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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2018 May 30;315(4):F977–F985. doi: 10.1152/ajprenal.00182.2018

Prevention of the progression of renal injury in diabetic rodent models with preexisting renal disease with chronic endothelin A receptor blockade

Denisha Spires 1, Bibek Poudel 1, Corbin A Shields 1, Alyssa Pennington 1, Brianca Fizer 1, Lateia Taylor 1, Kasi C McPherson 1, Denise C Cornelius 1, Jan M Williams 1,✉
PMCID: PMC6230747  PMID: 29846112

Abstract

The endothelin (ET) system has emerged as a therapeutic target for the treatment of diabetic nephropathy (DN). The present study examined whether chronic endothelin A (ETA) receptor blockade with atrasentan prevents the progression of renal injury in two models of DN with preexisting renal disease that exhibit an increased renal ET-1 system compared with nondiabetic rats: streptozotocin-treated Dahl salt-sensitive (STZ-SS) and type 2 diabetic nephropathy (T2DN) rats. Nine week-old SS rats were treated with (STZ; 50 mg/kg ip) to induce diabetes. After 3 wk of diabetes, proteinuria increased to 353 ± 34 mg/day. The rats were then separated into two groups: 1) vehicle and 2) atrasentan (5 mg·kg−1·day−1) via drinking water. After 6 wk of treatment with atrasentan, mean arterial pressure (MAP) and proteinuria decreased by 12 and 40%, respectively, in STZ-SS rats. The degree of glomerulosclerosis and renal fibrosis was significantly reduced in the kidneys of atrasentan-treated STZ-SS rats compared with vehicle STZ-SS rats. Interestingly, treatment with atrasentan did not affect GFR but significantly increased renal blood flow by 33% and prevented the elevations in filtration fraction and renal vascular resistance by 23 and 20%, respectively, in STZ-SS rats. In contrast to the STZ-SS study, atrasentan had no effect on MAP or proteinuria in T2DN rats. However, treatment with atrasentan significantly decreased glomerular injury and renal fibrosis and prevented the decline in renal function in T2DN rats. These data indicate that chronic ETA blockade produces advantageous changes in renal hemodynamics that slow the progression of renal disease and also reduces renal histopathology in the absence of reducing arterial pressure and proteinuria.

Keywords: atrasentan, Dahl S rats, diabetic nephropathy, renal injury, TDN rats, type 1 and type 2 diabetes

INTRODUCTION

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) in the world (22) and is characterized by functional and structural changes, including early glomerular hyperfiltration, which leads to mesangial expansion, thickening of the glomerular basement membrane, and increased proteinuria, eventually progressing to a loss in renal function. While there are effective therapies to delay the progression of DN such as glucose-lowering agents (19, 20, 40), antihypertensive and antidyslipidemic drugs (3, 6, 37), and inhibitors of the renin angiotensin aldosterone system (19, 20), the prevalence of end-stage renal disease continues to rise. Thus there is a critical need to identify novel, alternative approaches to prevent the progression of renal injury in patients with diabetes.

Over the last decade, the endothelin (ET) system has received high attention due to its implication in DN. ET-1 is a potent renal vasoconstrictor (44) that also functions as a strong inflammatory and profibrotic mediator (26, 30–33, 35). Previous studies have demonstrated that ET-1 gene transcription and expression are elevated during DN (9, 10). Moreover, the use of ET receptor antagonists has proven beneficial for the treatment of DN by attenuating the development of renal injury by reducing proteinuria and glomerular and tubular abnormalities (1, 2, 12, 16, 17, 31–35). Endothelin A receptor (ETA) antagonists, in particular, have shown great promise for slowing the progression of chronic kidney disease including DN. However, the beneficial effects of ETA blockade alone remain unclear but may involve their anti-inflammatory and antifibrotic properties (31–33, 35). While selective ETA blockade has not proven to inhibit glomerular hyperfiltration and hyperperfusion observed during the early stages of DN, to our knowledge studies examining the effects of selective ETA antagonists on renal hemodynamics in the later stages of DN are limited. Recently, we reported on two novel models of DN, the 1) type 1 diabetic streptozotocin-treated Dahl salt-sensitive (STZ-SS) rat (36) and 2) type 2 diabetic nephropathy (T2DN) rat (18, 20) that develop many characteristics of DN including glomerular hyperfiltration, mesangial expansion, thickening of the glomerular basement membrane, progressive proteinuria, tubulointerstitial fibrosis, and later decline in glomerular filtration rate (GFR). Therefore, in the current study, we compared the effects of chronic ETA blockade with atrasentan, a selective ETA antagonist, on the progression of renal injury and renal hemodynamics in STZ-SS (type 1 diabetes) and T2DN (type 2 diabetes) rats with preexisting renal disease.

