAV-LacZ-treated diabetic rats (Fig. 1AGo). Equivalent levels of ß-actin mRNA were detected in tissues of HK and LacZ groups. We also observed abundant HK protein expression in rAAV-HK-treated rats compared with rAAV-LacZ-treated rats, indicating that the increase in HK mRNA levels is accompanied by abundant HK protein expression (Fig. 1BGo). Immunoreactivity observed in rAAV-LacZ lanes is likely cross-reactivity of our antibodies with endogenous rat tissue kallikrein. Together, these results indicate that HK is stably expressed in multiple tissues related to cardiovascular and renal function after rAAV-HK gene transfer in rats.


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 1. Expression of kallikrein. A, RT-PCR analysis in diabetic rats treated with rAAV-HK (HK) or rAAV-LacZ (LacZ). Total RNA was prepared from lung (L), liver (Li), kidney (K), skeletal muscle (SK), heart (H), and adipose tissue (A). B, Representative immunoblotting results showing increased HK expression in heart, kidney, and liver of rAAV-HK-treated diabetic rats compared with rAAV-LacZ-treated diabetic rats. Experiments were repeated three or four times.

 
We also measured kallikrein protein levels in urine by ELISA. Low levels of immunoreactive kallikrein were detected in the urine of saline-treated nondiabetic normal control rats and rAAV-LacZ-treated diabetic animals. In contrast, in rAAV-HK-treated diabetic animals, the urinary level of immunoreactive kallikrein was 1.91 ± 0.73 ng/ml 2 wk after injection and was maintained at high levels (1.92 ± 0.49 ng/ml) throughout the duration of the experiment, which were significantly higher than those in nondiabetic normal control and rAAVLacZ-treated diabetic rats (Fig. 2Go). These results demonstrate that rAAV-mediated gene transfer achieves HK gene expression in vivo for an extended period of time.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 2. Urinary expression of kallikrein by ELISA in nondiabetic rats (normal), diabetic rats treated with rAAV-LacZ (LacZ), and diabetic rats treated with rAAV-HK (HK) at various time points after treatment. Values are mean ± SEM (n = 6 for each group); **, P < 0.01 vs. normal; ##, P < 0.01 vs. LacZ.

 
rAAV-HK reduces blood pressure in diabetic rats
Blood pressure was measured at baseline, after treatment with streptozotocin and high-fat diet, and after injection of saline, rAAV-LacZ, and rAAV-HK. The results shown in Fig. 3Go indicate that systolic blood pressure was significantly higher in diabetic rats than in control rats. Moreover, rAAV-HK treatment significantly reduced systolic blood pressure within 2 wk of injection and normalized systolic blood pressure from the fourth week after injection until the end of the experiment (16 wk). In contrast, the rAAV-LacZ-treated animals remained significantly hypertensive throughout the study period (Fig. 3Go). These results indicate that rAAV-HK gene delivery induces a sustained reduction in blood pressure in diabetic rats.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 3. Systolic blood pressure in nondiabetic rats injected with saline (control), diabetic rats injected with rAAV-LacZ, and diabetic rats injected with rAAV-HK. Values are mean ± SEM (n = 16 per group); **, P < 0.01 vs. control; ##, P < 0.01 vs. LacZ.

 
rAAV-HK attenuates diabetic nephropathy
Biochemical tests indicated that creatinine clearance was increased in the rAAV-LacZ group compared with normal, consistent with enhanced glomerular filtration with streptozotocin treatment. Treatment with rAAV-HK significantly prevented the increase in creatinine clearance (Fig. 4AGo). In addition, rAAV-LacZ-injected streptozotocin-treated rats had reduced urine osmolarity, suggesting renal tubular damage. In contrast, urine osmolarity was not significantly different from normal in rAAV-HK-treated rats (Fig. 4BGo). Moreover, urinary microalbumin levels were significantly decreased in the rAAV-HK group compared with the rAAV-LacZ group (Fig. 4CGo). Together, these results suggest that HK gene delivery significantly preserves renal glomerular and tubular functions after induction of diabetes in rats.


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 4. Creatinine clearance (A), urine osmolarity (B), and urinary microalbumin (C) in nondiabetic rats injected with saline (control), diabetic rats injected with rAAV-LacZ, and diabetic rats injected with rAAV-HK. Values are mean ± SEM (n = 6 for each group); #, P < 0.05 vs. LacZ; ##, P < 0.01 vs. LacZ; **, P < 0.01 vs. control.

