Abstract

Hyperfiltration, highly prevalent early in sickle cell disease (SCD), is in part driven by an increase in ultrafiltration coefficient (Kf). The increase in Kf may be due to enlarged filtration surface area and/or increased glomerular permeability (Palb). Previous studies have demonstrated that endothelin-1 (ET-1) contributes to Palb changes in models of diabetes and SCD. Thus, we performed longitudinal studies of renal function to determine the relationship between ET-1 and glomerular size and Palb that may contribute to hyperfiltration in humanized sickle cell (HbSS) and control (HbAA) mice at 8–32 weeks of age. HbSS mice were characterized by significant increases in plasma and glomerular ET-1 expression in both sexes although this increase was significantly greater in males. HbSS glomeruli of both males and females presented with a progressive and significant increase in glomerular size, volume, and Kf. During the onset of hyperfiltration, plasma and glomerular ET-1 expression were associated with a greater increase in glomerular size and Kf in HbSS mice, regardless of sex. The pattern of Palb augmentation during the hyperfiltration was also associated with an increase in glomerular ET-1 expression, in both male and female HbSS mice. However, the increase in Palb was significantly greater in males and delayed in time in females. Additionally, selective endothelin A receptor (ETA) antagonist prevented hyperfiltration in HbSS, regardless of sex. These results suggest that marked sex disparity in glomerular hyperfiltration may be driven, in part, by ET-1-dependent ultra-structural changes in filtration barrier components contributing to glomerular hyperfiltration in HbSS mice.

Introduction

Sickle cell disease (SCD)-associated renal complications occur in childhood [1,2] and often progress to proteinuria, chronic kidney disease [3,4] and eventually end-stage renal disease at the average age of 23 years [5,6]. One of the earliest manifestations of renal involvement is elevated glomerular filtration rate (GFR), termed as the hyperfiltration phase, which putatively plays a primary role in the initiation of SCD kidney disease. Hyperfiltration has been associated with the development and progression of SCD nephropathy and a poor prognosis in humans and mouse model of SCD [1,2,7,8,9]. Although a high prevalence of hyperfiltration is well documented in SCD [3,8,10,11,12], the mediators and underlying pathophysiological mechanisms are not well understood.

GFR is determined by the transcapillary hydraulic pressure gradient across the membrane, plasma oncotic pressure and the glomerular ultrafiltration coefficient (Kf). Changes in any of these parameters may result in hyperfiltration presented with increased estimated GFR > 140 ml/min/1.73m2 in SCD adults [13,14] and above 180 ml/min/1.73m2 in pediatric SCD patients [1]. The mechanisms of glomerular hyperfiltration most likely vary according to the underlying clinical condition. In SCD, an elevated GFR at a single nephron level appears to be the result of increased glomerular perfusion and/or Kf [15]. Kf is determined by a total filtering surface area and/or glomerular hydraulic permeability, and typically represents changes in the glomerular membrane structure and function. As glomerular hypertrophy may reflect increased filtration surface area, it is important to identify potential mediators underlying these changes as they occur in SCD nephropathy.

Endothelin–1 (ET–1), a vasoactive peptide ubiquitously produced in the body, signals through two counteracting receptor subtypes, endothelin–A (ETA) and endothelin–B (ETB). ET–1 has been widely reported as a mechanistic mediator of various proteinuric kidney diseases [16–19] including SCD nephropathy [20–23]. Augmented synthesis of ET–1 is stimulated by the SCD milieu [24]. Elevated plasma and urinary ET–1 levels occur in humans and mouse models of SCD [21,23,25,26] raising the possibility of a direct effect of ET–1 on kidney structure and function. We recently determined the time course of SCD nephropathy, characterized by peak hyperfiltration phase at 12 weeks of age in male and 20 weeks of age in female humanized sickle cell (HbSS) mice, followed by subsequent kidney function decline and injury [9]. Also our long–term treatment studies with a selective ETA receptor antagonist revealed robust renal protection with preserved GFR and glomerular structure at the level of non–disease controls [21]. Moreover, ET–1 has been shown to directly increase glomerular permeability to albumin (Palb) in the isolated, intact glomeruli, independent of hemodynamic changes, thereby suggesting a non–hemodynamic effect of ET–1 on the structure of the glomerular filtration barrier [19,21]. Therefore, the current study sought to determine the relationship between ET–1 and the glomerular filtration determinants that may contribute to hyperfiltration in HbSS mice. As we previously observed a distinct sex difference in renal phenotype in HbSS mice, we utilized an approach using gender in studying renal involvement in SCD.