METHODS

General

Experiments were performed on 8-wk-old male SS rats and 12-mo-old T2DN rats. SS and T2DN rats were obtained from our in-house colonies in the Laboratory Animal Facility at the University of Mississippi Medical Center, which is approved by the American Association for the Accreditation of Laboratory Animal Care. Additional experiments were performed on Sprague Dawley (SD) rats that were purchased from Envigo (Indianapolis, IN). SS rats are a well-established model of hypertension-induced renal disease (7, 23–25, 29, 41–43). Recently, we observed that inducing diabetes in SS rats with STZ causes progressive renal injury similar to patients with DN (36). The T2DN rat is a genetic model of DN that was derived by introducing alleles from the Fawn Hooded Hypertensive rat that are predisposed to develop renal injury into the genetic background of the Goto-Kakizaki rat that spontaneously develops type 2 diabetes but is resistant to renal disease (28). The rats had free access to food and water throughout the study. Rats were fed a 1% NaCl diet (TD8640; Harlan Laboratories, Madison, WI) to minimize the development of hypertension. All protocols are approved by the Institutional Animal Care Use Committee of the University of Mississippi Medical Center.

Measurement of Renal ET-1 Levels

To determine whether the renal ET-1 system was elevated in diabetic STZ-SS and T2DN rats compared with nondiabetic control rats we anesthetized rats with isoflurane and harvested kidneys at the appropriate time period 1) SS and STZ-SS after 9 wk of diabetes and 2) SD and T2DN rats at 12 mo of age. Kidneys were homogenized in 1 ml of RIPA buffer (Sigma, St. Louis, MO) for measurement of the expression of ET-1 levels. The homogenates were centrifuged at 5,000 g for 5 min and 11,000 g for 15 min, and the supernatant was collected and frozen in liquid N2 and stored at −80°C. The levels of ET-1 in the kidney homogenates were measured by using an ET-1 ELISA kit (R&D Systems, Minneapolis, MN). The protein concentration of the kidney homogenates was measured using a protein assay with γ-globulin standards (Bio-Rad Laboratories, Hercules, CA), and the data are expressed as nanograms per milligrams of protein.

Study 1: Type 1 Diabetes-Induced Renal Injury. Comparison of Time Courses Changes in Mean Arterial Pressure and Proteinuria in STZ-SS Rats Chronically Treated with ETA Antagonist Atrasentan

Experiments were performed on 8 wk-old SS rats that were placed in restrainers for 3 consecutive days for 15–30 min to become acclimated for the measurement of arterial pressure by tail-cuff plethysmography (MC4000 BP Analysis System; Hatteras Instruments, Cary, NC). Mean arterial pressure (MAP) was recorded at 9 wk of age to obtain a baseline arterial pressure measurement. The rats were placed in metabolic cages for a 24-h urine collection to determine baseline protein (Bradford method; Bio-Rad) excretion. Blood samples were also taken from the tail for the measurement of blood glucose levels. Then, the rats were injected with streptozotocin (STZ; 50 mg/kg ip) to induce diabetes and given one long-acting insulin implant (low dose, 2 U/day sc, recombinant human insulin; Linshin, Ontario, Canada) to maintain blood glucose levels between 400 and 600 mg/dl. After 3 wk of diabetes (3 wk post-STZ injection), rats were divided into two treatment groups 1): vehicle (drinking water) and 2) ETA receptor antagonist, atrasentan (5 mg·kg−1·day−1; AbbVie, Abbott Park, IL), and rats were treated for 6 wk. MAP, protein excretion, and blood glucose were measured every 3 wk until 9 wk of diabetes was completed.

Clearance experiments.

