apoE Expression in Glomerulus and Correlation with Glomerulosclerosis Induced by Adriamycin in Rats

Abstract

Lipid deposition in glomerulus plays an important role in the progression of glomerulosclerosis (GS), and apolipoprotein E (apoE) is an important protein in cholesterol homeostasis. This investigation was performed to explore whether there exists an association between apoE and GS susceptibility. Eighty Wistar rats were randomly divided into two groups: sham operation group and glomerulosclerosis model group, n = 40. The GS disease in rat was induced by uninephrectomy and injecting adriamycin (5 mg/kg) through the tail vein. At the end of 9 and 13 weeks, 20 rats in each group were killed and the relative samples were collected. Serum creatinine, blood urea nitrogen, and 24-h urine protein were determined. Immunohistochemical analysis was performed on renal tissue to detect the expression of apoE, collagen IV, fibronectin, α-smooth muscle actin, and transforming growth factor-β1 in glomerulus. Real-time reverse transcription polymerase chain reaction was conducted to detect the apoE mRNA expression in renal tissue. Compared with sham operation group at the end of 9/13 weeks, glomerulosclerosis model group exhibited levels of serum creatinine, blood urea nitrogen, 24-h urine protein, and a glomerulosclerosis index that were significantly elevated (  p < 0.01), and collagen IV, fibronectin, α-smooth muscle actin, and transforming growth factor-β1 protein expression and apoE expression (protein and mRNA) were significantly upregulated (p < 0.01). In conclusion, apoE can increase the accumulation of extracellular matrix in glomerulus and may take part in the progression of GS.

• Apolipoprotein E
• extracellular matrix
• glomerulosclerosis

INTRODUCTION

Apolipoprotein E (apoE), a 35-kDa plasma protein synthesized in liver and other organs such as kidney, regulates the lipid catabolism of triglyceride-depleted remnants of chylomicrons and very low density lipoprotein.^1,2 Lipid deposition in glomerulus is an important characteristic of nephritic syndrome. The role of apoE in kidney has been controversial since the first investigation was reported. Investigators reported that the apoE took a protective role for kidney.^3,4 Some other investigations found that the apoE expression in renal glomerulus was elevated when there were some renal diseases.^5–8 However, the available evidence reported to date is weak, due to small sample number in those studies to some extent, especially for some clinical studies. The investigation to explore the association of apoE with the accumulation of extracellular matrix (ECM) in glomerulosclerosis (GS) rats is rare. We performed this experiment to investigate the relation between apoE and GS in rats, with the intention to provide a much more reliable finding on the significance of the association.

MATERIALS AND METHODS

Animal Model

Eighty healthy male rats, 180–200 g, of Wistar backgrounds were purchased from the Experimental Animal Center of Guangxi Medical University, Nanning, China. All the procedures and protocols were approved by the Institutional Animal Care and Use Committee. The rats were divided into two groups randomly: sham operation group (SHO, n = 40) and GS model group without treatment (GS, n = 40). The SHO group was subjected to a sham operation and tail vein injection of normal saline solution alone. GS model in the GS group was induced by uninephrectomy, and adriamycin (ADR) (Wanle Pharmaceutical Co., Shenzhen, China) was injected in single tail vein after 1 week at a dose of 5 mg/kg, after dissolving in sterilized water. At the end of 9 and 13 weeks, 20 rats in each group were sacrificed; serum and urine were collected and stored at –20°C, and their kidneys were collected for histological and molecular biological determination.

At the end of 9 and 13 weeks, 24-h urine specimens were collected from 20 rats in each group. Blood specimens were collected before the rats were sacrificed. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were detected with a 7170-A biochemistry test kit (Hitachi Co., Tokyo, Japan). Urinary protein concentration from 24-h urine sample was measured by the sulfosalicylic acid method.

Renal Morphology

Renal tissues were fixed in 10% neutral formaldehyde, and they were dehydrated through a graded ethanol series and embedded in paraffin. Sections of 4 µm thickness were prepared on a microtome and stained with hematoxylin and eosin. Renal damage was viewed by light microscopy, and the severity of the renal lesion was defined by the glomerulosclerosis index (GSI). The GSI was calculated according to the method of Raij et al.⁹ The severity of the lesions in a minimum of 40 glomeruli (range 40–80) in each specimen was examined, graded from 0 to 4 points in accordance with the percentage of morphological changes on each glomerulus (0 = 0%, 1  +=  1–25%, 2  +=  26–50%, 3  +=  51–75%, 4  +=  76–100%). The number of glomeruli showing a lesion of 0 was n₀, of 1+ was n₁, of 2+ was n₂, of 3+ was n₃, of 4+ was n₄. Forty (range 40–80) glomeruli were examined independently, and the average score of each specimen was then calculated.