Materials and methods

Animals

Studies utilized male and female humanized sickle cell (HbSS) and genetic control (HbAA) mice originally generated and characterized by Townes et al. [27]. All studies were conducted at the University of Alabama at Birmingham (UAB), Birmingham, Alabama, U.S.A. Homozygous experimental SCD knockout, knockin mice, with notation: B6; 129–Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J, harbor a mutant β globin gene construct expressing human hemoglobin S. Homozygous control mice were derived from the same colony and harbor wild–type β globin construct expressing human hemoglobin A. Mice were housed under conditions of constant temperature, humidity, 12–h light/dark cycle and provided with water and food (Harlan Teklad, Indianapolis, IN) ad libitum. Studies were performed in HbSS and age–matched HbAA control mice at 8, 12, 20 and 32 weeks of age. All mice were maintained and studied in accordance with the National Institutes of Health Guide for the Care and use of Laboratory Animals following a protocol reviewed and approved by the UAB Institutional Animal Care and Use Committees. For tissue collection, animals were anesthetized with isoflurane (2.5%). Blood was collected from the abdominal aorta into an ethylenediaminetetraacetic acid (EDTA)–rinsed syringe and plasma was separated by centrifuging for 10 min at 1000×g, snap–frozen in liquid nitrogen and stored in −80°C until analyzed. Kidneys were removed, de–capsulated, and placed in ice–cold phosphate buffered saline (PBS) solution, pH 7.4 (137 mM sodium chloride, 8.1 mM sodium phosphate dibasic, 1.5 mM potassium phosphate monobasic, 2.7 mM potassium chloride, 0.49 mM magnesium dichloride, 0.9 mM calcium dichloride, 5.6 mM glucose). The left kidney was used for glomeruli isolation.

Treatment protocol

To confirm a potential contribution of ET–1 signaling in the development of hyperfiltration, a separate set of HbSS and control mice was utilized. Male and female HbSS and HbAA mice were treated with selective ETA receptor antagonist, ambrisentan (10 mg/kg/day; Gilead Sciences., Foster City, CA) for 16 weeks, beginning at 4 weeks of age [21].

GFR measurements

GFR was measured every 4 weeks, starting at 8 weeks of age, using transcutaneous measurement of fluorescein isothiocyanate–labeled sinistrin technique as previously described [9,28].

Isolated glomeruli experiments

Renal cortical tissue was immediately harvested after kidney dissection and used for glomeruli isolation by a gradual sieving technique as previously described with minor modifications [29,30]. Briefly, minced renal cortical tissue was pressed through a series of filters (with decreasing pore size of 150, 100, and 50 μm) resulting in suspension of decapsulated, intact glomeruli in ice–cold PBS solution. The collected glomeruli suspension was centrifuged for 10 min (3000×g) and the resulting pellet was re–suspended in fresh ice–cold PBS solution. Contamination with tubular fragments was less than 5% as assessed by the light microscopy. Isolated glomeruli were snap–frozen in liquid nitrogen for molecular analysis or re–suspended in 5% bovine serum albumin (BSA) solution. Glomeruli were affixed to poly–l–lysine covered wells in a six–well plate and incubated with fresh 5% BSA solution for 10 min. Unattached glomeruli were removed by gentle washing with fresh 5% BSA solution. Adherent glomeruli were used to measure Palb.

Measurements of glomerular permeability characteristics were performed in freshly isolated glomeruli as previously described with minor modification [29,31]. Briefly, changes in glomerular volume (V) were measured in response to an oncotic gradient (Δπ) induced by 5 and 1% BSA solutions (Δπ = 12 mmHg). The glomerular area (A) was measured to calculate glomerular volume using CellSens Dimension Software (Olympus, Japan) and formula V = [4/3 A√(A/π)]/10−6. The albumin reflection coefficient (σalb) was calculated as the ratio of the ΔV for the initial glomeruli volume to the ΔV of the final glomeruli volume in response to identical oncotic gradients (σalb = ΔVinitial/ΔVfinal). Palb, expressed as the reflection coefficient of albumin (convectional Palb), was calculated as Palb = 1 − σalb. At least five glomeruli from five or more mice per group were studied in each experiment. Kf was calculated as previously described [32]. Briefly, in isolated glomeruli preparation filtration is driven by the oncotic gradient. Changes of glomerular volume (ΔV) in time (Δt) recorded in response to an oncotic gradient were calculated as Kf = ΔV/Δt × Δπ.

Analytical methods

Plasma concentrations of ET–1 were measured using ELISA according to manufacturer’s instructions (QuantiGlo ET–1 Kit; R&D Systems, MN). Isolated glomeruli were homogenized and total RNA was extracted using Ambion RNAqueous–Micro Total RNA Isolation Kit (Ambion, Austin, TX). Reverse transcription was performed with 1 μg of mRNA using iScript cDNA synthesis kit (Bio–Rad, Hercules, CA). Real–time amplification was performed with ABI 7300 Real–Time PCR System using iTaq Universal Probes Supermix (Bio–Rad, Hercules, CA) and TaqMan primer gene expression assay (Applied Biosystems, Foster City, CA) of ET–1 (Mn00438656_m1), Wilms’ tumor antigen 1 (WT–1) (Mn0001337048_m1), and synaptopodin (Mn03413333_m1) according to the manufacturer’s instructions. The comparative method of relative quantification (2−ΔΔCT) was used to calculate the expression level of target gene, normalized to β actin or 8–week–old HbSS males.

