Renoprotective effects of empagliflozin in type 1 and type 2 models of diabetic nephropathy superimposed with hypertension

Abstract

Diabetes, hypertension, and aging are major contributors to cardiovascular and chronic kidney disease (CKD). Sodium/glucose cotransporter 2 (SGLT2) inhibitors have become a preferred treatment for type II diabetic patients since they have cardiorenal protective effects. However, most elderly diabetic patients also have hypertension, and the effects of SGLT2 inhibitors have not been studied in hypertensive diabetic patients or animal models. The present study examined if controlling hyperglycemia with empagliflozin, or given in combination with lisinopril, slows the progression of renal injury in hypertensive diabetic rats. Studies were performed using hypertensive streptozotocin-induced type 1 diabetic Dahl salt-sensitive (STZ-SS) rats and in deoxycorticosterone-salt hypertensive type 2 diabetic nephropathy (T2DN) rats. Administration of empagliflozin alone or in combination with lisinopril reduced blood glucose, proteinuria, glomerular injury, and renal fibrosis in STZ-SS rats without altering renal blood flow (RBF) or glomerular filtration rate (GFR). Blood pressure and renal hypertrophy were also reduced in rats treated with empagliflozin and lisinopril. Administration of empagliflozin alone or in combination with lisinopril lowered blood glucose, glomerulosclerosis, and renal fibrosis but had no effect on blood pressure, kidney weight, or proteinuria in hypertensive T2DN rats. RBF was not altered in any of the treatment groups, and GFR was elevated in empagliflozin-treated hypertensive T2DN rats. These results indicate that empagliflozin is highly effective in controlling blood glucose levels and slows the progression of renal injury in both hypertensive type 1 and type 2 diabetic rats, especially when given in combination with lisinopril to lower blood pressure.

Introduction

Diabetes is a major public health concern that contributes to cardiovascular and chronic kidney disease (CKD). In 2019, 37 million patients, or 11.3% of the population, have diabetes. This percentage increases to 29% of the population > 65 years old [1]. Diabetic nephropathy (DN) develops in about 45% of the patients. It is characterized by renal hypertrophy and glomerular hyperfiltration that lead to mesangial expansion, podocyte effacement, progressive proteinuria, and loss of renal function [2]. Current therapy that includes glycemic control in combination with ACE inhibitors or angiotensin II receptor blockers slows but does not prevent the progression of CKD [3, 4]. Consequently, the number of patients with CKD and ESRD is still escalating. In 2021, 37 million American adults now have CKD, which accounts for 38% of people > 65 years old [5]. The risk for CKD increases with age and hypertension, especially in diabetic patients. Currently, 74% of diabetic patients > 18 years of age are hypertensive, versus 30% in the general population [6]. Hypertension has multiplicative detrimental effects on the progression of cardiovascular disease and DN [6]. In addition, the incidence of hypertension, even in nondiabetic patients, has become endemic with aging in the USA and now afflicts 60% of the population at midlife and 75% of those > 70 years old [7]. The Medicare costs for CKD in patients > 66 years of age were $70 billion in 2018, or 23.8% of Medicare spending in this age group [5]. Thus, there is a critical need for novel and alternative therapies that can slow and possibly reverse renal injury, especially in diabetic patients with hypertension.

The sodium-glucose cotransporter 2 (SGLT2) in the proximal tubule [8–11] plays a critical role in glucose homeostasis by reabsorbing 90% of the filtered load of glucose [12]. The expression of SGLT2 is increased in both type 1 and 2 diabetic patients [13] and animal models [9, 14] and plays an essential role in maintaining hyperglycemia [14]. SGLT2 inhibitors that lower plasma glucose levels by blocking glucose reabsorption in the proximal tubule have entered clinical practice for the treatment of hyperglycemia in type 2 diabetes [3, 15]. Clinical trials have indicated that SGLT2 inhibitors protect from major cardiovascular adverse events, including stroke, myocardial infarction, angina, and death [16–20]. In the EMPA-REG OUTCOME Trial, empagliflozin was also found to have some renoprotective effects in type 2 diabetic patients. It reduced the onset of albuminuria > 300 mg/g creatinine from 19 to 13%, the doubling of serum creatinine levels from 2.6 to 1.5%, and development of renal failure from 0.6 to 0.3% [4]. Similar renal protective effects have been reported using other SGLT2 inhibitors [3, 19, 21, 22]. However, the underlying mechanisms for renal protection have yet to be defined [9, 15, 23]. In addition, the previous cardiovascular outcome studies were prevention studies using relative healthy normotensive patients. The renoprotective effects observed were significant but not that impressive given the short observation period. Studies specifically examining the impact of SGLT2 inhibitors on renal injury in diabetic patients and animal models with hypertension are limited [3, 15, 24], even though the majority DN patients develop moderate to severe hypertension as they age [2, 5, 6].