An additional group of 9-wk-old SS rats were studied to obtain baseline renal function before the induction of diabetes. At the end of the treatment, rats were prepared for the measurement of renal function. On the day of the experiment, the rats were anesthetized with ketamine (30 mg/kg im; Phoenix Pharmaceutica, St. Joseph, MO) and Inactin (50 mg/kg ip; Sigma) and catheters were placed in the femoral artery and vein for the measurement of arterial pressure and the infusion of a 2% BSA solution containing FITC-labeled inulin (2 mg/m; Sigma) in a 0.9% NaCl solution at a rate of 6 ml/hr. Renal blood flow (RBF) was measured using a flowmeter (Transonic System, Ithaca, NY) and GFR was measured from the clearance of FITC-labeled inulin. After a 30-min equilibration, urine and plasma samples were collected during a 30-min collection period. At the end of the experiment, the left kidney was removed and weighed and the concentrations of inulin in the urine and plasma samples were determined with a microplate fluorometer (Bio Tek Instruments, Winooski, VT). Filtration fraction (FF) and renal vascular resistance (RVR) were calculated from the RBF and GFR measurements.

Assessment of renal injury.

After the completion of the clearance experiments, the kidneys were collected, weighed, and fixed in a 10% buffered formalin solution. Paraffin sections (3 μm) were prepared and stained with periodic acid-Schiff and Masson’s trichrome to assess the degree of glomerular injury and renal fibrosis, respectively, on ~30 images per section per rat. Thirty glomeruli per section were scored in a blinded fashion on a 0–4 scale with 0 representing a normal glomerulus, 1 representing a 25% of loss, 2 representing a 50% loss, 3 representing a 75% loss, and 4 representing >75% loss of capillaries in the tuft. Additional analysis was performed to determine the degree of renal fibrosis. Images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fi1 color camera (Nikon, Melville, NY) and analyzed for the percentage of the image stained blue (primarily collagen) in the Mason trichrome-stained sections by using the NIS-Elements D 3.0 software. Fifteen to twenty representative fields were analyzed per section.

Study 2: T2DN-Induced Renal Injury. Comparison of Time Courses Changes in MAP and Proteinuria in T2DN Rats Chronically Treated With Atrasentan

Experiments were performed on 12-mo-old T2DN rats. The rats were trained for tail-cuff plethysmography as described above. After the baseline measurement of MAP, rats were placed in metabolic cages for a 24-h urine collection to determine and blood samples were also taken from the tail for the measurement of glucose levels. After baseline measurements, the rats were separated into two groups 1) vehicle (drinking water) and 2) atrasentan. The rats were treated for 6 wk with atrasentan and MAP, protein excretion, and blood glucose levels were measured every 2 wk. At the end of the study, the rats were prepared for clearance experiments and the kidneys were collected to assess renal injury as previously described above.

Statistical Analysis

Mean values ± SE are presented. The significance of differences in control and experimental values within the same animal were determined by a paired t-test. The significance of differences in the mean values between groups was determined by either one-way or two-way repeated measures ANOVA followed by Holm-Sidek test. P < 0.05 was considered to be significant.

RESULTS

Comparison of Renal ET-1 Levels in Dahl SS- and STZ-Treated SS Rats

The measurement of renal ET-1 levels in SS and STZ-SS rats is presented in Fig. 1. ET-1 levels were twofold higher from the kidneys in diabetic STZ-SS rats compared with the values measured in nondiabetic SS rats (426 ± 61 and 197 ± 36 ng/mg of protein, respectively).

Fig. 1.

Fig. 1.

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Comparison of renal endothelin-1 (ET-1) levels in Dahl salt-sensitive (SS) and streptozotocin (STZ)-treated SS rats. Numbers in parentheses indicate the number of rats studied per group. Values are presented as means ± SE. †Significant difference from the corresponding value in SS rats.

Study 1: Effects of Atrasentan on the Progression of Renal Injury in STZ-SS Rats With Preexisting Renal Disease

Temporal changes in arterial pressure and protein excretion.

The effects of atrasentan on MAP and protein excretion in STZ-SS rats are presented in Fig. 2. After injection of STZ, blood glucose levels increased from 89 ± 3 to 524 ± 35 mg/dl in both groups and remained elevated throughout the protocol (data not shown). During the study, vehicle- and atrasentan-treated STZ-SS rats did not experience any weight loss after 9 wk of diabetes (322 ± 18 to 334 ± 11 and 321 ± 19 to 330 ± 10 g, respectively) (data not shown). As expected, MAP increased similarly in both groups after SS rats were given STZ (140 ± 4 and 141 ± 6 to 160 ± 6 and 163 ± 5 mmHg; Fig. 2A). However, treatment with atrasentan significantly prevented the rise in arterial pressure by the end of the study compared with rats treated with vehicle (159 ± 6 and 181 ± 8 mmHg, respectively). After the induction of diabetes with STZ, protein excretion increased by more than threefold in both groups (108 ± 6 and 107 ± 7 to 356 ± 41 and 353 ± 35 mg/day; Fig. 2B). At the end of the study, chronic treatment with atrasentan markedly reduced protein excretion by 43% compared with the values measured in vehicle-treated rats (404 ± 42 and 668 ± 81 mg/day, respectively).