 
Effect of rAAV-HK on renal histology
Histological analysis of kidneys revealed moderate mesangial cell proliferation and ECM accumulation in rats with streptozotocin-induced diabetes (Fig. 5Go, B and J). Severe interstitial lymphocyte infiltration, renal tubular vacuolation, protein casts in renal tubules, and hyaline caps were observed in rats with diabetes (Fig. 5Go, D and G). Moderate collagen deposition and blood capillary thrombosis was also observed in the diabetic rats. In contrast, rats treated with rAAV-HK displayed only mild mesangial cell proliferation, a small degree of interstitial lymphocyte infiltration, and nearly normal renal tubular morphology (Fig. 5Go, C, E, H, and K). To quantify collagen accumulation in kidneys, red-staining ECM was expressed as a percentage of the total tissue area and was used as an index of fibrosis. There was a significant difference in ECM accumulation between the rAAV-LacZ group (0.277 ± 0.069) and the HK group (0.209 ± 0.032) (P < 0.01). Together, these results suggest that HK gene delivery attenuates the renal injury associated with diabetes.


Figure 5
View larger version (118K):
[in this window]
[in a new window]

  		FIG. 5. Histological assessment of rat kidneys. Paraffin sections of kidney cortex from nondiabetic rats (A, F, and I) or from diabetic rats treated with rAAV-HK (C, E, H, and K) or rAAV-LacZ (B, D, G, and J) stained with PAS (A–C), Masson’s trichrome (D–H), or Sirius red reagent (I–K). Magnification, x400 (A–C and F–K) or x200 (D and E). PAS staining shows that rAAV-LacZ-treated rats had moderate mesangial cell proliferation (arrow) with severe blood vessel narrowing (downward-pointing arrow) (B), and rAAV-HK-treated rats had minimal glomerulosclerosis (arrow) (C), much milder than rAAV-LacZ-treated diabetic rats (B) and nearly normal blood vessels (A). Masson’s trichrome staining shows renal tubule vacuolation (arrow) in rAAV-LacZ treated rats (D) but almost normal renal tubules (arrow) in rAAV-HK-treated rats (E). Masson’s trichrome staining also showed significant capillary thrombosis (arrow) in rAAV-LacZ-treated diabetic rats (G) but minimal capillary thrombosis (arrow) in rAAV-HK-treated diabetic rats (H). Sirius red staining shows rAAV-LacZ-treated diabetic kidneys with moderate collagen deposition (J) but only mild collagen deposition in kidneys from rAAV-HK-treated diabetic rats (K). One section from each experimental animal was prepared, and representative slices are shown.

 
Kallikrein decreases hyperinsulinemia and insulin resistance
Fasting glucose and insulin levels were measured before rAAV-LacZ and rAAV-HK injections and at the end of the study. Fasting glucose was significantly higher in both the rAAV-HK group and the rAAV-LacZ group compared with the control group at the end of study, indicating that kallikrein expression did not alter this parameter (Table 2Go). However, fasting insulin levels were significantly lower in the rAAV-HK group than the rAAV-LacZ group despite lack of an observed change in fasting glucose (Table 2Go). These findings suggest that less insulin is required to maintain the same glucose level in the HK group than the LacZ group. Consistent with these data, insulin resistance (as measured via HOMA-IR) was significantly decreased in the rAAV-HK group compared with the rAAV-LacZ group (Table 2Go). We also found that HK gene delivery lowers circulating levels of triglycerides and cholesterol, important indicators of cardiovascular risk. Together, these data indicate that HK decreases hyperinsulinemia and insulin resistance and improves lipid profiles in streptozotocin-induced diabetic rats.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Physiological parameters in nondiabetic normal, diabetic-LacZ, and diabetic-HK rats 12 wk after introduction of rAAV-HK and rAAV-LacZ

 
Effects of kallikrein on the PI3-kinase/Akt and AMPK signaling pathways
PI3-kinase is recruited to IRS signaling complexes through binding of Src homology 2 domain in its 85-kDa regulatory subunit to specific phosphotyrosine residues in IRS (28). This process leads to activation of the PI3-kinase p110 catalytic subunit. To begin to investigate the signaling mechanisms through which HK attenuates insulin resistance, we evaluated the expression of PI3-kinase and Akt associated with the insulin signaling cascade in kidney, liver, and skeletal muscle. PI3-kinase p110 catalytic subunit expression was determined by immunoblotting with p110-specific antibodies. Results showed that PI3-kinase p110 catalytic subunit expression was significantly decreased in kidneys from rAAV-LacZ-treated rats compared with nondiabetic controls. In contrast, rAAV-HK treatment increased p110 expression to normal levels (Fig. 6Go, A and B). Similarly, rAAV-HK treatment restored partially PI3-kinase levels in liver and skeletal muscle compared with rAAV-LacZ-treated diabetic rats (Fig. 6Go, C and D).


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 6. Representative Western blots and densitometric quantification of at least three repeats for each experiment showing the effects of rAAV-mediated HK gene therapy on PI3-kinase/Akt and p-AMPK signaling pathways. A–D, Effects of HK gene therapy on the expression of PI3-kinase p110 catalytic subunit in kidney (A and B) and liver and skeletal muscle (C and D); E and F, effects of HK gene therapy on total and p-Akt at Thr-308 in kidney; G and H, effects of HK gene therapy on total and p-AMPK in kidney. Blots were scanned, and relative expression levels were normalized to total ß-actin. Values shown are the means ± SE of three independent experiments. *, P < 0.05 vs. rAAV-LacZ.