We previously observed that treatment with an SGLT2 inhibitor, luseogliflozin, reduced renal injury in streptozotocin-induced type 1 diabetic Dahl (STZ-SS) and type 2 diabetic nephropathy (T2DN) rats without altering blood pressure [25, 26]. These two rat models develop most of the characteristics of DN, including glomerular hyperfiltration, mesangial expansion, thickening of the glomerular basement membrane, Kimmelsteil-Wilson nodules, tubulointerstitial fibrosis, progressive proteinuria, and a fall in glomerular filtration rate (GFR) [25–27]. These results are consistent with a study by Gembardt et al. demonstrating that treatment with empagliflozin reduced blood glucose levels and albuminuria in control and angiotensin II hypertensive BTBR ob/ob mice independent of changes in arterial pressure, but it could not reduce renal hypertrophy, inflammation, or renal fibrosis in the hypertensive diabetic animals [28]. Other studies, however, suggest that the renoprotective effects of SGLT2 inhibitors are often linked to reductions in blood pressure [29]. For example, canagliflozin lowered systolic blood pressure (SBP) in type 2 diabetic patients with CKD in the CREDENCE trial [22]. Vallon and colleagues observed chronic treatment with an SGLT2 inhibitor reduced SBP, glomerular hyperfiltration, renal hypertrophy, and albuminuria in diabetic Akita mice [30]. In the SACRA trial, empagliflozin significantly reduced SBP without altering glucose and HbA1c levels in patients with well-controlled hyperglycemia but uncontrolled nocturnal hypertension [31].

Thus, it remains to be determined to what extent the renal protection afforded by SGLT2 inhibitors is due to their hypoglycemic, antihypertensive, or renal hemodynamic effects. The question as to whether SGLT2 inhibitors can preserve renal function in diabetic patients or animal models with hypertension is unknown. Therefore, in the present study, we studied the long-term effects of the SGLT2 inhibitor, empagliflozin, given alone and in combination with the ACE inhibitor lisinopril, on the progression of renal injury in hypertensive STZ-SS rats (type 1 diabetes) and in T2DN rats with deoxycorticosterone acetate (DOCA)-salt hypertension.

Methods

General

Experiments were performed on 57 8-week-old male Dahl SS and 55 6-month-old male T2DN rats that were obtained from 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. The rats were maintained on a low-salt chow diet (Teklad 7034, 0.1% sodium chloride diet, Envigo, Indianapolis, IN) and had free access to food and water throughout the study. All protocols were approved by the Animal Care Committee of the University of Mississippi Medical Center.

Protocol 1. Effects of control of hyperglycemia using empagliflozin on the progression of renal injury in hypertensive type 1 diabetic STZ-SS rats

Experiments were performed on 8-week-old male SS rats that were placed in restrainers for 15–30 min on 3 consecutive days to become acclimated for the measurement of blood pressure by tail-cuff plethysmography (MC4000 Blood Pressure Analysis System; Hatteras Instruments, Cary, NC). Systolic blood pressure (SBP) was then recorded at 9 weeks of age to obtain a baseline measurement. The rats were then placed in metabolic cages to collect an overnight urine sample. Protein excretion was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Blood was collected from the tail vein to measure baseline blood glucose levels using a glucometer (Bayer HealthCare, Mishawaka, IN) and glycosylated hemoglobin (HbA1c; Bayer HealthCare, Sunnyvale, CA) levels. After collecting the baseline data, the rats were injected with STZ (50 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO) and switched to a high salt diet (HS) containing 4% NaCl (Teklad traditional diet TD. 92,034, Envigo) to induce type I diabetes and hypertension. Three days after the STZ injection, blood glucose levels were measured to confirm the onset of diabetes. Then, a low-dose silastic insulin pellet (LinShin Canada, Ontario, Canada) was implanted subcutaneously to maintain blood glucose levels between 300 and 500 mg/dL to prevent muscle wasting. The STZ-SS rats were randomly assigned to 5 groups. (1) Vehicle: rats were given tap water to drink. (2) Insulin: these rats received an additional insulin implant to normalize blood glucose levels. (3) Lisinopril: these rats received the ACE inhibitor lisinopril (10 mg/kg/day) in the drinking water. (4) Empagliflozin: these rats received the SGLT2 inhibitor empagliflozin (20 mg/kg/day) in the drinking water (Boehringer Ingelheim, Germany). (5) Combination therapy: these rats received both empagliflozin and lisinopril in the drinking water. Food and water intake, body weight, protein excretion, blood glucose, and HbA1C levels were measured every 3 weeks for 9 weeks. SBP was measured once a week after placing the rats in the tail-cuff device for 15–30 min on 3 consecutive days to become reacclimated.

Measurement of renal hemodynamics

At the end of the chronic study, the rats were prepared for clearance studies of renal function. An additional group of 9-week-old SS rats was also studied to obtain baseline renal function prior to the induction of diabetes and hypertension. The rats were anesthetized with ketamine (30 mg/kg, i.m., Phoenix Pharmaceutical Co., St. Joseph, MO) and inactin (50 mg/kg, i.p., Sigma-Aldrich) and placed on a warming table to maintain body temperature at 37 °C. The trachea was cannulated with PE-240 tubing to facilitate breathing. Catheters were placed in the right femoral artery and vein for the measurement of arterial pressure and infusion of saline solution containing 2% bovine serum albumin and 2 mg/ml fluorescein isothiocyanate (FITC)-labeled inulin (Sigma-Aldrich) at a rate of 6 ml/h. Renal blood flow (RBF) was measured using an ultrasonic flowmeter (Transonic System, Ithaca, NY). After a 30-min equilibration period, urine and plasma samples were collected during two 30-min clearance periods. The concentrations of inulin in the urine and plasma samples were determined with a microplate fluorometer (Bio-Tek Instruments, Winooski, VT) at wavelengths of 490 (excitation) and 520 nm (emission). GFR was determined from the clearance of FITC-labeled inulin as we previously described [25, 26, 32].

