# Altered Expression Profile of Renalα1D-Adrenergic Receptor in Diabetes and Its Modulation by PPAR Agonists

Authors: Xueying Zhao; Yuanyuan Zhang; Michelle Leander; Lingyun Li; Guoshen Wang; Nerimiah Emmett
Journal: Journal of Diabetes Research
 (2014)

---

## Abstract

Alpha
1
D-adrenergic receptor (α1D-AR) plays important roles in regulating physiological and pathological responses mediated by catecholamines, particularly in the cardiovascular and urinary systems. The present study was designed to investigate the expression profile of α1D-AR in the diabetic kidneys and its modulation by activation of peroxisome proliferator-activated receptors (PPARs). 12-week-old Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with fenofibrate or rosiglitazone for 8–10 weeks. Gene microarray, real-time PCR, and confocal immunofluorescence microscopy were performed to assess mRNA and protein expression of α1D-AR in rat kidney tissue. Using microarray, we found that α1D-AR gene was dramatically upregulated in 22-week-old ZD rats compared to ZL controls. Quantitative PCR analysis verified a 16-fold increase in α1D-AR mRNA in renal cortex from ZD animals compared to normal controls. Chronic treatment with fenofibrate or rosiglitazone reduced renal cortical α1D-AR gene. Immunofluorescence staining confirmed that α1D-AR protein was induced in the glomeruli and tubules of diabetic rats. Moreover, dual immunostaining for α1D-AR and kidney injury molecule-1 indicated that α1D-AR was expressed in dedifferentiated proximal tubules of diabetic Zucker rats. Taken together, our results show that α1D-AR expression is upregulated in the diabetic kidneys. PPAR activation suppressed renal expression of α1D-AR in diabetic nephropathy.


---

## Body

## 1. Introduction

Alpha1-adrenergic receptors (α1-ARs) are a heterogeneous family of G-protein-coupled receptors that present in most human and animal tissues, and there are considerable variations in the expression levels of α1-AR subtypes in various tissues of different species [1–4]. α1-ARs play important roles in regulating physiological and pathological responses mediated by catecholamines, particularly in the cardiovascular and urinary systems. In addition to mediating catecholamine-induced vasoconstriction, the α1-ARs in vascular wall have been shown to promote proliferation and hypertrophy of arterial smooth muscle cells and adventitial fibroblasts [5, 6]. In the urinary system, the demonstration of α1-AR expression in human prostate, bladder muscle, and smooth muscles has led to the treatment of bladder outlet obstruction and ureteral stones, via blocking these receptors [7, 8].Of the threeα1-AR subtypes (α1A, α1B, and α1D), the α1D-AR has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [9–11]. However, recent experiments performed in α1D-AR knockout models suggest that this α1-AR subtype plays an important role in the overall regulation of blood pressure [12]. Additionally, Armenia et al. reported that α1A- and α1D-ARs are the major functional subtypes of renal α1-ARs in both normal and streptozotocin-induced diabetic Sprague-Dawley rats [13].Using in situ hybridization, Kurooka et al. identified the gene expression of all threeα1-AR subtypes in human kidney cortex [14]. They found that intense α1-AR mRNA staining was apparent especially in the smooth muscle of arterial walls, whereas weak staining of each of the α1-AR mRNAs was observed in the glomeruli and renal tubules [14]. More recently, the presence and distribution of subtypes in human renal pelvis and calyces were evaluated, and α1D-AR was most dense in both followed by α1A and α1B [8]. However, the expression and distribution patterns of α1D-AR in normal and diabetic rat kidneys remain unknown.In the present study, we examined mRNA levels of threeα1-AR subtypes in the kidney cortex of normal Zucker lean (ZL) and Zucker diabetic (ZD) rats by microarray and real-time PCR analyses. Confocal immunofluorescence microscopy was performed to evaluate the distribution of α1D-AR in the kidney of Zucker rats. Furthermore, we investigated whether activation of peroxisome proliferator-activated receptor-α (PPARα) or PPARγ, known renoprotective intervention in animal models of type 2 diabetes, would affect renal expression of α1-AR subtypes.


## 2. Materials and Methods

### 2.1. Experimental Animals

Six-week-old male Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were purchased from Charles River Laboratories (Wilmington, MA, USA). Rats were housed in a temperature-controlled room with a 12 : 12-hour light-dark cycle and free access to Purina 5008 rat chow and water. Blood glucose was monitored using the Accu-chek glucometer by tail-vein blood sampling. In a previous study in which we characterized the time course of blood glucose in this model [15], we showed that blood glucose of the ZD rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter. The rats were housed in the animal care facility at the Morehouse School of Medicine that is AAALAC accredited. All animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the requirements stated in the National Institutes of Health Guide for the Care and Use Laboratory Animals.For fenofibrate treatment, 12-week-old Zucker rats were divided into 3 experimental groups: vehicle- (0.5% carboxymethylcellulose ig) treated ZL, vehicle-treated ZD, or fenofibrate- (150 mg/kg/day ig) treated ZD (F-ZD) rats for 10 weeks. For rosiglitazone treatment, 12-week-old ZD animals were treated with rosiglitazone (10 mg/kg/day in drinking water) or vehicles for 8 weeks.