Immunohistochemical Analysis of apoE, Collagen IV, Fibronectin, α-Smooth Muscle Actin, and Transforming Growth Factor-β1

The operation was implemented according to the instructions of the SP staining system. Renal tissue samples were fixed in 10% neutral formaldehyde, dehydrated with ethanol, and embedded in paraffin. Serial 4 µm sections were collected sequentially on glass slides. The paraffin was removed from the sections with xylene and rehydrated in graded ethanol. To retrieve antigenicity from formalin fixation, we incubated the sections for 10 min in 10 mmol/L sodium citrate buffer using a microwave oven. Endogenous peroxidase activity was blocked by further pretreatment with 3% hydrogen peroxide (H₂O₂) and methanol. Finally, the sections were incubated with primary monoclonal antibodies against apoE (1/150) (Bo Ao-Sen, Co., Beijing, China), Collagen IV (Col-IV; 1/100) (Shanghai ChangDao Co., Shanghai, China), fibronectin (FN; 1/100) (Beijing Zhongshan Co. Ltd., Beijing, China), α-smooth muscle actin (α-SMA) (ready-to-use kit) (Shanghai Changdao, Co.), and transforming growth factor-β1 (TGF-β1; 1/100) (Wuhan Boshide, Co., Wuhan, China) overnight at 4°C. The sections were washed thoroughly in phosphate-buffered saline solution and incubated with rabbit anti-mouse biotinylated second antibody immunoglobulin (Shanghai Changdao, Co.) for 30 min. Finally, the sections were stained with diaminobenzidine. We obtained negative controls by replacing specific antisera with phosphate-buffered saline solution. For determination of the expression of apoE, Col-IV, FN, α-SMA, and TGF-β1, a semi-quantitative evaluation was performed. Score 0 represents little or no positive staining, whereas 1–4 represent the disruptive redistribution of apoE, Col-IV, FN, α-SMA, and TGF-β1, involving <25%, 25–50%, 50–75%, and >75% of positive staining of glomerular area, respectively.^10,11 At least 40 randomly chosen glomeruli were evaluated at 400-folds original magnification for each mouse, and an average composite score was counted.

Real-Time Reverse Transcription Polymerase Chain Reaction to Detect apoE mRNA Expression

Renal tissue was homogenized and total RNA was extracted with TRIzol (Beijing Tiangen Co., Beijing, China). Ultraviolet spectrophotometer measured the absorbance, agarose gel electrophoresis confirmed that there had been no degradation of RNA. Primers for apoE and β-actin were designed according to primer design principles by Primer Premier 5.0. One microgram total RNA from the renal tissue of each rat was reverse transcribed into cDNA with an ExScript RT reagent kit (Takara Biotechnology Co., Dalian, China). ApoE was amplified with SYBR Premix Ex Taq (Beijing Tiangen Co.). The cycling parameters were denatured at 95°C for 5 s, with annealing at 61°C for 20 s and extension at 72°C for 15 s. A total of 35 cycles were performed within the linear amplification range. Gene expression of β-actin was also measured in each sample and used as an internal control for loading and reverse transcription efficiency. Each sample analysis was repeated three times. The average threshold cycle (CT, the cycles of template amplification to the threshold) was worked out as the value of each sample. Relatively quantitative 2^−ΔΔCT was used to compare the mRNA expression.¹²

Statistical Analysis

The data are shown as mean ± standard deviation (SD). The t-test for independent samples was performed to compare variables between groups, and Pearson's correlation coefficients were used to determine the relationships between the indicators for detection. A value of p < 0.05 was considered as significant. Statistical analysis was performed using the statistical package for social studies SPSS version 13.0 (SPSS, Chicago, IL, USA).

RESULTS

Changes in Renal Function

At the end of the 9/13 weeks, BUN and Scr levels of group GS were markedly higher than those in group SHO (p < 0.01). In GS group, BUN or Scr in 13 weeks was slightly increased compared with that of 9 weeks (BUN: 13/9 weeks = 1.25; Scr: 13/9 weeks = 1.15) (Table 1).