Fig. 2.

Fig. 2.

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Comparison of time course measurements of tail-cuff arterial pressure (TAP; A) and protein excretion (B) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats that received either vehicle or atrasentan (5 mg·kg−1·day−1). Numbers in parentheses indicate the number of rats studied per group. Values are presented as means ± SE. *Significant difference from the corresponding value within the group at baseline. †Significant difference from the corresponding value in vehicle SS rats.

Measurement of renal hemodynamics.

The effects of chronic treatment with atrasentan on RBF, GFR, FF, and RVR in STZ-SS rats are presented in Fig. 3. While rats were under anesthesia, MAP was significantly lower compared with the values measured in vehicle- and atrasentan-treated STZ-SS rats by tail-cuff. MAP was significantly reduced in STZ-SS rats vs. SS rats (118 ± 4 and 132 ± 6 mmHg, respectively) (data not shown). We observed a tendency for RBF to decrease in the vehicle-treated group compared with baseline SS rats (Fig. 3A). Chronic treatment with atrasentan prevented the decreased trend in RBF observed in vehicle STZ-SS rats. Surprisingly, we did not detect any significant changes in GFR among the groups (Fig. 3B). However, GFR was elevated in both diabetic groups, vehicle and atrasentan (1,430 ± 149 vs. 1,818 ± 220 and 1,925 ± 234 µl/min, respectively). FF (Fig. 3C) and RVR (Fig. 3D) were significantly increased in vehicle-treated STZ-SS rats compared with baseline SS rats and treatment with atrasentan prevented the increase in both FF and RVR.

Fig. 3.

Fig. 3.

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Effects of the treatment of atrasentan (5 mg·kg−1·day−1) on renal blood flow (RBF, ml/min; A), glomerular filtration rate (GFR, μl/min; B), filtration fraction (FF, %), (Panel C) and renal vascular resistance (RVR; mmHg·ml−1·min−1·g kidney wt−1; D) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats. *Significant difference from the corresponding value in baseline untreated 9-wk-old SS rats. †Significant difference from the corresponding value in vehicle SS rats.

Assessment of renal histopathology.

The effects of atrasentan on the degree of renal injury in STZ-SS rats are presented in Fig. 4. The kidneys from vehicle-treated rats displayed mesangial expansion and severe glomerulosclerosis (Fig. 4A). When examining renal fibrosis (the percentage of blue staining), we observed a significant amount of renal fibrosis in the renal cortex of vehicle-treated STZ-SS rats (Fig. 4, C and F). The glomerular injury score in vehicle-treated STZ-SS rats averaged 2.5 indicating that nearly 63% of the glomerular capillary area available for filtration was lost (Fig. 4E). However, treatment with atrasentan significantly reduced mesangial expansion, glomerular injury, and renal fibrosis (Fig. 4, B and D–F).

Fig. 4.

Fig. 4.

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Comparison of renal histology: periodic acid-schiff (PAS) staining (A and B), Masson’s Trichrome staining (C and D), glomerular injury score (E), and renal fibrosis (F) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats that received either vehicle or atrasentan (5 mg·kg−1·day−1). Numbers in parentheses indicate the number of rats/glomeruli studied per group for glomerular injury score and the number of rats/images studied per group for renal fibrosis. Values are presented as means ± SE. †Significant difference from the corresponding value in vehicle SS rats.

Comparison of Renal ET-1 Levels in SD and T2DN Rats

The measurement of renal ET-1 levels in SD and T2DN rats is presented in Fig. 5. ET-1 levels were markedly elevated from the kidneys in T2DN rats versus the values observed in nondiabetic SD rats (397 ± 81 and 118 ± 15 ng/mg of protein, respectively).

Fig. 5.

Fig. 5.

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Comparison of renal ET-1 levels in Sprague Dawley (SD) and type 2 diabetic nephropathy (T2DN) rats. Numbers in bars indicate the number of rats studied per group. Values are presented as means ± SE. †Significant difference from the corresponding value in SD rats.