### 2.2. Isolation of Glomeruli

Glomeruli were isolated from the kidney cortex of ZL and ZD rats at the age of 7, 12, and 20–22 weeks, respectively, by a modified procedure as described previously [16]. Briefly, the rats were anesthetized and the kidneys were rapidly removed and placed in Hanks’ balanced salt solution (HBSS) at pH 7.4. The renal cortex was dissected and cut into small pieces with a surgical blade. Glomeruli were isolated by passing the tissues successively through calibrated sieves (pore size: 200, 125, and 65 μm) and rinsed with HBSS. Isolated glomeruli, collected on the 65 μm sieve, were resuspended in HBSS. They were devoid of Bowman’s capsule. Tubular fragments were less than 3% of the total number of isolated glomeruli [16].


### 2.3. RNA Extraction

Total RNA was prepared from isolated glomeruli or kidney cortex by using ultrapure TRIzol reagent according to the manufacturer’s instructions (GIBCO-BRL, Grand Island, NY). The quality of the RNA samples was assessed using the Agilent 2100 Bioanalyzer (G2938A). RNA concentrations were determined spectrophotometrically (absorbance at 260 and 280 nm).


### 2.4. Microarray Analysis

Seven hundred and fifty nanograms of total RNA per sample were used for cRNA synthesis and amplification. Agilent Spike-In control RNA was included as an internal control. Cyanine-3-(Cy3-) labeled cRNA was purified and hybridized to Agilent Whole Rat Genome 44k Oligo Microarray chips (p/n G2519F-14870, Agilent Technologies) according to the manufacturer’s instructions. The processed microarrays were scanned with the Agilent G2565BA DNA Microarray Scanner (p/n G2505-A). The scanned images were analyzed with Agilent Feature software (version 9.5.1.1) using default parameters. The resulting text files were loaded into the Agilent GeneSpring GX software (version 7.3) for further analysis. Gene expression values of all datasets were normalized using median normalization. Significantly differentially expressedα-adrenergic receptor genes among ZL, ZD, and F-ZD groups were identified by a threshold of ≥2 fold change and P≤0.05.


### 2.5. Real-Time PCR forα1A-, α1B-, and α1D-AR mRNA Expression

Reverse transcription was performed on equal amounts of total RNA by using random hexanucleotide primers to produce a cDNA library for each sample. Real-time PCR reactions were run on an iCycler iQ Real-Time PCR Detection System by using Taqman Universal PCR Master Mix (Applied Biosystems, P/N 4304437).α1A-, α1B-, and α1D-AR and β-actin gene-specific Taqman probe and primer sets were obtained from Applied Biosystems as Assays-on-Demand (AOD) gene expression products. The AOD identification numbers were Rn00567876 for α1A-AR, Rn01471343 for α1B-AR, Rn00577931 for α1D-AR, and 4331182 for rat β-actin endogenous control. Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2-ΔΔCt) compared with controls.


### 2.6. Immunostaining

For immunofluorescent staining, 5μm thick cryostat sections of OCT-embedded kidney samples were used. To study the localization of α1D-AR in the rat kidney, the sections were incubated with a mixture of two antibodies overnight: rabbit anti-α1D-AR antibody (1 : 100, Sigma-Aldrich, St. Louis, MO), mouse anti-α-smooth muscle actin (α-SMA, 1 : 100, Santa Cruz Biotechnology, Dallas, TX), or goat anti-kidney injury molecule-1 (Kim-1, 1 : 100, R&D Systems, Minneapolis, MN). As a negative control, the sections were exposed to nonimmune IgG (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining occurred. The sections were observed and imaged by Leica confocal microscope.


### 2.7. Statistical Analysis

Data are expressed as mean ± SEM. Student’st-test was used for comparison between the two groups. Comparisons among multiple groups were performed by one-way ANOVA and Newman-Keuls post hoc test. P<0.05 was considered statistically significant.


## 2.1. Experimental Animals

Six-week-old male Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were purchased from Charles River Laboratories (Wilmington, MA, USA). Rats were housed in a temperature-controlled room with a 12 : 12-hour light-dark cycle and free access to Purina 5008 rat chow and water. Blood glucose was monitored using the Accu-chek glucometer by tail-vein blood sampling. In a previous study in which we characterized the time course of blood glucose in this model [15], we showed that blood glucose of the ZD rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter. The rats were housed in the animal care facility at the Morehouse School of Medicine that is AAALAC accredited. All animal protocols were approved by the Institutional Animal Care and Use Committee and were in accordance with the requirements stated in the National Institutes of Health Guide for the Care and Use Laboratory Animals.For fenofibrate treatment, 12-week-old Zucker rats were divided into 3 experimental groups: vehicle- (0.5% carboxymethylcellulose ig) treated ZL, vehicle-treated ZD, or fenofibrate- (150 mg/kg/day ig) treated ZD (F-ZD) rats for 10 weeks. For rosiglitazone treatment, 12-week-old ZD animals were treated with rosiglitazone (10 mg/kg/day in drinking water) or vehicles for 8 weeks.