Statistical analysis

Statistical analysis was performed using Prism 7.0 software (GraphPad, La Jolla, CA). Data were analyzed using two–way analysis of variance (ANOVA) with Tukey’s post hoc test or linear regression. Parametric distribution of data was confirmed with Shapiro–Wilk test. Results are expressed as means ± SEM, with P<0.05 being considered statistically significant.

Results

Plasma ET–1 and glomerular ET–1 in HbAA and HbSS mice

Our previous longitudinal studies of kidney function determined that male HbSS mice demonstrated a rapid hyperfiltration phase as early as at 12 weeks of age, while HbSS females displayed a more moderate and prolonged hyperfiltration phase with a peak at 20 weeks of age. Hyperfiltration was followed by sex–dependent progression and severity of subsequent renal injury and dysfunction that was more rapid in male mice compared with female [9]. At 8 weeks of age, HbSS mice exhibited no differences in plasma or glomerular ET–1 expression when compared with age–matched HbAA mice, regardless of sex (Figure 1A,B,D,E). A four–fold increase in plasma ET–1 and almost a two–fold increase in glomerular ET–1 expression were observed at 12 weeks of age in male HbSS mice (Figure 1A,D). In contrast, female HbSS mice had a 2-fold increase in plasma and a 1.5-fold increase in glomerular ET-1 expression at 20 weeks of age compared with earlier ages (Figure 1B,E). At subsequent ages, we observed significant increases in ET-1 expression in both male and female HbSS mice (Figure 1A,B,D,E). Interestingly, HbSS females had slightly higher, but not statistically significant, plasma ET-1 concentrations at 8 weeks of age. However, males presented with significantly greater increase in plasma and glomerular ET-1 expression throughout the time course of the study (Figure 1C,F). Control HbAA mice did not have any changes in ET-1 throughout the course of study (Figure 1A,B,D,E).

Longitudinal study of plasma and glomerular expression of ET-1 in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

Figure 1
Longitudinal study of plasma and glomerular expression of ET-1 in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of plasma ET-1 levels in male control and HbSS mice. (B) Trajectory of plasma ET-1 levels in female HbAA and HbSS mice. (C) Comparison of plasma ET-1 levels between male and female HbSS mice. (D) Trajectory of relative glomerular ET-1 expression in glomeruli from male HbAA and HbSS mice. (E) Trajectory of relative glomerular ET-1 expression in glomeruli from female HbAA and HbSS mice. (F) Glomerular ET-1 expression in glomeruli from female HbSS mice relative to male HbSS mice. Data are mean ± S.E.M; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. §P<0.05 versus age-matched male HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–F).

Figure 1
Longitudinal study of plasma and glomerular expression of ET-1 in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of plasma ET-1 levels in male control and HbSS mice. (B) Trajectory of plasma ET-1 levels in female HbAA and HbSS mice. (C) Comparison of plasma ET-1 levels between male and female HbSS mice. (D) Trajectory of relative glomerular ET-1 expression in glomeruli from male HbAA and HbSS mice. (E) Trajectory of relative glomerular ET-1 expression in glomeruli from female HbAA and HbSS mice. (F) Glomerular ET-1 expression in glomeruli from female HbSS mice relative to male HbSS mice. Data are mean ± S.E.M; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. §P<0.05 versus age-matched male HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–F).

Glomerular injury in HbAA and HbSS mice

To assess glomerular filtration barrier damage, we measured glomerular expression of WT-1 and synaptopodin, markers of podocyte injury. Expression of WT- 1 marker in glomeruli from HbSS mice was similar to HbAA controls prior to the hyperfiltration phase, regardless of sex (Figure 2A,B). Synaptopodin expression was significantly reduced in HbSS male mice beginning at 12 weeks of age, but was not significantly reduced in female HbSS mice until 32 weeks of age (Figure 2C,D). Additionally, we observed no loss of glomeruli or podocyte number, nor other markers of glomerular injury prior to the hyperfiltration in HbSS mice [9]. Progressive glomerular damage, including glomerular sclerosis and congestion, occurred subsequent to hyperfiltration in both HbSS sexes [9]. None of the measured markers of podocyte injury changed significantly throughout the course of study in control HbAA mice (Figure 2).

Longitudinal study of podocyte injury in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

Figure 2
Longitudinal study of podocyte injury in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of WT-1 expression in glomeruli from male control and HbSS mice. (B) Trajectory of WT-1 expression in glomeruli from female control and HbSS mice. (C) Trajectory of synaptopodin expression in glomeruli from male control and HbSS mice. (D) Trajectory of synaptopodin expression in glomeruli from female control and HbSS mice. Data are mean ± S.E.M.; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–D).