Table 1. Parameters in two groups (mean ± SD)

Parameters	9 weeks		13 weeks
	SHO	GS	SHO	GS
BUN (mmol/L)	7.83 ± 1.87	13.11 ± 2.76	8.10 ± 1.65	16.39 ± 3.37
Scr (µmol/L)	30.67 ± 5.13	43.17 ± 7.96	31.33 ± 4.86	49.83 ± 8.38
24UTP (mg/24 h)	16.42 ± 2.13	50.24 ± 6.32	17.59 ± 3.28	65.09 ± 7.64
GSI (%)	1.25 ± 0.21	17.64 ± 3.52	1.36 ± 0.27	23.63 ± 4.63
Col-IV (%)	17.52 ± 6.31	40.08 ± 8.32	18.24 ± 6.42	56.27 ± 8.53
FN (%)	7.26 ± 1.47	15.34 ± 4.53	7.73 ± 1.33	23.87 ± 7.63
α-SMA (%)	0.56 ± 0.13	23.42 ± 5.42	0.56 ± 0.28	34.32 ± 6.03
TGF-β1 (%)	0.46 ± 0.29	21.36 ± 4.84	0.51 ± 0.31	30.25 ± 5.78

Notes: SHO, sham operation group; GS, glomerulosclerosis model group; BUN, blood urea nitrogen; Scr, serum creatinine; 24UTP, 24-h urine protein excretion; GSI, glomerulosclerosis index; Col-IV, collagen IV; FN, fibronectin; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1.

*p < 0.01 compared with SHO.

24-h Urinary Protein Excretion

When compared with SHO group, 24-h urine protein in GS showed a more significant elevation (p < 0.01), especially at the end of 13 weeks (GS group, 13/9 weeks = 1.30) (Table 1).

Glomerular Morphology

Light microscopy was performed to observe the changes of glomerular morphology. Glomerular morphologies were normal in sham group (A₁ and A₂; Figure 1). Extensive damage was observed in GS group, such as glomerular hypertrophy, balloon adhesion, and wall thickening (A₃ and A₄; Figure 1), especially at 13 weeks (A₄; Figure 1). Quantitative analysis showed that the GSI of group GS at week 9/13 was much higher than that in SHO group (p < 0.01), and the GSI in 13 weeks in GS group was a little elevated when compared with that of 9 weeks (13/9 weeks = 1.34) (Table 1).

Figure 1. Glomerular morphology was normal in sham group (A₁: 9 weeks; A₂: 13 weeks; HE). Proliferation occurring in the majority of glomerular mesangial cells and extracellular matrix in GS group, and degeneration of glomerular epithelial cells and infiltration of widespread mononuclear cells were shown (A₃: 9 weeks; A₄: 13 weeks; HE), especially in 13 weeks of GS group (A₄). Representative samples of immunohistochemical staining for glomerular Col-IV (SHO: B₁: 9 weeks, B₂: 13 weeks; GS: B₃: 9 weeks and B₄: 13 weeks), FN (SHO: C₁: 9 weeks, C₂: 13 weeks; GS: C₃: 9 weeks and C₄: 13 weeks), α-SMA (SHO: D₁: 9 weeks, D₂: 13 weeks; GS: D₃: 9 weeks and D₄: 13 weeks), TGF-β1 (SHO: E₁: 9 weeks, E₂: 13 weeks; GS: E₃: 9 weeks and E₄: 13 weeks), and apoE (SHO: F₁: 9 weeks, F₂: 13 weeks; GS: F₃: 9 weeks and F₄: 13 weeks) were observed in all groups. Sham group (B₁, B₂, C₁, C₂, D₁, D₂, E₁, E₂, F₁, and F₂): positive staining (in brown) was faint in glomerulus, glomerular endothelial cells, glomerular basement membrane, mesangial cells, and visceral epithelial cells. GS group (B₃, B₄, C₃, C₄, D₃, D₄, E₃, E₄, F₃, and F₄): positive staining was strong in most glomerulus, glomerular endothelial cells, glomerular basement membrane, mesangial cells, and visceral epithelial cells, especially in those of 13 weeks (B₄, C₄, D₄, E₄, and F₄). Magnification 400×.

Notes: SHO, sham operation group; GS, glomerulosclerosis model group; HE, hematoxylin and eosin; FN, fibronectin; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1; apoE, apolipoprotein E.