## 2.2. Isolation of Glomeruli

Glomeruli were isolated from the kidney cortex of ZL and ZD rats at the age of 7, 12, and 20–22 weeks, respectively, by a modified procedure as described previously [16]. Briefly, the rats were anesthetized and the kidneys were rapidly removed and placed in Hanks’ balanced salt solution (HBSS) at pH 7.4. The renal cortex was dissected and cut into small pieces with a surgical blade. Glomeruli were isolated by passing the tissues successively through calibrated sieves (pore size: 200, 125, and 65 μm) and rinsed with HBSS. Isolated glomeruli, collected on the 65 μm sieve, were resuspended in HBSS. They were devoid of Bowman’s capsule. Tubular fragments were less than 3% of the total number of isolated glomeruli [16].


## 2.3. RNA Extraction

Total RNA was prepared from isolated glomeruli or kidney cortex by using ultrapure TRIzol reagent according to the manufacturer’s instructions (GIBCO-BRL, Grand Island, NY). The quality of the RNA samples was assessed using the Agilent 2100 Bioanalyzer (G2938A). RNA concentrations were determined spectrophotometrically (absorbance at 260 and 280 nm).


## 2.4. Microarray Analysis

Seven hundred and fifty nanograms of total RNA per sample were used for cRNA synthesis and amplification. Agilent Spike-In control RNA was included as an internal control. Cyanine-3-(Cy3-) labeled cRNA was purified and hybridized to Agilent Whole Rat Genome 44k Oligo Microarray chips (p/n G2519F-14870, Agilent Technologies) according to the manufacturer’s instructions. The processed microarrays were scanned with the Agilent G2565BA DNA Microarray Scanner (p/n G2505-A). The scanned images were analyzed with Agilent Feature software (version 9.5.1.1) using default parameters. The resulting text files were loaded into the Agilent GeneSpring GX software (version 7.3) for further analysis. Gene expression values of all datasets were normalized using median normalization. Significantly differentially expressedα-adrenergic receptor genes among ZL, ZD, and F-ZD groups were identified by a threshold of ≥2 fold change and P≤0.05.


## 2.5. Real-Time PCR forα1A-, α1B-, and α1D-AR mRNA Expression

Reverse transcription was performed on equal amounts of total RNA by using random hexanucleotide primers to produce a cDNA library for each sample. Real-time PCR reactions were run on an iCycler iQ Real-Time PCR Detection System by using Taqman Universal PCR Master Mix (Applied Biosystems, P/N 4304437).α1A-, α1B-, and α1D-AR and β-actin gene-specific Taqman probe and primer sets were obtained from Applied Biosystems as Assays-on-Demand (AOD) gene expression products. The AOD identification numbers were Rn00567876 for α1A-AR, Rn01471343 for α1B-AR, Rn00577931 for α1D-AR, and 4331182 for rat β-actin endogenous control. Each sample was run in triplicate, and the comparative threshold cycle (Ct) method was used to quantify fold increase (2-ΔΔCt) compared with controls.


## 2.6. Immunostaining

For immunofluorescent staining, 5μm thick cryostat sections of OCT-embedded kidney samples were used. To study the localization of α1D-AR in the rat kidney, the sections were incubated with a mixture of two antibodies overnight: rabbit anti-α1D-AR antibody (1 : 100, Sigma-Aldrich, St. Louis, MO), mouse anti-α-smooth muscle actin (α-SMA, 1 : 100, Santa Cruz Biotechnology, Dallas, TX), or goat anti-kidney injury molecule-1 (Kim-1, 1 : 100, R&D Systems, Minneapolis, MN). As a negative control, the sections were exposed to nonimmune IgG (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining occurred. The sections were observed and imaged by Leica confocal microscope.