Renal histology

After 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 Masson’s trichrome to evaluate the degree of glomerular injury and renal fibrosis. Thirty glomeruli per rat were scored for the degree of glomerulosclerosis by a blinded observer using a microscope on a 0–4 scale with 0 representing a normal glomerulus; 1, 2, 3, and 4 representing a loss of 1–25%, 26–50%, 51–75%, and > 75% of the capillaries in the glomerular tuft, respectively [33, 34]. Fibrosis was scored in 10 or more low power (10X objective) images in the outer medulla per animal. These images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fil1 color camera (Nikon Instruments Inc., Melville, NY) and analyzed for the percentage of the image stained blue (primarily collagen) in Mason trichrome–stained sections using the NIS-Elements D 4.6 software (Nikon) as we previously described [35].

Protocol 2. Effects of control of hyperglycemia with empagliflozin on the progression of renal injury in type 2 diabetic T2DN rats treated with DOCA-salt

These experiments were performed on 6-month-old male T2DN rats. The T2DN rat is a substrain of Goto-Kakizaki (GK) rats that carry the mitochondrial genome of fawn hooded hypertensive (FHH) rats [36, 37]. Unlike the GK rats and most other rodent models of diabetes, the T2DN strain develops progressive proteinuria and exhibits all of the renal changes associated with DN in humans, including thickening of glomerular and tubular basement membranes, glomerular hypertrophy, mesangial matrix expansion, nodular glomerular lesions, and renal fibrosis [26, 36, 38]. The rats were trained for several days to measure blood pressure using tail-cuff plethysmography. After obtaining baseline measurements, a long-acting DOCA pellet (200 mg/90 day, Innovative Research of America, Sarasota, FL) was implanted subcutaneously, the right kidney was removed, and the rats were given 1% NaCl in the drinking water to induce hypertension, as we previously described [34]. The DOCA-salt–treated T2DN rats were randomly assigned into 5 groups as described in the “Protocol 1. Effects of control of hyperglycemia using empagliflozin on the progression of renal injury in hypertensive type 1 diabetic STZ-SS rats” section: (1) vehicle; (2) insulin; (3) lisinopril; (4) empagliflozin; and (5) combination therapy. Food and water intake, body weight, SBP, protein excretion, and blood glucose levels were measured every 2 weeks for 12 weeks as described in the “Protocol 1. Effects of control of hyperglycemia using empagliflozin on the progression of renal injury in hypertensive type 1 diabetic STZ-SS rats” section. At the end of the experiment, the rats were prepared for clearance studies to measure RBF and GFR. The kidneys were removed and fixed in 10% formalin for assessment of renal injury. An additional group of 6-month-old T2DN rats was also studied to assess baseline renal function and renal injury.

Statistics

Mean values ± standard error (SE) are presented. The significance of differences in mean values within and between groups in the time course studies was determined using a two-way ANOVA for repeated measures and the Holm-Sidak post-hoc test. Between-group comparisons of RBF, GFR, kidney weight, and MAP in the clearance studies, and assessment of the degree of renal injury were performed using a one-way ANOVA and Holm-Sidak test. A P value < 0.05 was considered to be statistically significant.

Results

Protocol 1. Effects of control of hyperglycemia using empagliflozin on the progression of renal injury in hypertensive type 1 diabetic STZ-SS rats

Body weight, blood glucose, and HbA1c levels

A summary of the effects of the various treatments on body weight, blood glucose, and HbA1c levels in hypertensive type 1 diabetic STZ-SS rats is presented in Fig. 1. All of the groups maintained or gained weight throughout the study (Fig. 1A). Body weight was significantly higher in the rats treated with insulin relative to the levels seen in vehicle-treated rats. Baseline blood glucose levels were similar and averaged approximately 100 mg/dL in all of the groups. Three days after administration of STZ and the low dose insulin pellet, blood glucose levels averaged 419 ± 24, 417 ± 26, 418 ± 21, 421 ± 14, and 419 ± 12 mg/dL in the vehicle, insulin, lisinopril, empagliflozin, and combination therapy groups, respectively. Blood glucose remained elevated (400–500 mg/dL) in HS-fed STZ-SS rats treated with vehicle or lisinopril (Fig. 1B). Chronic administration of insulin, empagliflozin, and combination therapy effectively normalized blood glucose levels to < 150 mg/dl in hypertensive STZ-SS rats. Similarly, HbA1c levels were elevated throughout the study in the vehicle (9.5 ± 0.25%) and lisinopril-treated (9.7 ± 0.30%) hypertensive STZ-SS rats, while insulin (5.13 ± 0.18%), empagliflozin (5.16 ± 0.20%), and combination therapy (5.32 ± 0.13%) effectively controlled HbA1c levels.