Col-IV, FN, α-SMA, and TGF-β1 Protein Expression in Glomerulus

Immunohistochemical staining for Col-IV, FN, α-SMA, and TGF-β1 was performed. Weak staining was observed in some glomerulus, glomerular endothelial cells, glomerular basement membrane, mesangial cells, and visceral epithelial cells of sham rats (B₁, B₂, C₁, C₂, D₁, D₂, E₁, and E₂; Figure 1). Staining was markedly enhanced in the majority of glomerulus, glomerular endothelial cells, glomerular basement membrane, mesangial cells, and visceral epithelial cells of GS rats (B₃, B₄, C₃, C₄, D₃, D₄, E₃, and E₄; Figure 1), especially for those in 13 weeks (B₄, C₄, D₄, and E₄; Figure 1). In the SHO group, the glomerular staining for Col-IV, FN, α-SMA, and TGF-β1 was markedly lower than the GS group (p < 0.01) in 9/13 weeks. Those of 13 weeks in GS group were remarkably elevated when compared with 9 weeks (Col-IV: 13/9 weeks = 1.40; FN: 13/9 weeks = 1.56; α-SMA: 13/9 weeks = 1.47; TGF-β1: 13/9 weeks = 1.42) (Table 1).

apoE Protein in Glomerulus and mRNA Expression of Renal Tissue

The apoE protein expressed in glomerular endothelial cells, glomerular basement membrane, mesangial cells, and visceral epithelial cells (Figure 1). Control kidneys showed mild expression of apoE in glomerulus (F₁ and F₂; Figure 1). However, when compared to the kidneys of SHO, remarkable deposition of apoE was observed in the region of glomerular endothelial cells, mesangial cells, and visceral epithelial cells in GS (F₃ and F₄; Figure 1), especially in that of 13 weeks (F₄; Figure 1). Consistently lower mRNA expression of renal tissue and alleviation of apoE staining in glomerulus in the SHO rats were observed when compared with those of GS rats at the end of 9/13 weeks (all p < 0.01), and those of 13 weeks in GS group were significantly increased when compared with 9 weeks (apoE protein: 13/9 weeks = 1.20; apoE mRNA: 13/9 weeks = 1.28) (Table 2).

Table 2. Protein expression of apoE in glomerulus and mRNA expression of apoE in renal tissue of the two groups (mean ± SD)

Groups	n	apoE protein		9 weeks		13 weeks
		9 weeks	13 weeks	ΔCT	Fold	ΔCT	Fold
SHO	20	17.69 ± 5.63	18.91 ± 5.43	3.43 ± 0.50	1	3.40 ± 0.49	1.02
GS	20	28.47 ± 6.42	34.17 ± 8.28	1.20 ± 0.10	4.69	0.84 ± 0.09	6.02

Notes: SHO, sham operation group; GS, glomerulosclerosis model group.

*p < 0.01 compared with SHO.

Correlation Analysis

Correlation analysis between apoE protein and Col-IV, FN, α-SMA, TGF-β1 protein or GSI in glomeruli was conducted for our investigation. Significant positive correlation was observed between apoE protein and Col-IV, FN, α-SMA, TGF-β1 protein, or GSI (r = 0.901, r = 0.893, r = 0.842, r = 0.826, r = 0.932, respectively; p < 0.01 for each).

DISCUSSION

Over-depositing of ECM such as FN and Col-IV is an important characteristic of GS. α-SMA is a specific marker for myofibroblasts and takes part in the development and progression of GS.^13,14 Of all the cytokines and growth factors involved, TGF-β1 plays the most important role and the elevation of TGF-β1 expression is closely correlated with GS.^15,16 TGF-β1 is known to be one of the major mediators that lead to GS by inducing the production of α-SMA and ECM (Col-IV and FN) in glomerulus.^15,17,18

In renal tissue, apoE is mainly synthesized by mesangial cell under normal physiologic conditions.¹⁹ Investigators reported that the apoE took a protective role for kidney.^3,4 Some other investigations found that the apoE expression in renal glomerulus was elevated when compared with that of the normal group;^5–7 however, those studies did not explore the relation between apoE and Col-IV, FN, TGF-β1, and α-SMA. The deposition of lipids in glomeruli, as an important pathogenetic factor, involves in glomerular sclerosis and leads to progression of renal disease. As a key apolipoprotein to mediate the catabolism of lipids deposition, the role and mechanism of apoE in GS is complicated, and there are rare reports that explore the association between apoE and the protein expression of Col-IV, FN, TGF-β1, or α-SMA. In this context, the aim of our study was to determine whether apoE expression correlates with the protein expression of Col-IV, FN, TGF-β1, and α-SMA in glomerulus and to compare these relationships with controls.