## 2.7. Statistical Analysis

Data are expressed as mean ± SEM. Student’st-test was used for comparison between the two groups. Comparisons among multiple groups were performed by one-way ANOVA and Newman-Keuls post hoc test. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. Differential Gene Expression of Alpha-Adrenergic Receptor Subtypes in the Diabetic Kidney

We performed microarray analysis to assess gene expression levels ofα-adrenergic receptor subtypes in the kidney cortex of 22-week-old normal ZL (blood glucose: 108±8 mg/dL) and diabetic ZD (blood glucose: 425±35 mg/dL) rats. The expression profile of each experimental group was determined in three animals per group. Table 1 shows gene expression levels of α1- and α2-adrenergic receptor subtypes detected in rat kidney tissue. The rank order of expression levels of the three α1-AR mRNAs in normal rat kidney cortex was α1B>α1D>α1A. Among these genes, α1D-AR was significantly upregulated in the kidney cortex of diabetic animals. Compared to normal ZL controls, renal cortical α1D-AR gene was increased by 553% in 22-week-old ZD rats (Table 1).Table 1
Alpha-adrenergic receptor gene expression in the kidney cortex of Zucker lean (ZL), untreated Zucker diabetic fatty (ZD), and fenofibrate-treated diabetic fatty (F-ZD) rats.
Accession number
Gene name
mRNA expression ZL
ZD versus ZL
F-ZD versus ZD
Fold change
P value
Fold change
P value
NM_017191
α
1A-adrenergic receptor (α1A-AR)
10.4 ± 2.9
NA
NA
NA
NA
NM_016991
α
1B-adrenergic receptor (α1B-AR)
1372.2 ± 101.8
1.49
NS
1.26
NS
NM_024483
α
1D-adrenergic receptor (α1D-AR)
273.3 ± 142.9
6.53
<0.001
−2.75
<0.001
NM_012739
α
2A-adrenergic receptor (α2A-AR)
53.2 ± 15.2
−1.09
NS
1.02
NS
NM_138505
α
2B-adrenergic receptor (α2B-AR)
116.8 ± 22.8
−1.18
NS
1.05
NS
NM_138506
α
2C-adrenergic receptor (α2C-AR)
33.1 ± 7.9
1.19
NS
1.07
NS
Data are shown in two ways: mRNA expression level presenting the relative abundance of different alpha-adrenergic receptor subtypes in normal rat kidney cortex and fold change showing the differential expression of these genes among ZL, ZD, and F-ZD groups. There is significant (P<0.001) upregulation in renal expression of α1D-AR gene in the ZD animals, which is markedly prevented by fenofibrate treatment (P<0.001). NA: not analyzed due to its low expression level in ZL, ZD, and F-ZD animals; NS: not significant.To verify the relative transcript levels ofα1A-, α1B-, and α1D-AR subtypes derived from the microarray experiment, we performed quantitative real-time PCR (qPCR) assay. The relative expression levels of α1A-, α1B-, and α1D-AR mRNAs normalized against β-actin in rat kidney cortex are shown in Figure 1. In consistency with the microarray results, qPCR analysis showed that renal cortical α1D mRNA was increased by 16-fold in 22-week-old ZD rats compared to ZL controls.mRNA expression of renal corticalα1A-, α1B-, and α1D-AR subtypes was measured by quantitative real-time PCR in 22-week-old Zucker lean (ZL), vehicle-treated Zucker diabetic (ZD), and fenofibrate-treated Zucker diabetic (F-ZD) rats. Fold changes of α1A- (a), α1B- (b), and α1D- (c) AR genes were calculated using β-actin as an internal control. Values are mean ± SEM. n=5-6 animals/group. P***<0.001 versus ZL; P###<0.001 versus ZD group.
(a)
(b)
(c)


### 3.2. Activation of PPARα or PPARγ Inhibited the Upregulation of α1D-AR mRNA Expression in the Diabetic Kidneys

We have previously shown that PPAR activation protects against kidney injury in Zucker diabetic fatty rats [15, 17]. Here, we further examined the effect of PPAR activation on the expression level of α1-AR mRNAs in the diabetic kidneys. Gene microarray analysis revealed that PPARα activation inhibits the upregulation of α1D gene in the diabetic kidneys. Chronic administration of fenofibrate, a PPARα agonist, resulted in a decrease in α1D-AR mRNA by 64% compared to vehicle-treated ZD animals (Table 1). qPCR analysis confirmed a reduction of α1D-AR mRNA in the kidney of F-ZD rats (Figure 1). In contrast, both microarray and qPCR analyses indicated that mRNA expression of α1A- and α1B-AR subtypes was not affected by fenofibrate in the diabetic kidneys.Additionally, the effect of PPARγ activation on gene expression of α1-AR subtypes was examined in the ZD rats after rosiglitazone treatment. Compared to vehicle-treated ZD rats, renal cortical α1D-AR mRNA was significantly lower when rosiglitazone was administered for 8 weeks (Figure 2). Similar to PPARα activation, α1A- and α1B-AR mRNA expression in the diabetic rats was not affected by rosiglitazone treatment.Effect of PPARγ activationon renal expression of α1A-, α1B-, and α1D-AR subtype mRNAs. Bar graphs present the real-time PCR results of renal cortical α1A- (a), α1B- (b), and α1D- (c) AR mRNAs in 20-week-old ZD rats following treatment with rosiglitazone for 8 weeks. Values are mean ± SEM. n=5-6 animals/group. P#<0.05 versus vehicle-treated ZD group.
(a)
(b)
(c)