 
The expression of Akt, a serine/threonine kinase that lies downstream of PI3-kinase, was also assessed in kidneys of the experimental animals. Because phosphorylation of Akt is required for its activation (29), levels of p-Akt at position Thr-308 were determined by immunoblotting with a phosphospecific anti-Akt antibody. A significant decrease of p-Akt was noted in kidneys of diabetic rats treated with rAAV-LacZ compared with normal control animals, but rAAV-HK treatment restored p-Akt to control levels (Fig. 6Go, E and F). There were no changes in total Akt levels between the groups. Together, these data indicate that reduced insulin resistance in rAAV-HK-treated rats was associated with partial activation of the PI3-kinase/Akt signaling pathway.

We also probed the phosphorylation state of AMPK. We found that AMPK phosphorylation was not down-regulated in our model of type 2 diabetes. However, rAAV-HK treatment enhanced phosphorylation of AMPK, whereas the total level of AMPK was not changed by rAAV-HK treatment (Fig. 6Go, G and H).

Effects of kallikrein on activity of p42/44 MAPK and IR-ß
The p42/44 MAPK are important signaling molecules activated during injury in multiple cells and tissues. We found that in rAAV-LacZ-treated diabetic rats, p-MAPK levels were markedly reduced in kidney relative to control rats. Importantly, rAAV-HK therapy returned p-MAPK to normal levels (Fig. 7Go, A and B). We also investigated IR-ß expression and activity in kidneys of normal and rAAV-LacZ- and rAAV-HK-treated rats. Figure 7Go, C and D, shows that IR-ß expression was comparable in all groups; however, the extent of IR-ß phosphorylation was reduced in rAAV-LacZ-treated diabetic rats. In contrast, rAAV-HK treatment restored IR-ß phosphorylation to normal levels. Together, these results suggest that reversal of diabetic pathology in rAAV-HK-treated rats is associated with activation of both p42/p44 MAPK and IR-ß.


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 7. Representative Western blots and densitometric quantification of at least three experiments showing effects of rAAV-mediated HK gene therapy on phosphorylation of p42/44 MAPK and IR-ß. Results show decreased phosphorylation of MAPK in diabetic kidney is reversed by HK overexpression (A and B) and that decreased phosphorylation of IR-ß in diabetic treated rats is also reversed by rAAV-HK treatment (C and D). Results are representative of three independent experiments. Blots were scanned, and relative p-ERK (B) and p-IR-ß levels (D) were normalized to total ERK and total IR-ß, respectively. Values shown are the means ± SE of three independent experiments. *, P < 0.05 vs. rAAV-LacZ.

 
Kallikrein decreases renal apoptosis in diabetic rats
Tubular and interstitial cell apoptosis is known to occur in streptozotocin-induced diabetic rats (30, 31). Hence, we examined the effects of rAAV-HK treatment on renal apoptosis and expression of apoptosis-related genes in diabetic rats. Consistent with previous literature, we found that the number of apoptotic cells, as assessed by TUNEL staining, in kidneys from rAAV-LacZ-treated diabetic rats was significantly increased compared with control rats (Fig. 8Go, A and B). Apoptotic cells were observed in the renal tubules but not in the glomerulus in diabetic rats. Interestingly, rAAV-HK treatment significantly attenuated this effect (Fig. 8Go, A and B).


Figure 8
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 8. Effects of kallikrein on renal apoptosis. A, TUNEL staining of kidney showing significantly increased TUNEL-positive cells in diabetic rat renal tubules from rAAV-LacZ rats compared with nondiabetic controls and rAAV-HK-treated rats; B, the number of TUNEL-staining apoptotic cells per high-power field (HPF) is quantified: *, P < 0.01 vs. normal control; #, P < 0.01 vs. rAAV-LacZ; C, representative Western blots showing expression of antiapoptotic protein Bcl-2 and proapoptotic protein Bax in kidney; D and E, blots were scanned and Bcl-2 and Bax levels (D and E, respectively) were normalized to ß-actin. Values shown are the means ± SE of three independent experiments. *, P < 0.05 vs. rAAV-LacZ.

 
Apoptosis-related protein levels were assessed using Western blotting. Levels of the antiapoptotic protein Bcl-2 was decreased, whereas levels of the proapoptotic protein Bax was increased in rAAV-LacZ-treated diabetic rats (Fig. 8Go, C–E). Interestingly, rAAV-HK treatment reversed these changes (Fig. 8CGo). Together, these results indicate renal tubular apoptosis is an important component of diabetic nephropathy and that this is attenuated by HK treatment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our previous studies, iv delivery of a plasmid carrying the HK cDNA was found to reduce blood pressure and reverse hyperinsulinemia in fructose-induced hypertensive rats. However, these effects persisted for only 3–4 wk, likely due to the transient nature of the pcDNA3.1-HK expression system. In the current study, we delivered the HK cDNA in a rAAV-based vector to type 2 diabetic rats induced by streptozotocin and high-fat diet. A single injection of rAAV-HK resulted in persistent and stable expression of HK and caused significant reductions in blood pressure and insulin resistance in diabetic rats for up to 12 wk. In contrast, injection of the control rAAV-LacZ did not affect blood pressure or reduce circulating insulin levels in diabetic rats. We also found that HK gene therapy may elicit these changes by altering a number of signal transduction pathways associated with diabetes. Indeed, mechanistic studies revealed an enhancement in the expression of PI3-kinase and increased phosphorylation of Akt suggesting activation of the PI3-kinase/protein kinase B (Akt) pathway in kidney, liver, and skeletal muscle. Moreover, we observed activation of MAPK and enhanced phosphorylation of IR-ß in rAAV-HK-treated rats.