Fig. 1.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on body weight (panel A) and non-fasting blood glucose levels (panel B) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats fed a high salt (HS) diet. Data are presented as mean values ± standard error (SE). N = 7–11 rats per group. * indicates P < 0.05 from the corresponding value in the baseline control values in the rats prior to induction of diabetes with STZ and hypertension with an HS diet containing 4% NaCl. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Temporal changes in SBP and proteinuria

The effects of the various treatments on systolic blood pressure and proteinuria in hypertensive type 1 diabetic STZ-SS rats are presented in Fig. 2. SBP increased in all groups after SS rats were given STZ and switched to an HS diet (Fig. 2A). However, combination therapy with empagliflozin and lisinopril attenuated the development of hypertension compared to rats treated with vehicle, lisinopril, or empagliflozin alone. Proteinuria increased in all the treatment groups after the induction of hypertension and diabetes (Fig. 2B). Hypertensive STZ-SS rats treated with insulin, empagliflozin, or combination therapy to control hyperglycemia significantly lowered proteinuria relative to values observed in vehicle-treated rats. However, there were some differences in that combination therapy that normalized glucose levels and lowered blood pressure were more effective than empagliflozin, which did not reduce blood pressure. Insulin that prevented hyperglycemia but did reduce blood pressure had the greatest effect on proteinuria. In contrast, lisinopril did not reduce proteinuria in this mixed model of diabetes and salt-sensitive hypertension.

Fig. 2.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on systolic blood pressure (SBP, panel A) and proteinuria (panel B) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats fed a high salt (HS) diet. Data are presented as mean values ± standard error (SE). N = 7–11 rats per group. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to induction of diabetes with STZ and hypertension with an HS diet containing 4% NaCl. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Renal hypertrophy and hemodynamics

The effects of the various treatments on MAP, renal hypertrophy, GFR, and RBF in STZ-SS rats after the induction of diabetes and hypertension are presented in Fig. 3. Consistent with the chronic tail-cuff measurements of SBP, lisinopril or insulin had no significant effect on MAP measured under ketamine and inactin anesthesia. MAP was significantly reduced in the rats treated with empagliflozin or combination therapy (Fig. 3A). Left kidney weight, an index of renal hypertrophy, increased by 50–70% in rats treated with either vehicle or lisinopril in comparison to baseline kidney weight measured before the induction of diabetes and hypertension (Fig. 3B). Controlling hyperglycemia with insulin prevented renal hypertrophy even though the rats became hypertensive. Combination treatment with empagliflozin and lisinopril, but not empagliflozin or lisinopril alone, significantly reduced the degree of renal hypertrophy in comparison to the values seen in rats receiving vehicle. The induction of diabetes caused a twofold increase in GFR (hyperfiltration) in vehicle-treated rats compared to values measured at baseline (Fig. 3C). However, none of the treatments had a significant effect on GFR. Moreover, other than a slight increase in the insulin-treated group, RBF was not significantly different in any of the treatment groups compared to the value measured in the baseline group (Fig. 3D).

Fig. 3.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on mean arterial pressure (MAP, Panel A), left kidney weight (panel B), glomerular filtration rate (GFR, panel C), and renal blood flow (RBF, Panel D) in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats fed a high salt (HS) diet for 8 weeks. Data are presented as mean values ± standard error (SE). N = 6–10 rats per group. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to induction of diabetes with STZ and hypertension with an HS diet containing 4% NaCl. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Assessments of renal histopathology

The effects of the various treatments on the degree of renal injury in STZ-SS rats are presented in Figs. 4 and 5. The kidneys from vehicle-treated rats exhibited severe glomerulosclerosis compared to the baseline group, and the glomerular injury scores (GIS) were 3.08 ± 0.10 versus 1.36 ± 0.18, respectively (Fig. 4). Control of hyperglycemia with insulin and empagliflozin or administration of lisinopril alone significantly reduced the degree of glomerular injury with GIS of 2.61 ± 0.07, 2.43 ± 0.19, and 2.76 ± 0.08 compared to rats treated with vehicle. Combination therapy that controlled hyperglycemia and lowered systolic pressure was most effective and reduced the GIS to 1.81 ± 0.02. The induction of diabetes and hypertension in vehicle-treated SS rats markedly increased renal medullary fibrosis relative to the baseline group (Fig. 5). Control of hyperglycemia with insulin did not reduce the degree of fibrosis. Lisinopril which did not lower plasma glucose or blood pressure had no significant effect on renal medullary fibrosis. However, administration of empagliflozin, alone or in combination with ACE inhibitor, reduced the degree of renal medullary fibrosis compared with that seen in vehicle-treated hypertensive STZ-SS rats.