### 3.3. Glomerular and Cortical Expression ofα1D-AR mRNA in Zucker Rats of Various Ages

In a previous study [15], we showed that blood glucose levels were not different between normal ZL and diabetic ZD rats at the age of 7 weeks. Blood glucose of the ZD rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter. To analyze the temporal pattern of renal expression of α1D-AR receptors, renal cortical and glomerular α1D-AR mRNA levels were compared in the Zucker rats at the ages of 7, 12, and 20–22 weeks. As shown in Figure 3, ZD rats at week 7 had slightly lower α1D-AR mRNA level in the glomeruli compared to their ZL littermates, whereas renal cortical α1D-AR mRNAs were not different between the two groups. At the age of 12, both renal glomerular (2.4-fold) and cortical (1.7-fold) α1D-AR mRNA levels were significantly higher in the ZD animals, which were further increased by 3.4-fold and 12.9-fold, respectively, at the age of 20–22 weeks.mRNA expression ofα1D-AR in the glomeruli and cortex of normal ZL and diabetic ZD rats at different time points. α1D-AR gene was progressively increased in both glomeruli (a) and cortex (b) of ZD rats compared to normal ZL littermates. mRNA fold changes of α1D-AR were calculated using β-actin as an internal control. Values are mean ± SEM. n=5-6 animals/group. P*<0.05, P**<0.01 versus ZL control group.
(a)
(b)


### 3.4. Expression and Distribution ofα1D-AR Protein in the Kidney of Zucker Rats

We performed immunofluorescence staining to correlateα1D-AR gene expression results with its protein level and distribution in the kidney of Zucker animals. As expected, α1D-AR protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals (Figure 4). The intense staining was primarily in the smooth muscle of renal arterial walls as evidenced by its colocalization with α-SMA. In normal ZL rats, weak α1D-AR staining was also detected in the glomeruli, whereas there was no obvious staining in the renal tubules (Figure 4). In consistency with the mRNA expression results, α1D-AR protein was largely induced in the diabetic ZD kidneys. Immunofluorescence staining identified increased α1D-AR signal in the glomeruli of diabetic rats, which was partially colocalized with α-SMA (Figure 4). Moreover, intense tubulointerstitial staining of α1D-AR was apparent in the diabetic kidneys. As shown in Figure 5, α1D-AR was expressed in both tubular epithelial cells and activated interstitial fibroblasts, which was positive for α-SMA staining.Figure 4
Representative confocal immunofluorescence images ofα1D-AR and α-smooth muscle actin (α-SMA) in the glomeruli of ZL and ZD rats. In normal control, colocalization of α1D-AR (green) with α-SMA (dark blue) was apparent in the renal vasculature (white arrow), whereas weak staining of α1D-AR was detected in the glomeruli. An increase in α1D-AR staining in the diabetic glomeruli was accompanied by an increase in α-SMA signal.Figure 5
Increased tubulointerstitialα1D-AR staining in the kidney of 20-week-old Zucker diabetic rats. In the diabetic kidneys, an induction of α1D-AR protein (green) was detected in the epithelium of dilated tubules as well as the surrounding interstitial cells, which are α-SMA-positive (dark blue). Images in (a′), (b′), and (c′) are enlarged from the boxed areas in (a), (b), and (c).To further characterizeα1D-AR expression in renal tubules of diabetic animals, we performed double staining for α1D-AR and Kim-1, a sensitive tubular injury marker. Dual labeling revealed a spatial relationship between Kim-1 and α1D-AR in the diabetic kidneys. As shown in Figure 6, virtually all dilated tubules expressing α1D-AR were also Kim-1-positive, suggesting that α1D-AR was expressed in the injured dedifferentiated proximal tubules. In these tubules, α1D-AR expression was predominantly cytoplasmic, whereas Kim-1 staining was prominent at the apical membrane.Figure 6
Representative confocal images of double staining forα1D-AR and Kim-1 in the tubules of 20-week-old Zucker diabetic rats. Colocalization of α1D-AR (green) and Kim-1 (red) was seen in the dilated proximal tubules of diabetic rats. Images in (a′), (b′), and (c′) are enlarged from the boxed areas in (a), (b), and (c).