Study 2: Effects of Atrasentan on the Progression of Renal Injury in T2DN Rats With Preexisting Renal Disease

Temporal changes in arterial pressure and protein excretion.

The effects of atrasentan on MAP and protein excretion in T2DN rats are presented in Fig. 6. At baseline, blood glucose levels averaged 315 ± 35 mg/dl in both groups of T2DN rats and we did not observe any differences between the groups during treatment with atrasentan (data shown). Similarly, we did not detect any differences in MAP between the groups at baseline or during chronic atrasentan treatment (Fig. 6A). Protein excretion was not different between both, vehicle and atrasentan groups, at baseline (272 ± 25 and 270 ± 33 mg/day, respectively), and we did not observe any differences after 6 wk of atrasentan (428 ± 73 and 413 ± 68 mg/day, respectively) (Fig. 6B).

Fig. 6.

Fig. 6.

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Comparison of time course measurements of tail-cuff arterial pressure (TAP; A) and protein excretion (B) in type 2 Diabetic Nephropathy (T2DN) rats that received either vehicle or atrasentan (5 mg·kg−1·day−1). Numbers in bars indicate the number of rats studied per group. Values are presented as means ± SE. *Significant difference from the corresponding value within the group at baseline.

Measurement of renal hemodynamics.

The effects of chronic treatment with atrasentan on RBF, GFR, FF, and RVR in T2DN rats with preexisting renal injury are presented Fig. 7. In contrast to STZ-SS rats, we did not observe an overall decrease in arterial pressure in T2DN rats by tail-cuff vs. under anesthesia. MAP was similar in both vehicle- and atrasentan-treated T2DN rats (159 ± 5 and 153 ± 5 mmHg, respectively) (data not shown). We observed a marked decline in RBF in the vehicle-treated T2DN rats compared with the values measured in baseline 12-mo-old T2DN rats (5.9 ± 0.4 vs. 11.9 ± 0.5 ml/min, respectively; Fig. 7A). Treatment with atrasentan for 6 wk prevented the reduction in RBF observed in vehicle-treated T2DN rats (8.3 ± 1.0 ml/min). We observed similar results when measuring GFR. GFR decreased by more than 50% in vehicle-treated rats compared with baseline rats (736 ± 69 vs. 1,554 ± 68 µl/min, respectively), and chronic ETA blockade with atrasentan normalized GFR (1,505 ± 113 µl/min) (Fig. 7B). FF was similar between baseline and vehicle-treated T2DN rats (Fig. 7C). However, treatment with atrasentan significantly increased FF by 57% when compared with vehicle-treated T2DN rats. RVR increased by more than twofold in vehicle T2DN rats compared with the baseline group (64 ± 4 vs. 23 ± 2 mmHg·ml−1·min−1·g−1, respectively; Fig. 7D), and atrasentan treatment significantly decreased RVR in T2DN rats with preexisting renal disease.

Fig. 7.

Fig. 7.

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Effects of the treatment of atrasentan (5 mg·kg−1·day−1) on renal blood flow (RBF, ml/min; A), glomerular filtration rate (GFR, μl/min; B), filtration fraction (FF, %; C) and renal vascular resistance (RVR, mmHg·ml−1·min−1·g kidney wt−1; D) in type 2 diabetic nephropathy (T2DN) rats. Numbers in parentheses indicate the number of rats studied per group. Values are presented as means ± SE. *Significant difference from the corresponding value in baseline T2DN rats. †Significant difference from the corresponding value in vehicle-treated T2DN rats.

Assessment of renal histopathology.

The effects of atrasentan on the degree of renal injury in T2DN rats are presented in Fig. 8. The kidneys from vehicle-treated T2DN rats exhibited prominent mesangial expansion (Fig. 8A), glomerulosclerosis (Fig. 8A), and renal fibrosis (Fig. 8C). In contrast, kidneys from T2DN rats treated with atrasentan displayed reduced expansion of the mesangial matrix and glomerular injury (Fig. 8B) and decreased renal fibrosis (Fig. 8D). Glomerular injury scores and renal fibrosis (%blue staining) were markedly reduced in T2DN rats chronically treated with atrasentan (Fig. 8, E and F).

Fig. 8.

Fig. 8.