Figure 2
Longitudinal study of podocyte injury in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of WT-1 expression in glomeruli from male control and HbSS mice. (B) Trajectory of WT-1 expression in glomeruli from female control and HbSS mice. (C) Trajectory of synaptopodin expression in glomeruli from male control and HbSS mice. (D) Trajectory of synaptopodin expression in glomeruli from female control and HbSS mice. Data are mean ± S.E.M.; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–D).

Glomerular permeability in HbAA and HbSS mice

At 8 weeks of age, HbSS mice exhibited no significant differences in Palb when compared with HbAA mice, regardless of sex (Figure 3A,B). Glomeruli from male HbSS mice displayed a significant increase in Palb at 12 weeks of age (0.487 ± 0.065 vs. 0.209 ± 0.050), whereas glomeruli from control mice presented with no significant changes (Figure 3A). Also, a significant increase in Palb was observed at 20 weeks of age in glomeruli from female HbSS mice compared with HbAA (0.325 ± 0.047 vs. 0.128 ± 0.018, respectively; Figure 3B). Interestingly, the pattern of Palb augmentation was consistent with the increase in ET-1, specifically glomerular ET-1 expression, in both male and female HbSS mice and stayed elevated throughout the time course of the study. Although significantly higher than age-matched HbAA controls, female HbSS mice demonstrated significantly lower Palb when compared with male mice (Figure 3C).

Longitudinal study of Palb in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

Figure 3
Longitudinal study of Palb in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of Palb in male control and HbSS mice. (B) Trajectory of Palb in female control and HbSS mice. (C) Comparison of Palb between male female HbSS mice. Data are mean ± S.E.M; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. §P<0.05 versus age-matched male HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–C).

Figure 3
Longitudinal study of Palb in male and female genetic control (HbAA) and humanized sickle mice (HbSS)

(A) Trajectory of Palb in male control and HbSS mice. (B) Trajectory of Palb in female control and HbSS mice. (C) Comparison of Palb between male female HbSS mice. Data are mean ± S.E.M; *P<0.05 versus age-matched HbAA mice; P<0.05 versus 8-week HbSS mice. §P<0.05 versus age-matched male HbSS mice. Analysis by two-way ANOVA with Tukey’s post hoc analysis (A–C).

Association of ET-1 with increased glomerular permeability and Kf in HbSS mice

To further elucidate if ET-1 could contribute to the rise in GFR resulting from glomerular changes observed in HbSS mice, we determined if glomerular ET-1 expression is associated with increased glomerular filtration surface area and Kf. Characteristics, including renal morphometric measurement of male and female HbSS and control mice are displayed in Table 1 and 2, respectively. During the time when hyperfiltration was developing, 8–12 weeks of age in males and 8–20 weeks of age in females, glomerular expression of ET-1 was significantly correlated with glomerular size (Figure 4A,B) and Kf (Figure 4C,D) in HbSS mice, regardless of sex. Interestingly, plasma ET-1 concentrations were also positively associated with the degree of glomerular size and Kf in both male and female HbSS mice (Figure 4E–H).

Table 1
Characteristics of experimental (HbSS) and control (HbAA) male mice
 HbAA HbSS 
 8 weeks 12 weeks 20 weeks 32 weeks 8 weeks 12 weeks 20 weeks 32 weeks 
Body weight [g] 24.8 ± 0.5 25.7 ± 0.6 27.5 ± 1.4 33.3 ± 0.7 24.4 ± 1.0 26.2 ± 0.9 29.7 ± 0.8 31.9 ± 0.6*† 
Kidney/body weight [mg/g] 4.2 ± 1.1 5.7 ± 0.8 4.6 ± 0.1 5.3 ± 0.2 5.7 ± 0.1 5.1 ± 0.2 5.8 ± 0.1 5.1 ± 1.3 
Glomerular area [mm22.5 ± 0.1 2.6 ± 0.1 2.9 ± 0.1 3.0 ± 0.1 3.4 ± 0.2* 4.0 ± 0.1*† 4.5 ± 0.1*† 5.2 ± 0.1*† 
Glomerular volume [nl] 0.09 ± 0.01 0.10 ± 0.01 0.12 ± 0.00 0.13 ± 0.01 0.15 ± 0.01* 0.20 ± 0.01*† 0.23 ± 0.01*† 0.28 ± 0.00*† 
Kf [nl/min × mmHg] 0.042 ± 0.001 0.046 ± 0.001 0.048 ± 0.001 0.046 ± 0.001 0.082 ± 0.001 0.12 ± 0.001 0.32 ± 0.001 0.028 ± 0.001*† 
 HbAA HbSS 
 8 weeks 12 weeks 20 weeks 32 weeks 8 weeks 12 weeks 20 weeks 32 weeks 
Body weight [g] 24.8 ± 0.5 25.7 ± 0.6 27.5 ± 1.4 33.3 ± 0.7 24.4 ± 1.0 26.2 ± 0.9 29.7 ± 0.8 31.9 ± 0.6*† 
Kidney/body weight [mg/g] 4.2 ± 1.1 5.7 ± 0.8 4.6 ± 0.1 5.3 ± 0.2 5.7 ± 0.1 5.1 ± 0.2 5.8 ± 0.1 5.1 ± 1.3 
Glomerular area [mm22.5 ± 0.1 2.6 ± 0.1 2.9 ± 0.1 3.0 ± 0.1 3.4 ± 0.2* 4.0 ± 0.1*† 4.5 ± 0.1*† 5.2 ± 0.1*† 
Glomerular volume [nl] 0.09 ± 0.01 0.10 ± 0.01 0.12 ± 0.00 0.13 ± 0.01 0.15 ± 0.01* 0.20 ± 0.01*† 0.23 ± 0.01*† 0.28 ± 0.00*† 
Kf [nl/min × mmHg] 0.042 ± 0.001 0.046 ± 0.001 0.048 ± 0.001 0.046 ± 0.001 0.082 ± 0.001 0.12 ± 0.001 0.32 ± 0.001 0.028 ± 0.001*† 