In this investigation, we found the expression of apoE in glomerulus might reflect the seriousness of GS in rats. The protein expressions of Col-IV, FN, α-SMA, and TGF-α1 in glomerulus were increased when compared with those in controls. The apoE protein expression in glomerulus of GS group was also much higher compared with that in SHO group. The correlation analysis was performed to explore the association between apoE protein and Col-IV, FN, α-SMA, TGF-β1 protein, or GSI in glomeruli and significant positive correlation was observed between apoE protein and Col-IV, FN, α-SMA, TGF-β1 protein, or GSI. The expression of apoE mRNA was elevated in GS when compared with that in control group. We also measured the protein expression of apoE in tubular interstitium and found the apoE expression in tubular interstitium was less when compared with that in glomerulus in SHO group or group of GS (data not shown). In other words, the increased apoE expression in glomerulus was more remarkable than that in tubular interstitium. So, in disease state of GS, the elevated expression of apoE mRNA in renal tissue might be mainly contributed by the glomerulus. We speculated that the apoE was a risk factor for GS, but the role and mechanism of apoE in the pathological process of GS was ambiguous.

Endothelial, mesangial, and visceral epithelial cells in glomerulus can take up the lipids by receptor-independent mechanism. Those lipoproteins could be rapidly removed by the glomerular cells and lead to lipid deposition in glomerulus.²⁰ apoE, as an important apolipoprotein, mediates the lipoproteins combining with the glomerular cells for catabolism. However, the over-upgraded apoE mediates too much lipid depositing in glomerular cells which also can cause the progression of GS. In our study, we found the apoE was much markedly accumulated in glomerular of GS rats when compared with that in SHO. We draw a conjecture that the apoE mediated the lipoproteins integrating with endothelial, mesangial, and visceral epithelial cells and induced the accumulation of lipids in glomerulus. The direct negative effects of lipid over-accumulation on the renal parenchymal cells was due to the formation of free radicals because of oxidized low-density lipoprotein (LDL),⁷ and oxidized LDL can induce mesangial cells and macrophages to release cytokines and growth factors (such as TGF-β1), which may increase mesangial proliferation^21,22 and lead to ECM accumulation and α-SMA over-expression. The role of apoE taking part in the pathological mechanism of GS might be that the apoE mediated too much lipoprotein depositing in glomerulus and leaded to GS.

How to regress or repair the GS is a difficult problem to solve at present. There were some reports observed that some new agents, such as angiotensin II blockers, all-trans retinoic acid, and so on, could alleviate the pathological changes of ADR-induced GS in the past years.^23–25 Statins, as an important agent to mediate the lipid metabolism, can reduce renal injury via diminishing the proliferation of mesangial and vascular smooth muscle cells, and inhibiting macrophage infiltration and the production of cytokines.²⁶ Some investigations have found that statins can play a protective role in patients with renal disease or animal model accompanying renal lesion.^25,27,28 Whether the statins can alleviate the lesion of GS by regulating the expression of apoE, it might be an interesting issue to be explored in times to come.

In conclusion, although the relationship between apoE and GS is till now not fully elucidated, these observations in our study may suggest that apoE is actively involved in the pathogenesis of GS by upregulating the protein expressions of Col-IV, FN, TGF-β1, and α-SMA in glomerulus in rats. Considering the widespread interest in apoE and their interactions in the field of GS, our investigation might have significant influence on future research as well as making some appropriate interventions for GS. However, more studies by cell culture or other experimental models of renal disease are needed to explore the molecular mechanisms and confirm the role of apoE in the pathogenesis of GS in the future.

ACKNOWLEDGMENTS

This study was supported by the Natural Science Foundation of the Guangxi Zhuang Autonomous Region (no. 0640103) and the Education Department of Guangxi Zhuang Autonomous Region (no. 0810). The authors gratefully acknowledge the most helpful comments on this article received from Professor Liang Rong, Department of Pediatric-Neonatology, Baylor College of Medicine, Houston, TX, USA.

Declaration of interest: The authors report no conflicts of interest. The author alone is responsible for the content and writing of the paper.