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Comparison of renal histology: periodic acid-schiff (PAS) staining (A and B) and Masson’s trichrome staining (C and D), glomerular injury score (Panel E) and renal fibrosis (F) in type 2 diabetic nephropathy (T2DN) rats that received either vehicle or atrasentan (5 mg·kg−1·day−1). Numbers in parentheses indicate the number of rats, glomeruli studied per group for glomerular injury score and the number of rats, images studied per group for renal fibrosis. Values are presented as means ± SE. †Significant difference from the corresponding value in vehicle SS rats.

DISCUSSION

Preclinical studies have demonstrated that chronic blockade of ETA reduces proteinuria and renal injury in models of diabetes independent of decreases in arterial pressure by preserving the glomerular permeability barrier (31–33). While the protective effect of ETA blockade on the preservation of the glomerular permeability barrier is not due to the prevention of renal hyperfiltration observed in the early stages of DN, the effects of ETA blockade on renal hemodynamics during the later stages of DN have not been thoroughly examined. Therefore, in the current study, we examined the effects of chronic ETA blockade with atrasentan on the progression of renal injury and renal hemodynamics during the later stages of DN in STZ-SS (type 1 diabetes) and T2DN (type 2 diabetes) rats with preexisting renal disease. We observed that both models, STZ-SS and T2DN rats, have increased renal ET-1 levels during the progression of renal injury associated with diabetes. In STZ-SS rats, treatment with atrasentan significantly prevented the rise in arterial pressure and proteinuria and reduced glomerular injury and renal fibrosis. While we did not observe any differences in GFR after atrasentan treatment in STZ-SS rats, chronic ETA blockade significantly increased RBF and prevented the elevations in RVR and FF. In contrast to STZ-SS rats, treatment with atrasentan did not have any effects on arterial pressure or the progression of proteinuria in T2DN rats. However, chronic ETA blockade significantly reduced glomerular injury and renal fibrosis and prevented the decline in renal function in T2DN rats.

ET is an endothelial cell-derived peptide that binds to the ETA receptor on vascular smooth muscle to stimulate vasoconstriction and elevations in arterial pressure (13–16). Therefore, preventing the increase in arterial pressure in response to chronic ETA receptor blockade in STZ-SS rats was not that surprising. More recently, Jin et al. (12) observed that chronic ETA blockade with atrasentan markedly attenuated the development of hypertension in diabetic SS rats. The decrease in arterial pressure in STZ-SS rats in response to atrasentan may be attributed to the lack of ETA-stimulated vasoconstriction and increased ETB-mediated vasodilation. While we observed reductions in arterial pressure in STZ-SS rats, chronic ETA blockade had no effect on arterial pressure in T2DN rats, which was rather unexpected. Our laboratory recently reported that T2DN rats are sensitive to treatment with angiotensin-converting enzyme inhibitors causing a decrease in arterial pressure (20). Furthermore, previous studies have demonstrated angiotensin II stimulates intrarenal ET-1 synthesis (5), and ET-1 is considered to be an amplifier of the pressor effects of angiotensin II via activation of the ETA receptor (8). Then again, the arterial pressure-lowering effect of an ETA antagonist during DN may occur only in the presence of elevations in arterial pressure or hypertension (>20–30 mmHg). In the current study, we observed a mild increase in arterial pressure (≈10 mmHg) in T2DN rats throughout the study. These results confirm previous findings that SS rats are sensitive to chronic ETA blockade and indicate that further studies are needed to investigate the role of ET-1 in arterial pressure regulation in T2DN rats.

One of the major therapeutic effects of ETA antagonists is preventing glomerular leakage to albumin and decreasing proteinuria during DN. Sasser et al. (35) observed that ETA blockade markedly reduced albuminuria along with arterial pressure in STZ-induced diabetes. Similarly, treatment with atrasentan attenuated the development of hypertension and significantly decreased proteinuria in diabetic SS-fed high-salt/high-fat diet (12). In the current study, we observed that the atrasentan-mediated decrease in proteinuria was associated with a 20-mmHg decline in arterial pressure in STZ-SS rats. These studies indicate that chronic ETA blockade slows the development of proteinuria in an arterial pressure-dependent manner. However, other preclinical studies have demonstrated that treatment with selective ETA antagonists prevent the development and progression of proteinuria independent of lowering arterial pressure (4, 13, 14, 27, 31). Interestingly, chronic ETA blockade did not reduce the progression of proteinuria in T2DN rats with preexisting renal disease. One possible explanation for the lack of an effect of ETA blockade on the progression of proteinuria in T2DN rats is the decline in renal function in vehicle-treated T2DN rats preventing any further increase in proteinuria. Therefore, a reduction in proteinuria cannot be observed in atrasentan-treated T2DN rats. While we did not observe a decrease in proteinuria in T2DN rats, chronic ETA blockade reduced glomerular injury and renal fibrosis. Saleh et al. (31) recently demonstrated that treatment with a selective ETA antagonist restored the glomerular filtration barrier and had anti-inflammatory and antifibrotic properties independent of lowering arterial pressure indicating that the ETB receptor has renoprotective properties. Collectively, these data suggest that chronic ETA blockade prevents the progression of proteinuria and renal injury in an arterial pressure-dependent manner but also has the capabilities of reducing renal injury and inhibiting a decline in renal function independent of lowering arterial pressure and proteinuria.