Data are means ± S.E.M. (n=5–6 in HbSS groups and n=5 in HbAA groups).

*P<0.05 versus age-matched HbAA.

P<0.05 versus 8-week-old HbSS.

P<0.05 versus 8-week-old HbAA

Table 2
Characteristics of experimental (HbSS) and control (HbAA) female mice
 HbAA HbSS 
 8 weeks 12 weeks 20 weeks 32 weeks 8 weeks 12 weeks 20 weeks 32 weeks 
Body weight [g] 17.5 ± 0.3 23.0 ± 0.8 24.8 ± 0.3 22.8.3 ± 0.8 20.3 ± 0.6 24.2 ± 0.8 22.8 ± 1.0 25.5 ± 0.8 
Kidney/body weight [mg/g] 4.6 ± 0.1 4.6±0.2 4.0 ± 0.2 4.6 ± 0.2 5.3 ± 0.3 5.2 ± 0.1 5.4 ± 0.2 5.5 ± 0.4 
Glomerular area [mm22.5 ± 0.1 2.9 ± 0.1 3.2 ± 0.1 3.5 ± 0.1 2.5 ± 0.1 3.0 ± 0.1*† 3.4 ± 0.1* 5.2 ± 0.1* 
Glomerular volume [nl] 0.10 ± 0.01 0.12 ± 0.01 0.14 ± 0.01 0.16 ± 0.01 0.10 ± 0.01 0.13 ± 0.01*† 0.15 ± 0.01*† 0.28 ± 0.01*† 
Kf [nl/min × mmHg] 0.034 ± 0.001 0.037 ± 0.002 0.036 ± 0.004 0.033 ± 0.005 0.035 ± 0.005 0.057 ± 0.003*† 0.077 ± 0.006*† 0.028 ± 0.006 
 HbAA HbSS 
 8 weeks 12 weeks 20 weeks 32 weeks 8 weeks 12 weeks 20 weeks 32 weeks 
Body weight [g] 17.5 ± 0.3 23.0 ± 0.8 24.8 ± 0.3 22.8.3 ± 0.8 20.3 ± 0.6 24.2 ± 0.8 22.8 ± 1.0 25.5 ± 0.8 
Kidney/body weight [mg/g] 4.6 ± 0.1 4.6±0.2 4.0 ± 0.2 4.6 ± 0.2 5.3 ± 0.3 5.2 ± 0.1 5.4 ± 0.2 5.5 ± 0.4 
Glomerular area [mm22.5 ± 0.1 2.9 ± 0.1 3.2 ± 0.1 3.5 ± 0.1 2.5 ± 0.1 3.0 ± 0.1*† 3.4 ± 0.1* 5.2 ± 0.1* 
Glomerular volume [nl] 0.10 ± 0.01 0.12 ± 0.01 0.14 ± 0.01 0.16 ± 0.01 0.10 ± 0.01 0.13 ± 0.01*† 0.15 ± 0.01*† 0.28 ± 0.01*† 
Kf [nl/min × mmHg] 0.034 ± 0.001 0.037 ± 0.002 0.036 ± 0.004 0.033 ± 0.005 0.035 ± 0.005 0.057 ± 0.003*† 0.077 ± 0.006*† 0.028 ± 0.006 

Data are means ± S.E.M. (n=5–6 in HbSS groups and n=5 in HbAA groups).

*P<0.05 versus age-matched HbAA.

P<0.05 versus 8-week-old HbSS.

P<0.05 versus 8-week-old HbAA.