There is considerable controversy as to the relative roles of insulin resistance and insulin secretion in the pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that insulin resistance can be observed in nondiabetic first-degree relatives of patients with type 2 diabetes (32). Normal glucose tolerance is maintained in these subjects because the pancreas is able to secrete sufficient insulin to overcome the insulin resistance, and frank hyperglycemia does not occur until this compensatory response fails. Previous studies have shown that fructose-fed or high-fat-fed rodents become hyperinsulinemic and hypertensive but are still able to maintain normal glucose levels (33, 34), a condition that is similar to the aforementioned prediabetic state in humans. Administration of relatively low doses of streptozotocin to such animals leads to significant hyperglycemia, whereas the same dose does not decrease the insulin secretory capacity enough to cause hyperglycemia in the rodents that were fed with conventional chow. In this study, we used high-fat feed and low-dose streptozotocin injection to induce a diabetic state characterized by hyperglycemia, hyperinsulinemia, and hypertension, a state that closely resembles the metabolic abnormalities of patients with type 2 diabetes.

The majority of individuals with essential hypertension also display insulin resistance and hyperinsulinemia (35). Angiotensin-converting enzyme (ACE) inhibitors are frequently used for the treatment of high blood pressure. ACE inhibitors not only reduce blood pressure but also improve insulin action and peripheral glucose utilization, as shown in both animal studies (36) and clinical investigations (37). ACE inhibitors have at least two effects at the tissue level: 1) inhibition of the conversion of angiotensin I to angiotensin II and 2) enhancement of bradykinin levels through inhibition of kininase II-mediated degradation of this peptide (4). There is evidence to suggest that bradykinin itself may have an effect on the enhanced insulin action and insulin signaling at the skeletal muscle level (38). Tissue kallikrein is a serine protease that converts kininogen to the peptide hormone bradykinin. Considering these data, we hypothesized that HK gene therapy would reduce blood pressure and improve insulin resistance in diabetic rats.

In the present study, we used a rAAV vector that can drive efficient and prolonged transgene expression via persistent episomal concatamers (39, 40) or by genomic integration (41, 42). High levels of HK protein in urine was detected by ELISA at 2 wk after iv injection of rAAV-HK, and this level of HK protein was maintained for up to 12 wk. Moreover, RT-PCR analysis revealed HK mRNA was abundant in heart, lung, liver, skeletal muscle, fat, and kidney from rAAV-HK-treated diabetic rats at 12 wk after injection. This suggests that rAAV mediates stable and long-term expression of the HK gene in numerous target organs in diabetic rats.

The blood pressure in rAAV-HK-injected rats was decreased within 2 wk after initiating therapy. This is consistent with the increased urinary concentrations of HK at 2 wk after injection and suggests that the reduction of blood pressure was caused by the increased HK expression, as we have previously shown (13). In diabetic subjects, hypertension and hyperinsulinemia may be caused by related physiological processes, with insulin resistance being a common feature. In the present study, insulin resistance was attenuated in rats treated with rAAV-HK, although the fasting glucose levels were not significantly reduced compared with control rats.

A previous study (43) demonstrated that bradykinin administration significantly reduced fasting insulin levels by 20%, whereas fasting glucose was not significantly altered. In that study, the glucose areas under the curve was reduced by 21% during an oral glucose tolerance test, which suggests that glucose metabolism was significantly improved. The model used by Henriksen et al. (43) used chronic administration of bradykinin (twice daily for 14 consecutive days at a dose of 40 µg/kg body weight). Not even a very high level of HK gene expression can achieve high kinin levels in vivo. For example, Emanueli et al. (44) used a mouse model of adenovirus-mediated HK delivery (1.8 x 109 plaque-forming units per rat) to induce high-level expression of HK (350 ng/d in urine) and observed only a modest increase in urine kinin levels of 80–100 ng/d. More recently, Montanari et al. (8) reported adenovirus-mediated HK delivery that resulted in a very high expression level of HK, but only a moderate reduction in postprandial blood glucose (575.8 ± 28.3 to 450.8 ± 29.9 mg/dl). Furthermore, Montanari et al. (8) used a type 1 diabetes model that produces very high postprandial blood glucose levels, whereas our study used a type 2 diabetes model with insulin resistance that produces much lower glucose levels (13–15 mmol/liter = 234–270 mg/dl). Comparatively, our rAAV-mediated long-term HK gene delivery system resulted in levels of approximately 2 ng/ml in urine and approximately 1.2 ng/ml in serum. These differences may explain why glucose levels were not reduced by rAAV-HK therapy.