Fig. 4.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on the degree of glomerular injury in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats fed a high salt (HS) diet for 8 weeks. Data are presented as mean values ± standard error (SE). N = 7–11 rats per group. Individual dots represent mean values obtained from each animal. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to induction of diabetes with STZ and hypertension with an HS diet containing 4% NaCl. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Fig. 5.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on renal medullary fibrosis in streptozotocin (STZ)-treated Dahl salt-sensitive (SS) rats fed a high salt (HS) diet for 8 weeks. Data are presented as mean values ± standard error (SE). N = 5–8 rats per group. Individual dots represent mean values obtained from each animal. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to induction of diabetes with STZ and hypertension with an HS diet containing 4% NaCl. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Protocol 2. Effects of control of hyperglycemia with empagliflozin on the progression of renal injury in hypertensive type 2 diabetic T2DN rats

Body weight, blood glucose, and HbA1c levels

The effects of the various treatments on body weight, blood glucose, and HbA1c levels in hypertensive type 2 diabetic T2DN rats are presented in Fig. 6. Body weight remained relatively constant in the rats treated with either vehicle or lisinopril. However, body weight decreased by 8% and 10%, respectively, in rats treated with empagliflozin alone or given in combination with lisinopril. Body weight increased significantly by 13% in T2DN rats in which blood glucose concentration was controlled with insulin (Fig. 6A).

Fig. 6.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on body weight (panel A) and non-fasting blood glucose levels (panel B) in type 2 diabetic nephropathy (T2DN) rats treated with deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water. Data are presented as mean values ± standard error (SE). N = 5–13 rats per group. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to treatment with DOCA-salt. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Blood glucose concentration decreased slightly in vehicle- or lisinopril-treated hypertensive T2DN rats, possibly due to reduced food intake after uninephrectomy and giving the animals 1% NaCl in the drinking water to induce hypertension, but it remained within the diabetic range at 250 mg/dL throughout the study (Fig. 6B). Insulin and empagliflozin, given alone or in combination with lisinopril, markedly decreased blood glucose concentration in DOCA-salt–treated T2DN rats into the normoglycemic range. Similarly, elevated HbA1c levels were observed throughout the study in the vehicle- (8.05 ± 0.29%) and lisinopril-treated (9.25 ± 0.69%) hypertensive T2DN rats. In contrast, insulin (6.00 ± 0.39%), empagliflozin (5.26 ± 0.15%), and combination therapy (5.32 ± 0.15%) effectively reduced HbA1c levels.

Temporal changes in SBP and proteinuria

The effects of various treatments on SBP and proteinuria in hypertensive type 2 diabetic T2DN rats are presented in Fig. 7. SBP increased significantly in the vehicle-treated group after administration of DOCA-salt (Fig. 7A). Control of hyperglycemia with insulin, empagliflozin, or combination therapy did not affect SBP compared with vehicle-treated hypertensive T2DN rats, and it remained over 150 mmHg throughout the study. Treatment with lisinopril significantly decreased SBP when compared to the values measured in the vehicle-treated group (141 ± 4 vs. 167 ± 5 mmHg, respectively). Combination therapy was less effective than the administration of lisinopril alone. Proteinuria increased in all the treatment groups throughout the study in DOCA-salt–treated T2DN rats (Fig. 7B). At the end of the experiment, proteinuria was 538 ± 77, 443 ± 79, 504 ± 65, 318 ± 85, and 356 ± 55 mg/day in vehicle, insulin, empagliflozin, lisinopril, and combination-treated hypertensive T2DN rats, respectively. However, the changes in protein excretion were not significant from those seen in the vehicle-treated group.

Fig. 7.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on systolic blood pressure (SBP, panel A) and proteinuria (panel B) in type 2 diabetic nephropathy (T2DN) rats treated with deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water. Data are presented as mean values ± standard error (SE). N = 5–13 rats per group. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to treatment with DOCA-salt. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Renal hypertrophy and hemodynamics

The effects of the various treatments on MAP, renal hypertrophy, GFR, and RBF that were measured at the end of the study in hypertensive type 2 diabetic T2DN rats are presented in Fig. 8. MAP measured under ketamine and inactin anesthesia was only significantly reduced in the rats treated with lisinopril. Kidney weight increased in all groups after induction of hypertension compared to baseline levels in 6-month-old T2DN rats (Fig. 8A). Administration of empagliflozin alone or in combination with lisinopril did not affect renal hypertrophy compared to vehicle-treated hypertensive T2DN rats. In contrast, treatment with lisinopril significantly reduced renal hypertrophy. Chronic treatment with empagliflozin given alone but not in combination with ACE inhibitor increased GFR by 35% over that seen in vehicle-treated rats. However, GFR was not altered in rats treated with vehicle, lisinopril, or combination therapy compared with baseline (Fig. 8C). RBF was not significantly altered in any of the treatment groups compared to baseline values (Fig. 8D).