ET-1 correlates with glomerular size and glomerular Kf during hyperfiltration phase in male and female humanized sickle mice (HbSS)

Figure 4
ET-1 correlates with glomerular size and glomerular Kf during hyperfiltration phase in male and female humanized sickle mice (HbSS)

(A) Correlation of glomerular ET-1 expression with glomerular size during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (B) Correlation of glomerular ET-1 expression with glomerular size during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (C) Correlation of glomerular ET-1 expression with Kf during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (D) Correlation of glomerular ET-1 expression with Kf during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (E) Correlation of plasma ET-1 levels with glomerular size during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (F) Correlation of plasma ET-1 levels with glomerular size during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (G) Correlation of plasma ET-1 levels with Kf during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (H) Correlation of plasma ET-1 levels with Kf during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). Data are mean ± S.E.M. Analysis by linear regression (A–H).

Figure 4
ET-1 correlates with glomerular size and glomerular Kf during hyperfiltration phase in male and female humanized sickle mice (HbSS)

(A) Correlation of glomerular ET-1 expression with glomerular size during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (B) Correlation of glomerular ET-1 expression with glomerular size during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (C) Correlation of glomerular ET-1 expression with Kf during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (D) Correlation of glomerular ET-1 expression with Kf during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (E) Correlation of plasma ET-1 levels with glomerular size during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (F) Correlation of plasma ET-1 levels with glomerular size during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). (G) Correlation of plasma ET-1 levels with Kf during the development of hyperfiltration in male HbSS mice (age: 8–12 weeks). (H) Correlation of plasma ET-1 levels with Kf during the development of hyperfiltration in female HbSS mice (age: 8–20 weeks). Data are mean ± S.E.M. Analysis by linear regression (A–H).

Evidence for ET-1 involvement in the development of hyperfiltration in HbSS mice

To further support our hypothesis that ET-1 contributes to the onset of glomerular hyperfiltration in HbSS mice we examined the effect of selective ETA receptor antagonist, ambrisentan, on the development of hyperfiltration. Our previous studies demonstrated that HbSS male mice peaked at 12 weeks of age and the magnitude of rise in GFR from 8 to 12 weeks of age was 63 ± 9 μl/min [9]. However, the change in GFR from 8 to 12 weeks of age in HbSS male mice treated with ambrisentan was not significant, a change of 18 ± 3 μl/min compared with a change of 1 ± 4 μl/min in HbAA ambrisentan-treated mice. Unlike males, HbSS females had a slower rise in GFR with a maximum increase at 20 weeks of age [9]. Ambrisentan treatment in female HbSS mice did not display an increase in GFR from 8 to 20 weeks of age, an average change of 9 ± 4 μl/min compared with 4±7 μl/min in HbAA female mice treated with ambrisentan.

Discussion

Hyperfiltration is a well-documented risk factor for CKD in SCD [33,34] although mechanisms underlying this phenomenon in SCD and other forms of nephropathy are not well understood. Identifying the mechanisms that cause increased GFR at the onset of SCD nephropathy is of great importance for our understanding of the pathogenesis of resulting CKD. Our data reveal that increases in circulating ET-1 are associated with the increases in GFR that are likely related to increases in the glomerular Kf. Furthermore, selective ETA receptor antagonist prevented the increase in GFR during the hyperfiltration phase in HbSS mice. Also several recent studies have demonstrated that ETA receptor blockade can prevent/reduce renal injury in mouse models of SCD [21,22], highlighting the support for use of ET-1 receptor antagonism for SCD nephropathy.

An obvious mechanism for hyperfiltration in SCD is an increase in glomerular capillary pressure through pre-glomerular vasodilation [34,15]. However, given that SCD is associated with large changes in glomerular permeability [21], hyperfiltration may also be attributed to increases in Kf to increase the effective glomerular filtration surface area and/or glomerular permeability. Our results demonstrate that Kf significantly increased prior to the peak of hyperfiltration in both male and female HbSS mice. Moreover, we identified that HbSS mice with higher plasma concentrations and increased glomerular expression of ET-1 have a larger increase in Kf regardless of sex. We propose that the significant correlation between ET-1 and the magnitude of rise in Kf during hyperfiltration phase is associated with an increase in both glomerular filtration area and permeability. Measurements of glomerular size and calculation of glomerular volume, conducted on the isolated glomeruli, confirmed an enlargement of glomeruli in these mice during the development of hyperfiltration. We have reported increases in albumin permeability in SCD mice that is an indication of increased hydraulic conductivity. Our murine data are in agreement with a predominant increase in glomerular surface area and volume previously reported in SCD patients [35,36].