## 3.1. Differential Gene Expression of Alpha-Adrenergic Receptor Subtypes in the Diabetic Kidney

We performed microarray analysis to assess gene expression levels ofα-adrenergic receptor subtypes in the kidney cortex of 22-week-old normal ZL (blood glucose: 108±8 mg/dL) and diabetic ZD (blood glucose: 425±35 mg/dL) rats. The expression profile of each experimental group was determined in three animals per group. Table 1 shows gene expression levels of α1- and α2-adrenergic receptor subtypes detected in rat kidney tissue. The rank order of expression levels of the three α1-AR mRNAs in normal rat kidney cortex was α1B>α1D>α1A. Among these genes, α1D-AR was significantly upregulated in the kidney cortex of diabetic animals. Compared to normal ZL controls, renal cortical α1D-AR gene was increased by 553% in 22-week-old ZD rats (Table 1).Table 1
Alpha-adrenergic receptor gene expression in the kidney cortex of Zucker lean (ZL), untreated Zucker diabetic fatty (ZD), and fenofibrate-treated diabetic fatty (F-ZD) rats.
Accession number
Gene name
mRNA expression ZL
ZD versus ZL
F-ZD versus ZD
Fold change
P value
Fold change
P value
NM_017191
α
1A-adrenergic receptor (α1A-AR)
10.4 ± 2.9
NA
NA
NA
NA
NM_016991
α
1B-adrenergic receptor (α1B-AR)
1372.2 ± 101.8
1.49
NS
1.26
NS
NM_024483
α
1D-adrenergic receptor (α1D-AR)
273.3 ± 142.9
6.53
<0.001
−2.75
<0.001
NM_012739
α
2A-adrenergic receptor (α2A-AR)
53.2 ± 15.2
−1.09
NS
1.02
NS
NM_138505
α
2B-adrenergic receptor (α2B-AR)
116.8 ± 22.8
−1.18
NS
1.05
NS
NM_138506
α
2C-adrenergic receptor (α2C-AR)
33.1 ± 7.9
1.19
NS
1.07
NS
Data are shown in two ways: mRNA expression level presenting the relative abundance of different alpha-adrenergic receptor subtypes in normal rat kidney cortex and fold change showing the differential expression of these genes among ZL, ZD, and F-ZD groups. There is significant (P<0.001) upregulation in renal expression of α1D-AR gene in the ZD animals, which is markedly prevented by fenofibrate treatment (P<0.001). NA: not analyzed due to its low expression level in ZL, ZD, and F-ZD animals; NS: not significant.To verify the relative transcript levels ofα1A-, α1B-, and α1D-AR subtypes derived from the microarray experiment, we performed quantitative real-time PCR (qPCR) assay. The relative expression levels of α1A-, α1B-, and α1D-AR mRNAs normalized against β-actin in rat kidney cortex are shown in Figure 1. In consistency with the microarray results, qPCR analysis showed that renal cortical α1D mRNA was increased by 16-fold in 22-week-old ZD rats compared to ZL controls.mRNA expression of renal corticalα1A-, α1B-, and α1D-AR subtypes was measured by quantitative real-time PCR in 22-week-old Zucker lean (ZL), vehicle-treated Zucker diabetic (ZD), and fenofibrate-treated Zucker diabetic (F-ZD) rats. Fold changes of α1A- (a), α1B- (b), and α1D- (c) AR genes were calculated using β-actin as an internal control. Values are mean ± SEM. n=5-6 animals/group. P***<0.001 versus ZL; P###<0.001 versus ZD group.
(a)
(b)
(c)


## 3.2. Activation of PPARα or PPARγ Inhibited the Upregulation of α1D-AR mRNA Expression in the Diabetic Kidneys

We have previously shown that PPAR activation protects against kidney injury in Zucker diabetic fatty rats [15, 17]. Here, we further examined the effect of PPAR activation on the expression level of α1-AR mRNAs in the diabetic kidneys. Gene microarray analysis revealed that PPARα activation inhibits the upregulation of α1D gene in the diabetic kidneys. Chronic administration of fenofibrate, a PPARα agonist, resulted in a decrease in α1D-AR mRNA by 64% compared to vehicle-treated ZD animals (Table 1). qPCR analysis confirmed a reduction of α1D-AR mRNA in the kidney of F-ZD rats (Figure 1). In contrast, both microarray and qPCR analyses indicated that mRNA expression of α1A- and α1B-AR subtypes was not affected by fenofibrate in the diabetic kidneys.Additionally, the effect of PPARγ activation on gene expression of α1-AR subtypes was examined in the ZD rats after rosiglitazone treatment. Compared to vehicle-treated ZD rats, renal cortical α1D-AR mRNA was significantly lower when rosiglitazone was administered for 8 weeks (Figure 2). Similar to PPARα activation, α1A- and α1B-AR mRNA expression in the diabetic rats was not affected by rosiglitazone treatment.Effect of PPARγ activationon renal expression of α1A-, α1B-, and α1D-AR subtype mRNAs. Bar graphs present the real-time PCR results of renal cortical α1A- (a), α1B- (b), and α1D- (c) AR mRNAs in 20-week-old ZD rats following treatment with rosiglitazone for 8 weeks. Values are mean ± SEM. n=5-6 animals/group. P#<0.05 versus vehicle-treated ZD group.
(a)
(b)
(c)