Published data consistent with our findings suggest that kallikrein and bradykinin additively increase adipocyte hexose transport under conditions of maximal intrinsic insulin stimulation, and this action occurs at post-insulin-binding sites (46). Miyata et al. (47) also reported that bradykinin infusion could increase glucose uptake in peripheral tissues. This effect could be explained by the ability of bradykinin to up-regulate insulin receptor tyrosine kinase activity, which stimulates phosphorylation of IRS-1 and increases glucose transporter 4 (GLUT4) translocation. However, this effect was only when found insulin was present and not in the absence of insulin. This could also partially explain why the fasting glucose was not different between rAAV-HK and rAAV-LacZ groups.

A growing body of evidence indicates that defects in the early and intermediate steps of the insulin signaling pathway, including the IRS-1 and PI3-kinase/Akt pathways, contribute to insulin resistance in skeletal muscle of diabetic rodents and humans (38, 48). Data indicate that activation of PI3-kinase is a necessary, albeit not sufficient, step for insulin-induced glucose transport. The present study aimed to determine whether the impairment in PI3-kinase activation in diabetes could be reversed by HK gene therapy. We found that the levels of the PI3-kinase p110 catalytic subunit were significantly decreased in skeletal muscle and liver of diabetic rats and that rAAV-HK treatment corrected this defect. Recent evidence indicates that the serine/threonine protein kinase B (PKB/Akt) mediates many of the downstream events controlled by PI3-kinase. After the activation of PI3-kinase, Akt isoforms are recruited from the cytosol to the plasma membrane through interactions with phosphatidylinositol-3,4,5-trisphosphate and/or phosphatidylinositol-4,5-bisphosphate, where they are thought to undergo a conformational change and become activated by phosphorylation at two residues. Akt isoforms are phosphorylated at the T-loop (Thr-308 in PKB{alpha}) by 3-phosphoinositide-dependent protein kinase 1 (PDK1), and this phosphorylation appears to be crucial for activation of Akt (49). Buren et al. (50) have demonstrated that high glucose levels combined with high insulin produces impaired sensitivity to insulin stimulation of Akt activity. In this regard, we evaluated Akt phosphorylation on Thr-308 in skeletal muscle and liver of diabetic rats. We found that Akt phosphorylation on Thr-308 was decreased in diabetic rats, but this was enhanced by HK gene therapy. These data suggest that the improvement of insulin resistance after HK gene therapy could be caused, at least in part, by increased activation of PI3-kinase and Akt phosphorylation in skeletal muscle and liver. We found that HK gene treatment also enhanced AMPK phosphorylation, which has been shown to ameliorate insulin resistance (51).

Diabetic nephropathy occurs in approximately 30% of all patients with diabetes and has become the leading cause of end-stage renal disease in Western societies; it is also becoming more prevalent in China (52, 53, 54). Diabetic nephropathy is characterized by persistent albuminuria, elevated blood pressure, glomerular hyperfiltration at early stages, and a relentless decline in the glomerular filtration rate at later stages as well as a high risk for cardiovascular morbidity and mortality (55). Diabetic nephropathy involves progressive injury to both the glomerulus and the tubulointerstitium that is accompanied by increased protein excretion and ultimately a decline in renal function (56). In the present study, diabetic rats developed features similar to human diabetic nephropathy, including severe glomerulosclerosis, tubulointerstitial abnormalities, and albuminuria. We also found significant apoptosis in the renal tubules of diabetic rats. HK gene delivery significantly decreased urinary albumin excretion, increased urinary osmolarity, and attenuated renal tubule apoptosis, possibly by modifying expression of apoptosis-related proteins Bcl-2 and Bax. Morphological studies showed that both glomerular sclerotic lesions and tubular damage were reversed by HK gene transfer. These results demonstrate that persistent expression of HK gene protects against nephropathy in diabetic rats.

The mechanisms by which kallikrein prevents renal complications in diabetic rats are poorly understood. Previous studies have shown that bradykinin is able to reduce blood pressure, attenuate endothelial cell apoptosis, increase tissue plasminogen activator and increase matrix metalloproteinases (57). Together, these effects may contribute to the protection of the kidney from injury induced by the diabetic state. MAPK and AMPK activity is essential for cell survival. Our study demonstrated that HK gene delivery significantly enhanced phosphorylation of renal p42/p44 MAPK and AMPK, supporting the notion of a protective role of kallikrein in the kidney (58). The role of bradykinin and related products of the plasma kallikrein-kinin system in diabetic renal damage are currently undefined. Other researchers reported that renal tissue kallikrein and kinin increased in type 1 diabetic patients at risk for developing nephropathy (59). Treatment of diabetic rats with aprotinin, a kallikrein inhibitor, reduced glomerular filtration rate and renal plasma flow, indicating that tissue kallikrein and kinins help to mediate renal hyperfiltration in diabetes (60, 61). Some published data indicated that bradykinin can attenuate vascular disease associated with diabetes when there is an intact endothelium but could aggravate vascular disease when the endothelium is damaged (45, 59, 62). In the present study, HK gene delivery during the early period of diabetes appears to exert several protective effects in the kidney.