One of the well-known characteristics of DN is the elevation of GFR (renal hyperfiltration) during the early stages followed by a reduction in GFR during the later stages of this devastating disease (21, 38, 39, 45). Most of the studies investigating the effects of chronic ETA blockade have been performed during the early stages of DN. However, studies examining the impact of chronic ETA blockade on renal function during the later stages of DN are limited. In the current study, there was a twofold increase in GFR after 6 wk of hyperglycemia compared with the values measured at baseline, indicating that the STZ-SS rats developed renal hyperfiltration. This may be due to the hyperglycemia-mediated stimulation of the renin angiotensin aldosterone system to constrict the efferent arteriole, since we found that FF was elevated after 9 wk of hyperglycemia in STZ-SS rats compared with baseline SS rats. Chronic ETA blockade with atrasentan had no overall effect on GFR but did inhibit the increase in RVR by possibly shifting ET-1 to bind to ETB on the efferent arteriole stimulating dilation in STZ-SS rats, since FF was significantly decreased. Inscho et al. (11) observed similar results when specifically stimulating ETB on efferent arterioles dose dependently with sarafotoxin. In T2DN rats, we observed a marked decline in RBF and GFR with a substantial increase in RVR in vehicle-treated T2DN rats compared with the values measured in baseline T2DN rats. Treatment with atrasentan prevented the decline in GFR by decreasing RVR and increasing RBF in T2DN rats indicating chronic ETA blockade prevented the fall in GFR by perhaps causing afferent arteriole dilation, since atrasentan significantly increased FF in T2DN rats.

In conclusion, treatment with atrasentan reduced proteinuria and renal injury in an arterial pressure-lowering manner in STZ-SS rats. However, in T2DN rats, chronic ETA blockade had no effect on arterial pressure and proteinuria but prevented the decline in GFR and markedly decreased renal histopathological abnormalities. When examining the effects of selective ETA blockade on renal hemodynamics in these two models of progressive renal disease, we observed that treatment with atrasentan had no overall effect on GFR but did inhibit the increase in RVR by possibly stimulating efferent arteriole dilation in type-1 diabetic STZ-SS rats. In T2DN rats, chronic ETA blockade prevented the fall in GFR by triggering afferent arteriole dilation. The difference in the effects of atrasentan on the progression of renal injury between these two models may be due to either the type of diabetes or the severity of DN. In the current study, we used older T2DN rats (12 mo of age) that are genetically susceptible to develop insulin resistance-induced type 2 diabetes and renal disease (proteinuria) by 12 wk of age (28), whereas SS rats were injected with STZ to produce type-1 diabetes and developed renal injury over a span of only 9 wk. Overall, these data suggest that treatment with this class of drugs could yield advantageous changes in renal hemodynamics that slow the progression of renal disease during DN. Moreover, to our knowledge, this is the first study to demonstrate that chronic ETA blockade reduces renal histopathology in the absence of reducing arterial pressure and proteinuria.

GRANTS

This work was financially supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant 1F31-DK-109571 (to K. C. McPherson); National Heart, Lung, and Blood Institute Grant HL-130456 and National Institute of General Medical Sciences (NIGMS) Obesity, Cardiorenal and Metabolic Diseases–COBRE Grant P20-GM-104357 (to D. C. Cornelius), NIGMS GM-115428 (to Michael J. Ryan of the Department of Physiology at the University of Mississippi Medical Center for summer undergraduate students; to A. Pennington and B. Fizer), and NIGMS Grant P20-GM-104357 and NIDDK Grant DK-109133 (to J. M. Williams).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

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

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