Fig. 8.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on mean arterial pressure (MAP, panel A), left kidney weight (panel B), glomerular filtration rate (GFR, panel C), and renal blood flow (RBF, panel D) in type 2 diabetic nephropathy (T2DN) rats treated with deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water for 12 weeks. Data are presented as mean values ± standard error (SE). N = 6–10 rats per group. Individual dots represent mean values obtained from each animal. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to treatment with DOCA-salt. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Assessment of renal histology

The effects of the various treatments on renal injury in hypertensive type 2 diabetic T2DN rats are presented in Figs. 9 and 10. The 6-month-old T2DN rats exhibited mesangial matrix expansion (Fig. 9) and slightly elevated protein excretion at 50–60 mg/day (Fig. 7B) relative to the expected levels (< 20 mg/day) typically seen in nondiabetic rat models [36]. The kidneys from vehicle-treated hypertensive T2DN rats exhibited severe glomerulosclerosis compared to the baseline group, and the glomerular injury scores were 3.18 ± 0.10 versus 1.74 ± 0.02, respectively (Fig. 9). Control of hyperglycemia with insulin or empagliflozin significantly reduced the GIS to 2.84 ± 0.12 and 2.22 ± 0.16, respectively. Administration of lisinopril, which lowered blood pressure, and combination therapy also reduced the GIS to 2.07 ± 0.10 and 1.96 ± 0.07, respectively, relative to rats treated with vehicle. The induction of DOCA-salt hypertension in vehicle-treated T2DN rats markedly increased renal medullary fibrosis, particularly in vasa recta capillary bundles relative to the baseline group (Fig. 10). Controlling blood glucose concentration with insulin had no effect on the severity of renal medullary fibrosis compared with vehicle-treated rats. Treatment with empagliflozin, lisinopril, or combination therapy significantly attenuated renal medullary fibrosis compared with vehicle-treated hypertensive T2DN rats.

Fig. 9.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on the degree of glomerular injury in type 2 diabetic nephropathy (T2DN) rats treated with deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water for 12 weeks. Data are presented as mean values ± standard error (SE). N = 7–9 rats per group. Individual dots represent mean values obtained from each animal. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to treatment with DOCA-salt. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Fig. 10.

Effects of empagliflozin (Emp), lisinopril (Lis), combination therapy (Com), insulin (Ins), and vehicle (Veh) on renal medullary fibrosis in type 2 diabetic nephropathy (T2DN) rats treated with deoxycorticosterone acetate (DOCA) and 1% NaCl drinking water for 12 weeks. Data are presented as mean values ± standard error (SE). N = 6–9 rats per group. Individual dots represent mean values obtained from each animal. * indicates P < 0.05 from the corresponding value in the baseline control rats prior to treatment with DOCA-salt. † indicates P < 0.05 from the corresponding value in vehicle-treated rats

Discussion

Diabetes is the most common cause of CKD. Current therapies that include glycemic control using any of the available effective hypoglycemic agents in combination with ACE inhibitors and angiotensin receptor blockers slow but do not prevent the development of CKD. Consequently, the incidence of CKD is still rising, and this spurred the search for more effective therapies. SGLT2 inhibitors that block glucose reabsorption in the proximal tubule have emerged as a preferred treatment for diabetes since they have cardiovascular protective effects [8, 10, 24, 39–41]. Moreover, this novel class of hypoglycemic agents has been shown to reduce the degree of albuminuria and/or the rise in creatinine levels in diabetic animal models and type 2 diabetic patients [3, 15]. Recent clinical trials now suggest that SGLT2 inhibitors are renoprotective in both diabetic and nondiabetic subjects. For example, the EMPA-REG OUTCOME trial demonstrated that empagliflozin reduces the incidence of adverse cardiovascular events and CKD in type 2 diabetic patients [42]. The CREDENCE and CANVAS trials confirmed that SGLT2 inhibitors protect against the loss of renal function in type 2 diabetic patients [19, 21]. The DAPA-HF trial indicated that dapagliflozin protects against heart failure and renal injury independent of its glucose-lowering effects in well-controlled diabetic patients [20]. Finally, the DAPA-CKD trial [43] established that dapagliflozin has some renal protective effects even in CKD patients without diabetes. These collective findings have established that the addition of an SGLT2 inhibitor to the standard of care may provide superior renal protection [3]. However, the mechanism for renoprotection in diabetic nephropathy and nondiabetic models of CKD is unknown. It also remains to be determined if SGLT2 inhibitors are effective in hypertensive diabetic patients and animal models.

Hyperglycemia is associated with upregulation of renal SGLT2 expression, increased proximal tubular reabsorption, and oxygen consumption that promotes renal hypertrophy [30]. These changes have been linked to mitochondrial autophagy and oxidative stress [44], hypoxia [45], inflammation, and tubulointerstitial fibrosis [45]. Increased sodium reabsorption in the proximal tubule also reduces sodium delivery to the macula densa and promotes glomerular hyperfiltration by inhibiting tubuloglomerular feedback (TGF). Several mechanisms have been suggested to contribute to the renoprotective effects of SGLT2 inhibitors in diabetes. First, SGLT2 inhibitors prevent glomerular hyperfiltration following the onset of diabetes [9, 15]. They reduce Na and glucose reabsorption in the proximal tubule and increase the delivery of NaCl to the macula densa, which releases ATP and adenosine to activate TGF to reduce glomerular capillary pressure and GFR by promoting afferent arteriole constriction via activation of adenosine A1 receptors and efferent arteriole dilation by activation of adenosine A2 receptors [15, 46, 47]. The reduction in glomerular capillary pressure reduces podocyte injury, and the fall in the filtered load of glucose, sodium, and protein reduces tubular workload, tubular hypoxia, and fibrosis. Second, the inhibition of the reuptake of glucose reduces oxygen consumption and prevents proximal tubule hypoxia and damage [3]. Third, SGLT2 inhibitors cause caloric loss due to elevated glucose excretion and stimulate diuresis and natriuresis, leading to weight loss [25, 26] and reductions in arterial pressure [45]. Additionally, dapagliflozin was reported to protect against podocyte damage in protein overload conditions [48].