In summary, our studies demonstrate that rAAV-mediated HK gene delivery can efficiently attenuate insulin resistance, prevent diabetic nephropathy, and reduce blood pressure in streptozotocin-induced diabetic rats. HK gene therapy may mediate these effects by restoring activation of the insulin receptor, PI3-kinase/Akt, MAPK, and AMPK signaling pathways. These results suggest that rAAV-mediated HK gene administration may be a promising tool for the therapy of insulin resistance.


   Footnotes
 
This work was supported by grants from National 863 Plan project (2001AA217121), 973 project (G2000056901), National Nature Science Foundation Committee grant (30470824), Wuhan City International Collaboration project (997005135G), and funds from the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.

Disclosure Statement: All the authors have nothing to declare.

First Published Online February 1, 2007

1 G.Y. and J.D. contributed equally to this work. Back

Abbreviations: ACE, Angiotensin-converting enzyme; AMPK, AMP-activated protein kinase; ECM, extracellular matrix; HK, human tissue kallikrein; HOMA-IR, homeostasis model assessment for insulin resistance; IR-ß, insulin receptor ß; IRS-1, insulin receptor substrate-1; PAS, periodic acid Schiff; PI3-kinase, phosphatidylinositol 3-kinase; p-MAPK, phosphorylated MAPK; rAAV, recombinant adeno-associated virus; TUNEL, terminal dUTP nick-end labeling.

Received May 5, 2006.