Our previous findings suggest that targeting ET signaling pathway with ETA receptor blockade in SCD is beneficial for slowing the progression of renal injury [20,21,26]. Initiation of long-term selective antagonism of the ETA receptor at an early age prevents glomerular hypertrophy and preserves GFR in the normal range in humanized SCD mice [21]. Here we provided direct evidence that ET-1 contributes to the hyperfiltration as selective ETA receptor blockade prevented the rise in GFR during the period when hyperfiltration peaks in HbSS mice. Moreover, Sabaa et al. [22] also reported beneficial effects of bosentan, a dual ET receptor antagonist on the hypoxia-induced renal complication in SCD mice. Furthermore, 8-week administration of atorvastatin significantly reduces renal ET-1 expression and mean glomerular tuft area in SCD mice, suggesting that ET-1 could account for at least some of the glomerular hypertrophy observed in this model [37]. Thus, pre-clinical studies have provided some very convincing evidence for clinical studies to assess whether selective ET receptor antagonism could be considered as an early renoprotective intervention to prevent development and progression of hyperfiltration and subsequent renal injury in SCD. Importantly, the utility of ETA receptor blockade for treatment of glomerular disease most likely extends beyond SCD. For example, diabetic nephropathy is also associated with elevated glomerular ET-1 and hyperfiltration and pre-clinical studies showing the effectiveness of ETA blockade led to recent successful phase III clinical trials [38].

Our results are consistent with multiple changes in glomerular barrier properties, potentially mediated by ET-1, and associated with increased Kf. Previous studies have shown that ET-1, via an ETA receptor mediated mechanism, may induce cytoskeleton rearrangement and intracellular structural changes across the podocyte [39]. In particular, ET-1 alters the podocyte contractile apparatus via F-actin redistribution [39] that may lead to modification of filtration surface area. Enlargement of filtration surface area may result from ultrastructural changes in podocyte morphology and volume. Interestingly, sitaxsentan, a selective ETA receptor antagonist, ameliorated podocyte foot process architecture and cell volume in adriamycin-treated mice [40]. Moreover, ET-1 has been shown to cause phenotypic changes in cultured podocytes, evidenced by the decreased expression of synaptopodin, an F-actin associated protein. Activation of β-arrestin-1 signaling by an ETA receptor-dependent mechanism increased podocyte motility and plasticity, confirming ET-1 involvement in podocyte structure remodeling [40]. In the current study, we observed a significant reduction in synaptopodin in male HbSS mice, consistent with intraglomerular ET-1 increase leading to podocyte structure reorganization, increased cell volume, and resultant changes in filtration surface area. This is in agreement with our previous studies, which provided direct evidence for ET-1/ETA signaling involvement in podocyte health in HbSS mice [21]. Long-term treatment with a selective ETA receptor antagonist preserved structural integrity of podocyte foot processes and actin-associated cytoskeletal proteins in HbSS mice [21].

Size selectivity of the filtration barrier is a crucial component of glomerular filtration and damage to this barrier is associated with increased Palb and albuminuria [41,42,43]. Moreover, SCD patients have reduced glomerular glycocalyx volume [44]. Recent studies indicated that ET-1 induces heparinase release from podocytes and glycocalyx damage [45,16]. Furthermore, podocyte-specific deletion of ET-1 receptors prevented this damage in diabetic mice [45,16]. Our current data suggest that activation of ETA signaling in SCD glomeruli may influence filtration barrier properties during hyperfiltration. It is likely that endothelial activation or local release of ET-1 is triggered by hypoxia [46] and/or heme [47] to provide a stimulus for podocytes to release heparinase, an enzyme that cleaves negatively charged sulfated proteoglycans. The loss of proteoglycans results in easier passage of the filtrate, including larger proteins, that potentially alter hydraulic conductivity and increase Kf. Interestingly, selective ETA receptor blockade preserves the glomerular endothelial glycocalyx and glomerular charge barrier in vitro in diabetic milieu and diabetic apoE mice [48] as well as reduces proteinuria and improves Palb in streptozotocin-induced diabetic rats [49]. Our previous data confirmed that ETA receptor blockade has a direct protective effect on Palb in HbSS mice [21]. These pre-clinical observations suggest that ET-1 could also be involved in structural changes of filtration barrier thus ‘promoting’ hyperfiltration in SCD nephropathy.

A decrease in size selectivity and enhanced macroglomerular trafficking reported in SCD may account for increased Palb [15]. In the current study, we report increased Palb during the hyperfiltration phase in HbSS mice, regardless of sex. Although increased Palb is usually associated with glomerular injury, studies reported by Schmitt et al. [15] provide convincing evidence that increased Kf and Palb coexist in SCD patients at a very early stage. Similar to our findings in the humanized mouse model, SCD patients with more advanced glomerular dysfunction, a declining GFR and Kf was accompanied by a loss of glomerular permselectivity [33]. Thus, it appears that an increase in Kf and Palb represent the onset of early glomerular changes.