## 3.3. Glomerular and Cortical Expression ofα1D-AR mRNA in Zucker Rats of Various Ages

In a previous study [15], we showed that blood glucose levels were not different between normal ZL and diabetic ZD rats at the age of 7 weeks. Blood glucose of the ZD rats began to increase at week 8, reached a peak at week 12, and remained at this higher level thereafter. To analyze the temporal pattern of renal expression of α1D-AR receptors, renal cortical and glomerular α1D-AR mRNA levels were compared in the Zucker rats at the ages of 7, 12, and 20–22 weeks. As shown in Figure 3, ZD rats at week 7 had slightly lower α1D-AR mRNA level in the glomeruli compared to their ZL littermates, whereas renal cortical α1D-AR mRNAs were not different between the two groups. At the age of 12, both renal glomerular (2.4-fold) and cortical (1.7-fold) α1D-AR mRNA levels were significantly higher in the ZD animals, which were further increased by 3.4-fold and 12.9-fold, respectively, at the age of 20–22 weeks.mRNA expression ofα1D-AR in the glomeruli and cortex of normal ZL and diabetic ZD rats at different time points. α1D-AR gene was progressively increased in both glomeruli (a) and cortex (b) of ZD rats compared to normal ZL littermates. mRNA fold changes of α1D-AR were calculated using β-actin as an internal control. Values are mean ± SEM. n=5-6 animals/group. P*<0.05, P**<0.01 versus ZL control group.
(a)
(b)


## 3.4. Expression and Distribution ofα1D-AR Protein in the Kidney of Zucker Rats

We performed immunofluorescence staining to correlateα1D-AR gene expression results with its protein level and distribution in the kidney of Zucker animals. As expected, α1D-AR protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals (Figure 4). The intense staining was primarily in the smooth muscle of renal arterial walls as evidenced by its colocalization with α-SMA. In normal ZL rats, weak α1D-AR staining was also detected in the glomeruli, whereas there was no obvious staining in the renal tubules (Figure 4). In consistency with the mRNA expression results, α1D-AR protein was largely induced in the diabetic ZD kidneys. Immunofluorescence staining identified increased α1D-AR signal in the glomeruli of diabetic rats, which was partially colocalized with α-SMA (Figure 4). Moreover, intense tubulointerstitial staining of α1D-AR was apparent in the diabetic kidneys. As shown in Figure 5, α1D-AR was expressed in both tubular epithelial cells and activated interstitial fibroblasts, which was positive for α-SMA staining.Figure 4
Representative confocal immunofluorescence images ofα1D-AR and α-smooth muscle actin (α-SMA) in the glomeruli of ZL and ZD rats. In normal control, colocalization of α1D-AR (green) with α-SMA (dark blue) was apparent in the renal vasculature (white arrow), whereas weak staining of α1D-AR was detected in the glomeruli. An increase in α1D-AR staining in the diabetic glomeruli was accompanied by an increase in α-SMA signal.Figure 5
Increased tubulointerstitialα1D-AR staining in the kidney of 20-week-old Zucker diabetic rats. In the diabetic kidneys, an induction of α1D-AR protein (green) was detected in the epithelium of dilated tubules as well as the surrounding interstitial cells, which are α-SMA-positive (dark blue). Images in (a′), (b′), and (c′) are enlarged from the boxed areas in (a), (b), and (c).To further characterizeα1D-AR expression in renal tubules of diabetic animals, we performed double staining for α1D-AR and Kim-1, a sensitive tubular injury marker. Dual labeling revealed a spatial relationship between Kim-1 and α1D-AR in the diabetic kidneys. As shown in Figure 6, virtually all dilated tubules expressing α1D-AR were also Kim-1-positive, suggesting that α1D-AR was expressed in the injured dedifferentiated proximal tubules. In these tubules, α1D-AR expression was predominantly cytoplasmic, whereas Kim-1 staining was prominent at the apical membrane.Figure 6
Representative confocal images of double staining forα1D-AR and Kim-1 in the tubules of 20-week-old Zucker diabetic rats. Colocalization of α1D-AR (green) and Kim-1 (red) was seen in the dilated proximal tubules of diabetic rats. Images in (a′), (b′), and (c′) are enlarged from the boxed areas in (a), (b), and (c).