Accepted for publication January 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gu K, Cowie C, Harris M 1998 Mortality in adults with and without diabetes in a national cohort of the U.S. population, 1971–1993. Diabetes Care 21:1138–1145[Abstract]
  2. Yu Y, Suo L, Yu H, Wang C, Tang H 2004 Insulin resistance and endothelial dysfunction in type 2 diabetes patients with or without microalbuminuria. Diabetes Res Clin Pract 65:95–104[CrossRef][Medline]
  3. Martin BC, Warram J, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR 1992 Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 17:925–929
  4. Uehara M, Kishkawa H, Isami S, Kisanuki K, Ohkubo Y, Miyamura N, Miyata T, Yano T, Shichiri M 1994 Effect on insulin sensitivity of angiotensin converting enzyme inhibitors with or without a sulphydryl group: bradykinin may improve insulin resistance in dogs and humans. Diabetologia 37:300–307[Medline]
  5. Isami S, Kishkawa H, Araki E, Uehara M, Kaneko K, Shirotani T, Todaka M, Ura S, Motoyoshi S, Matsumoto K, Miyamura N, Shichiri M 1996 Bradykinin enhances GLUT4 translocation through the increase of insulin receptor tyrosine kinase in primary adipocytes: evidence that bradykinin stimulates the insulin signaling pathway. Diabetologia 39:412–420[Medline]
  6. Tschope C, Reinecke A, Seidl U, Yu M, Gavriluk V, Riester U, Gohlke P, Graf K, Bader M, Hilgenfeldt U, Pesquero JB, Ritz E, Unger T 1999 Functional, biochemical, and molecular investigations of renal kallikrein-kinin system in diabetic rats. Am J Physiol Heart Circ Physiol 277:H2333–H2340
  7. Zhao C, Wang P, Xiao X, Chao J, Chao L, Wang DW, Zeldin DC 2003 Gene therapy with human tissue kallikrein reduces hypertension and hyperinsulinemia in fructose-induced hypertensive rats. Hypertension 42:1026–1033[Abstract/Free Full Text]
  8. Montanari D, Yin H, Dobrzynski E, Agata J, Yoshida Hideaki, Chao J, Chao L 2005 Kallikrein gene delivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats. Diabetes 54:1573–1580[Abstract/Free Full Text]
  9. Xiao X, Li J, Samulski RJ 1998 Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72:2224–2232[Abstract/Free Full Text]
  10. Monahan PE, Samulski RJ 2000 AAV vectors: is clinical success on the horizon? Gene Ther 7:24–30[CrossRef][Medline]
  11. Duan D, Yue Y, Yan Z, Engelhardt JF 2000 A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Nat Med 6:595–598[CrossRef][Medline]
  12. Xiao X, Li J, Samulski R 1996 Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70:8098–8108[Abstract]
  13. Wang T, Li H, Zhao C, Chen C, Li J, Chao J, Chao L, Xiao X, Wang DW 2004 Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries. Gene Ther 3:1038
  14. Auricchio A, Hildinger M, Connor E, Gao GP, Wilson JM 2001 Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Hum Gene Ther 12:71–76[CrossRef][Medline]
  15. Giorgino F, Logoluso F, Davalli A, Napoli R, Laviola L, Hirshman M, Horton E, Weir G, Smith R 1999 Islet transplantation restores normal levels of insulin receptor and substrate tyrosine phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle and myocardium of streptozocin-induced diabetic rats. Diabetes 48:801–812[Abstract]
  16. Jian L, Josephine Q, Joyce T, Christina KH, Cynthia S, Richard H, Gerald MR 1998 Nongenetic mouse models of non-insulin dependent diabetes mellitus. Metabolism 47:663–668[CrossRef][Medline]
  17. Wang J, Xiong W, Yang Z, Davis T, Dewey MJ, Chao J, Chao L 1994 Human tissue kallikrein induces hypotension in transgenic mice. Hypertension 23:236–243[Abstract/Free Full Text]
  18. Srinivasan SR, Frontini MG, Berenson GS 2003 Longitudinal changes in risk variables of insulin resistance syndrome from childhood to young adulthood in offspring of parents with type 2 diabetes: The Bogalusa Heart Study. Metabolism 52:443–450[CrossRef][Medline]
  19. Thule PM, Campbell AG, Kleinhenz DJ, Olson DE, Boutwell JJ, Sutliff RL, Hart CM 2006 Hepatic insulin gene therapy prevents deterioration of vascular function and improves adipocytokine profile in STZ-diabetic rats. Am J Physiol Endocrinol Metab 290:E114–E122
  20. Tomie Furuya D, Binsack R, Onishi ME, Monteiro Seraphim P, Fabres Machado U 2005 Low ethanol consumption induces enhancement of insulin sensitivity in liver of normal rats. Life Sci 77:1813–1824[CrossRef][Medline]
  21. Lee HJ, Choi SS, Park MK, An YJ, Seo SY, Kim MC, Hong SH, Hwang TH, Kang DY, Garber AJ, Kim DK 2002 Fenofibrate lowers abdominal and skeletal adiposity and improves insulin sensitivity in OLETF rats. Biochem Biophys Res Commun 296:293–299[CrossRef][Medline]
  22. Gault MH, Cockcroft DW1975 Letter: creatinine clearance and age. Lancet 2:612–613
  23. Xiong W, Chao J, Chao L 1995 Muscle delivery of human kallikrein gene reduces blood pressure in hypertensive rats. Hypertension 25:715–719[Abstract/Free Full Text]
  24. Guesdon JL, Ternynck T, Avrameas S 1979 The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27:1131–1139[Abstract]
  25. Wang C, Dobrzynski E, Chao J, Chao L 2001 Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats. Am J Physiol Renal Physiol 280:F964–F971
  26. Maejima Y, Adachi S, ItoH, Nobori K, Tamamori-Adachi M, Isobe M 2003 Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity. Cardiovasc Res 59:308–320[Abstract/Free Full Text]
  27. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  28. Virkamaki A, Ueki K, Kahn CR 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943[Medline]
  29. Alessi DR, Cohen P 1998 Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev 8:55–62[CrossRef][Medline]
  30. Kumar D, Zimpelmann J, Robertson S, Burns KD 2004 Tubular and interstitial cell apoptosis in the streptozotocin-diabetic rat kidney. Nephron Exp Nephrol 96:e77–e88
  31. Susztak K, Ciccone E, McCue P, Sharma K, Bottinger EP 2005 Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy. PLoS Med 2:e45