Consistent with this hypothesis, Vallon and colleagues reported that the reduction in albuminuria, kidney hypertrophy, and inflammation was secondary to the glucose-lowering effect of empagliflozin in type 1 diabetic Akita mice, but the reduction in hyperfiltration was not [30]. Kidokoro et al. reported that empagliflozin constricted Af-art by ~ 15% and lowers glomerular hyperfiltration within 30 min in type 1 diabetic Akita mice. Blockade of the A1 receptor abolished the SGLT2 inhibitor–related Af-art constriction [49]. Cherney et al. reported that empagliflozin reduced Af-art diameters and RBF in type 1 diabetic patients [50]. Moreover, empagliflozin increased urinary adenosine excretion in type 1 diabetic mice and patients [49, 51]. Notably, urinary adenosine concentration was also increased in type 2 diabetic patients. Dapagliflozin reduced GFR and filtration fraction without increasing renal vascular resistance, suggesting that dilation of the Ef-Art may also contribute to the regulation of GFR by SGLT2 inhibitors [52, 53]. While the initial decrease in GFR may contribute to a reduction in proteinuria following administration of SGLT2 inhibitors, several studies have indicated that GFR returns to control within a month, perhaps as the filtered load of glucose and sodium delivery to the macula densa is reduced [3, 4, 8]. Over time, GFR may actually be higher in patients treated with SGLT2 inhibitors since they slow the decline in GFR. These findings suggest that SGLT2 inhibitors provide a promising new option for glycemic control but also for the prevention of diabetes-induced renal disease. On the other hand, most patients with diabetes develop moderate to severe hypertension as they age [2, 5], and the effects of SGLT2 inhibitors have yet to be studied in hypertensive diabetic patients or animal models.

Therefore, the present study examined the effect of chronic inhibition of SGLT2 with empagliflozin alone or in combination with the standard of care, lisinopril, in hypertensive type 1 and 2 rat models of DN. We found that empagliflozin was just as effective at normalizing blood glucose levels in hypertensive STZ-SS and T2DN rats as therapeutic doses of insulin (Figs. 1 and 6). The reason that empagliflozin was more effective at controlling hyperglycemia in our studies compared to those seen in clinical practice is due to the dose. We used 10 to 20 mg/Kg/day in rats, which completely blocked the SGLT2 cotransporter in vivo [25, 26]. The clinical dose is 10–25 mg or 0.1 to 0.3 mg/Kg/day daily.

Empagliflozin given alone or in combination with lisinopril reduced proteinuria, glomerular injury, and renal fibrosis in hypertensive SS-STZ rats without altering GFR or RBF. Blood pressure and renal hypertrophy were also significantly reduced with combination therapy. Control of hyperglycemia with insulin did not reduce proteinuria, renal hypertrophy, glomerular injury, or renal fibrosis in STZ-SS rats. The renoprotective effect of empagliflozin in hypertensive STZ-SS rats is most likely due to inhibition of proximal tubular glucose reabsorption, reduced oxygen consumption, and hypoxia that drives renal interstitial fibrosis and inflammation. In contrast, insulin stimulates proximal tubular reabsorption, oxygen consumption, and renal hypertrophy, which promotes renal fibrosis. The failure to see the expected TGF-mediated fall in GFR in the STZ-SS rats treated with empagliflozin is likely due to the marked elevation in blood pressure in the STZ-SS rats fed an HS diet since TGF and autoregulation of RBF are known to be impaired in SS rats [54].

Empagliflozin given alone or in combination with lisinopril reduced glomerulosclerosis and renal fibrosis but did not affect arterial pressure, kidney hypertrophy, or protein excretion in DOCA-salt hypertensive T2DN rats. Interestingly, RBF was not altered in any of the treatment groups. GFR was not significantly altered in hypertensive T2DN rats even though kidney weight doubled. This likely reflects the development of severe glomerulosclerosis secondary to the induction of hypertension in this model. Similarly, one might expect that inhibition of SGLT2 would have activated TGF and lowered GFR as it does in normotensive diabetic models. However, in hypertensive T2DN rats, the higher GFR at the end of the study in the empagliflozin group is probably due to reduced fibrosis and glomerular protection rather than TGF-mediated changes in renal hemodynamics. It is worth mentioning a recent study demonstrating that luseogliflozin improves the loss of the myogenic response and autoregulation of cerebral blood flow in elderly T2DN rats [10]. Thus, SGLT2 inhibitors might also enhance constriction of renal afferent arterioles, and autoregulation of RBF in diabetic animals.