Our laboratory has provided abundant evidence that ET signaling is associated with sex differences in normal renal physiology and multiple kidney-related diseases [50,21,51], including SCD [9]. While female mice are relatively protected and have a survival advantage in the humanized SCD mouse [52], how this could apply to SCD-associated kidney disease remains unclear. Our previous studies in SCD mice reported a rapid hyperfiltration followed by renal damage in males that was more severe compared with females indicating existence of protective mechanisms in female mice to delay the onset of nephropathy [9]. Here we observed clear sex differences in the ET-1 expression profile and glomerular integrity in HbSS mice, along with novel functional Palb data. These intriguing observations raise the question whether the delayed renal phenotype in females is associated with sex steroids, and/or whether SCD-stimulated ET-1 could explain why nephropathy in females is slower to develop. Ovarian hormones appear to protect against of a wide range of renal diseases [53,54,55]. Numerous studies have shown the benefits of ovarian hormones, specifically, estrogen or estrogen receptor agonists, are remarkably similar to those of ETA receptor antagonists. For example, Zimmerman et al. [56] have shown that long-term estrogen treatment in ovariectomized Long-Evans rats lowers blood pressure, improves vasodilator ability and reduces renal injury. Similar findings were observed in aging Fisher-344 rats [57]. While the assumption is that most of these effects are through classical estrogen steroid receptors, the G protein-coupled estrogen receptor, GPR30, is also important for reducing oxidative stress and related injury [58]. It is possible that the ETA protection and estrogen protection function through similar pathways, although this requires further study. Further, testosterone inhibits nitric oxide (NO) synthesis and NO production that could exacerbate the development of injury in males [59,60].

Arginase, an important endogenous substrate for NO production may also provide some explanation for the observed sex differences in SCD nephropathy [61]. Arginase as well as NO synthase use arginine as a substrate. Arginase is elevated in the patients with increased hemolytic rate [62], which could contribute to reduced NO bioavailability that has been reported in male SCD patients, while females appear to have increased basal NO production and NO-dependent relaxation [63]. A recent study examining a rodent model of chronic NO inhibition shows that females develop less severe chronic kidney disease than age-matched males [64]. Considering the effect of testosterone to also reduce NO and the adverse effects of NO deficiency on renal complications in SCD, we speculate that female sex can account for a milder renal phenotype reported in our HbSS mice due to better NO activity. To further confirm the effect of sex hormones on renal phenotype, studies utilizing ovariectomized and/or gonadectomized HbSS mice should be performed along with direct measurement of arginase activity. Another explanation for sex-related differences in the rate of renal disease progression in males versus females with SCD could be related to hemolysis and anemia status [65,52], fetal hemoglobin levels [66], or susceptibility to ischemia–reperfusion injury [67]. Although our previous studies have not shown any differences in anemia status in HbSS mice, we observed a greater degree of renal hypoxia in males, suggesting greater susceptibility to vascular stasis and recurrent acute kidney injury episodes [9].

In conclusion, the current studies offer a new perspective for understanding potential mediators involved in complex mechanisms underlying the onset of SCD nephropathy and raise the translational relevance of sex disparities and the usage of ET-1 receptor antagonists as a therapeutic value for treating SCD kidney disease.

Clinical perspectives

  • A clear sex difference exists in the rate of progression of nephropathy in the humanized sickle mouse model that requires clinical investigation.

  • ET-1-dependent ultrastructural changes in filtration barrier components leading to initial increased in GFR, appear to contribute to hyperfiltration in SCD.

  • ET-1 receptor antagonists may represent a therapeutic value for treating SCD-associated kidney disease.

Acknowledgments

The authors thank Binli Tao for his expertise in managing our animal colony.

Funding

This work was supported by the National Heart, Lung, and Blood Institute [grant number U01 HL117684 (to D.M.P.)]; an American Society of Nephrology Joseph A. Carlucci Research Award (to M.K.); and an American Heart Association Career Development Award [grant number 19CDA34660073 (to M.K.)].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

M.K. and D.M.P. designed the project. M.K. performed experiments. M.K. and D.M.P. analyzed and interpreted data, and prepared the manuscript.

Abbreviations

     
  • σalb

    albumin reflection coefficient

  •  
  • Δπ

    oncotic gradient

  •  
  • BSA

    bovine serum albumin

  •  
  • ET-1

    endothelin-1

  •  
  • ETA

    endothelin A receptor

  •  
  • GFR

    glomerular filtration rate

  •  
  • HbAA

    humanized genetic control mouse

  •  
  • HbSS

    humanized sickle cell mouse

  •  
  • Kf

    ultrafiltration coefficient

  •  
  • NO

    nitric oxide

  •  
  • Palb

    glomerular permeability to albumin

  •  
  • PBS

    phosphate buffered saline

  •  
  • SCD

    sickle cell disease

  •  
  • t

    time

  •  
  • UAB

    University of Alabama at Birmingham

  •  
  • V

    glomerular volume

  •  
  • WT-1

    Wilms’ tumor antigen 1

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