## 4. Discussion

In this study, the expression and distribution ofα1D-AR mRNA and protein were determined by the gene microarray, qPCR, and confocal immunofluorescence analyses. Although mRNA expression of all three α1-AR subtypes (α1B>α1D>α1A) was detected in rat kidney cortex, only α1D-AR gene was massively upregulated in the diabetic animals. Moreover, diabetes-related increase in α1D-AR mRNA was inhibited when the ZD rats were treated with fenofibrate or rosiglitazone. Immunostaining for α1D-AR confirmed that intense α1D-AR staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys. Weak α1D-AR protein staining was detected in the glomeruli of normal ZL controls, but there was no obvious staining in the normal tubular epithelium. In consistency with the gene expression results, α1D-AR protein was significantly increased in the glomeruli and proximal tubules of diabetic animals.The expression ofα1-AR subtype mRNAs has previously been studied in various animal and human organs, and the predominant subtype mRNA expressed differs among species and organs. For example, Kurooka et al. reported that α1A-AR gene was detected more than α1B-AR or α1D-AR in human kidney cortex [14]. In contrast, Karabacak et al. [8] recently evaluated α1-AR subtype protein expression in human renal pelvis and calyx tissues and found that α1D-AR was most dense in both followed by α1A- and α1B-AR subtypes, respectively, where the rate of α1B-AR was significantly lower than the other two. In the rat kidney, it was reported that the α1B-AR is predominant when detected by a radioligand binding assay [18] and RNase protection assay [19]. In consistency with these findings, we confirmed the expression of all three α1-AR subtype mRNAs (α1B>α1D>α1A) in rat kidney cortex by microarray and qPCR analyses.Of the threeα1-AR subtypes, the α1D-AR has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [9–11]. However, recent experiments performed in α1D-AR knockout models suggest that this α1-AR subtype plays an important role in the overall regulation of blood pressure [12]. Moreover, Armenia et al. reported that α1A- and α1D-ARs are the major functional subtypes of renal α1-ARs in both normal and streptozotocin-induced Sprague-Dawley rats [13]. Interestingly, among the three α1-AR subtypes detected in rat kidney cortex, we found that α1D-AR mRNA was markedly increased by 16-fold in the diabetic kidneys. Additionally, diabetes-associated upregulation of α1D-AR mRNA expression was inhibited when the diabetic animals were treated with PPAR agonists, known renoprotective interventions. Therefore, previous evidences and present results strongly suggest that α1D-AR may play an important role in renal physiology and/or pathophysiology.Diabetic nephropathy is characterized by a series of ultrastructural changes, including glomerular and tubular hypertrophy, mesangial expansion, glomerulosclerosis, and tubulointerstitial fibrosis. We have previously demonstrated a progressive loss of renal function in the diabetic animals as evidenced by an increase in urinary protein to creatinine ratio in the ZD rats between the ages of 7 and 20 weeks [15]. In the current study, we further observed a gradual increase in cortical and glomerular α1D-AR mRNA expression during disease progression in the diabetic animals. We speculate that increased α1D-AR may play a role in the development of diabetic renal hypertrophy and fibrosis. In fact, a role for α1D-AR in vascular hypertrophy and remodeling has been suggested by a recent report that a decrease in the lumen area and an increase in the wall thickness of arteries in hypoxic pulmonary hypertension were strongly inhibited in α1D-AR knockout mice [5]. In this study, we evaluated the expression and distribution of α1D-AR protein in the kidneys of ZL and ZD rats by immunofluorescence staining. As expected, intense α1D-AR staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys. Compared to low expression level of α1D-AR protein in the glomeruli of normal ZL controls, ZD rats demonstrate a significant increase in α1D-AR signal. Moreover, ZD kidneys displayed strong tubulointerstitial α1D-AR signal within fibrotic areas. α1D-AR protein was expressed in both tubular epithelial cells and interstitial cells. A colocalization of α1D-AR with α-SMA indicates that α1D-AR expressing interstitial cells are myofibroblasts. Future work in the field could be necessary to establish the exact role of α1D-AR in activation and proliferation of renal interstitial fibroblasts.Another interesting novelfinding in our set of data reported here is theα1D-AR expression in tubular epithelial cells of diabetic kidneys. Although there was no obvious staining in the normal tubules, α1D-AR staining was apparent in the dilated tubules in the fibrotic areas. To further characterize α1D-AR expression in renal tubules of diabetic animals, the spatial relationship between tubular α1D-AR and Kim-1, a tubular injury marker, was studied by double staining. In tubulointerstitial fibrotic areas, virtually all dilated tubules expressing α1D-AR were also Kim-1-positive. The selective increased expression by dedifferentiated epithelial cells and activated interstitial fibroblasts supports the potential importance of α1D-AR in renal tubulointerstitial injury. Moreover, recent studies on the pulmonary circulation suggest that catecholamines may participate in the excessive muscularization and fibrosis of the arteries through ERK1/2 signaling pathway in hypoxic pulmonary hypertension [5, 20–22]. Therefore, further studies are warranted to evaluate the functional consequences of α1D-AR induction in diabetic kidney injury.In summary, the current study highlights mRNA expression of the threeα1-AR subtypes in rat kidney cortex. An increase in renal expression of α1D-AR mRNA and protein was associated with glomerular and tubulointerstitial injury in diabetic nephropathy. Chronic treatment with PPAR agonists prevents the increase in α1D-AR mRNA in the diabetic kidneys. These findings may provide new insights into the prevention and early management of diabetic kidney disease.



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