We found that lisinopril, which is the standard of care to reduce blood pressure and glomerular injury, and proteinuria in type 2 diabetic patients, had no effect on SBP, proteinuria, renal injury, or GFR and RBF in hypertensive STZ-SS. These results are not surprising since feeding hypertensive STZ-SS rats an HS diet suppresses the renin–angiotensin–aldosterone system and mitigates the effects of the ACE inhibitor. Our previous findings that lisinopril attenuated the development of hypertension and proteinuria and reduced GFR, glomerular injury, and fibrosis in STZ-SS rats fed a low salt diet support this view [25]. In the present study, lisinopril attenuated the rise in arterial pressure and decreased glomerular injury and renal fibrosis in hypertensive T2DN rats. The differences in the effects of lisinopril in the two models might be due to differences in the level of hypertension. Blood pressure was 40 mmHg higher in STZ-SS rats fed an HS diet than the values measured in DOCA-salt–treated T2DN rats.

Chronic treatment with empagliflozin alone or in combination with lisinopril markedly reduced blood glucose levels in hypertensive STZ-SS and T2DN rats. We and others have demonstrated that the blockade of SGLT2 effectively controls blood glucose levels by increasing the fractional excretion of glucose [15, 25, 26]. Moreover, the glucose-lowering effect of empagliflozin was just as effective as that observed in hypertensive STZ-SS and T2DN rats treated with insulin. We previously observed that the key differences between an SGLT2 inhibitor and insulin were that water intake and urine flow increased in STZ-SS and T2DN rats treated with an SGLT2 inhibitor but decreased in both models treated with insulin [25, 26]. These data support the hypothesis that the SGLT2 inhibitor-mediated inhibition of proximal tubular reabsorption promotes volume depletion, whereas insulin increases sodium retention. In this regard, Tikkanen et al. reported that empagliflozin reduced blood pressure in type 2 diabetic patients with mild to moderate hypertension [55]. Similar results were obtained in clinical trials and in diabetic animal models treated with empagliflozin [30, 56]. However, in the present study, empagliflozin had no effect on blood pressure in STZ-SS rats fed an HS diet, or DOCA-salt–treated T2DN rats. It also failed to lower pressure in our previous studies [25, 26]. We suspect that it causes a compensatory activation of sympathetic tone and renin release that opposes the fall in blood pressure, similar to the effects of many diuretic agents. In support of this view, we found that combination therapy with empagliflozin and lisinopril markedly reduced arterial pressure in STZ-SS rats fed an HS diet.

Another interesting finding is that insulin was very effective at controlling hyperglycemia in both the STZ-SS and hypertensive T2DN diabetic models. It markedly reduced proteinuria in the STZ-SS rats, but the mechanism involved is unclear. It might have something to do with elevated levels of insulin altering the expression of megalin and stimulating the reuptake of filtered protein. Regardless, normalization of plasma glucose levels with insulin did not reduce glomerulosclerosis or renal fibrosis in either of our type 1 or 2 diabetic models with hypertension, whereas empagliflozin, especially when given with lisinopril, was remarkably effective. This leads one to question the control of hyperglycemia with insulin in elderly type 2 diabetics with beta-cell failure.

In conclusion, the current results indicate that treatment with the selective SGLT2 inhibitor empagliflozin was very effective in preventing hyperglycemia, but it had no effect on blood pressure in two novel models of DN with superimposed hypertension. Controlling hyperglycemia with empagliflozin reduced proteinuria to some extent in hypertensive STZ-SS but not in hypertensive T2DN rats. It did reduce glomerulosclerosis and renal fibrosis in both models. Combination therapy with empagliflozin and lisinopril was more effective at lowering blood pressure and preventing the development of proteinuria than either drug alone in a type 1 model of DN with hypertension. Combination therapy was very effective in reducing glomerulosclerosis and renal fibrosis in both models. Overall, these results suggest treatment with an SGLT2 inhibitor alone attenuates the development of glomerular injury and renal fibrosis in both type 1 and 2 diabetic animal models with hypertension. When given in combination with an ACE inhibitor or angiotensin II receptor blocker to lower blood pressure, it may prove to be very effective in slowing the progression of renal injury, especially in elderly hypertensive diabetic patients.

Author contribution

R.J.R conceived and designed research; S.R.M., J.M.W., and W.W. performed experiments; J.M.W., S.R.M., W.W., J.J.B., F.F., and R.J.R analyzed data; J.M.W., F.F., and R.J.R interpreted results of experiments; S.R.M., J.J.B., and F.F. prepared figures; J.M.W. and F.F. drafted the manuscript. All authors contributed to the article and approved the final version of the manuscript.

Funding

This study was partially supported by grants DK109133, AG057842, P20GM104357, and HL138685 from the National Institutes of Health and a collaborative research agreement DE 811138149 from Boehringer Ingelheim International GmbH. The views expressed in this manuscript are expressly those of the author(s) and were not influenced by the funding institutions.

Data availability

Copies of data files, images presented in this manuscript, and breeding pairs of the animal strains used will be made available upon written request and after a Material Transfer Agreement is completed by both institutions.

Declarations

Ethics approval

This study utilized Dahl salt-sensitive and type 2 diabetic nephropathy rats that were obtained from 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. All of the animal studies were performed in accordance with the US Public Health Service Policy on the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of the University of Mississippi Medical Center.

Conflict of interest

The authors declare no competing interests.