  32. Jackson S, Bagstaff S, Lynn S, Yeaman S, Turnbull D, Walker M 2000 Decreased insulin responsiveness of glucose uptake in cultured human skeletal muscle cells from insulin-resistant nondiabetic relatives of type 2 diabetic families. Diabetes 49:1169–1177[Abstract]
  33. Storlien LH, Pan DA, Kriketos AD, Baur LA 1993 High fat diet-induced insulin resistance. Lessons and implications from animal studies. Ann NY Acad Sci 683:82–90[Medline]
  34. Dai S, McNeill JH 1995 Fructose-induced hypertension in rats is concentration- and duration-dependent. J Pharmacol Toxicol Methods 33:101–107[CrossRef][Medline]
  35. Pollare T 1990 Insulin sensitivity and blood lipids during antihypertensive treatment with special reference to ACE inhibition. J Diabetes Complications 4:75–78[CrossRef]
  36. Jacob S, Henriksen E, Fogt DL, Dietze GJ 1996 Effects of trandolapril and verapamil on glucose transport in insulin-resistant rat skeletal muscle. Metabolism 45:535–541[CrossRef][Medline]
  37. Vuorinen-Markkola H, Yki-Jarvinen H 1995 Antihypertensive therapy with enalapril improves glucose storage and insulin sensitivity in hypertensive patients with non-insulin-dependent diabetes mellitus. Metabolism 44:85–89[CrossRef][Medline]
  38. Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB 1999 Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest 104:733–741[Medline]
  39. Yan Z, Zhang Y, Duan D, Engelhardt JF 2000 Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci USA 97:6716–6721[Abstract/Free Full Text]
  40. Yang J, Zhou W, Zhang Y, Zidon T, Ritchie T, Engelhardt JF 1999 Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination. J Virol 73:9468–9477[Abstract/Free Full Text]
  41. Samulski R, Sally M, Muzyczka N 1999 Adeno-associated viral vectors. In: Friedman T, ed. Development of human gene therapy; 131–172
  42. Wang Z, Ma H, Li J, Sun L, Zhang J, Xiao X 2003 Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther 10:2105–2111[CrossRef][Medline]
  43. Henriksen EJ, Jacob S, Fogt DL, Dietze GJ 1998 Effect of chronic bradykinin administration on insulin action in an animal model of insulin resistance. Am J Physiol 275:R40–R45
  44. Emanueli C, Salis MB, Chao J, Chao L, Agata J, Lin KF, Munao A, Straino S, Minasi A, Capogrossi MC, Madeddu P 2000 Adenovirus-mediated human tissue kallikrein gene delivery inhibits neointima formation induced by interruption of blood flow in mice. Arterioscler Thromb Vasc Biol 20:1459–1466[Abstract/Free Full Text]
  45. Greene EL, Velarde V, Jaffa AA 2000 Role of reactive oxygen species in bradykinin-induced mitogen-activated protein kinase and c-fos induction in vascular cells. Hypertension 35:942–947[Abstract/Free Full Text]
  46. Goldman J, Pfister D, Vukmirovich R 1987 Potentiation of insulin stimulation of hexose transport by kallikrein and bradykinin in isolated rat adipocytes. Mol Cell Endocrinol 53:183–191
  47. Miyata T, Taguchi T, Uehara M, Isami S, Kishikawa H, Kaneko K, Araki E, Shichiri M 1998 Bradykinin potentiates insulin-stimulated glucose uptake and enhances insulin signal through the bradykinin B2 receptor in dog skeletal muscle and rat L6 myoblasts. Eur J Endocrinol 138:344–352[Abstract]
  48. Bjornholm M, Kawano Y, Lehtihet M, Zierath J 1997 Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation. Diabetes 46:524–527[Abstract]
  49. Bevan P 2001 Insulin signaling. J Cell Sci 114:1429–1430[Abstract/Free Full Text]
  50. Buren J, Liu HX, Lauritz J, Eriksson JW 2003 High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes. Eur J Endocrinol 148:157–167[Abstract]
  51. Ido Y, Carling D, Ruderman N 2002 Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells. Inhibition by the AMP-activated protein kinase activation. Diabetes 51:159–167[Abstract/Free Full Text]
  52. Tseng C-H 2004 Mortality and causes of death in a national sample of diabetic patients in Taiwan. Diabetes Care 27:1605–1609[Abstract/Free Full Text]
  53. Mauer M, Drummond K 2002 The early natural history of nephropathy in type 1 diabetes: I. Study design and baseline characteristics of the study participants. Diabetes 51:1572–1579[Abstract/Free Full Text]
  54. Drummond K, Mauer M 2002 The early natural history of nephropathy in type 1 diabetes: II. Early renal structural changes in type 1 diabetes. Diabetes 51:1580–1587[Abstract/Free Full Text]
  55. Thomson SC, Deng A, Bao D, Satriano J, Blantz RC, Vallon V 2001 Ornithine decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes. J Clin Invest 107:217–224[Medline]
  56. Gilbert RE, Cooper ME 1999 The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int 56:1627–1637[CrossRef][Medline]
  57. Yoshida H, Zhang JJ, Chao L, Chao J 2000 Kallikrein gene delivery attenuates myocardial infarction and apoptosis after myocardial ischemia and reperfusion. Hypertension 35:25–31[Abstract/Free Full Text]
  58. Olsen GS, Hansen BF 2002 AMP kinase activation ameliorates insulin resistance induced by free fatty acids in rat skeletal muscle. Am J Physiol Endocrinol Metab 283:E965–E970
  59. Jaffa AA, Durazo-Arvizu R, Zheng D, Lackland DT, Srikanth S, Garvey WT, Schmaier AH 2003 Plasma prekallikrein: a risk marker for hypertension and nephropathy in type 1 diabetes. Diabetes 52:1215–1221[Abstract/Free Full Text]
  60. Jaffa AA, Rust PF, Mayfield RK 1995 Kinin, a mediator of diabetes-induced glomerular hyperfiltration. Diabetes 44:156–160[Abstract]
  61. Harvey JN, Jaffa AA, Margolius HS, Mayfield RK 1990 Renal kallikrein and hemodynamic abnormalities of diabetic kidney. Diabetes 39:299–304[Abstract]
  62. Douillet CD, Velarde V, Christopher JT, Mayfield RK, Trojanowska ME, Jaffa AA 2000 Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-ß and MAPK. Am J Physiol Heart Circ Physiol 279:H2829–H2837




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/5/2016    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission