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Nephroprotective effect of losartan in IgA model rat

Objective This study was performed to investigate the possible nephroprotective effects of losartan in a rat model of experimental IgA nephropathy (IgAN). Methods Thirty male Sprague–Dawley rats were randomly divided into three groups. The rats in the model group were treated with bovine serum albumin (oral gavage), lipopolysaccharide (tail vein injection), and carbon tetrachloride (subcutaneous injection); rats in the losartan group received treatments similar to those of the model group, and were orally gavaged with losartan; and rats in the control group received phosphate-buffered saline alone (both orally and intravenously). Results Losartan treatment lowered the 24-hour urinary protein, serum blood urea nitrogen, and serum creatinine levels. Proliferating mesangial cells with a variable increase in the mesangial matrix were detected in the model group, whereas injury in the losartan group was significantly attenuated. Immunohistochemistry revealed that the expression levels of transforming growth factor (TGF)-β1 and α-smooth muscle actin were significantly elevated in the model group but reduced in the losartan group. The expression levels of TGF-β1 and monocyte chemoattractant protein-1 were minimal in the control group, significantly increased in the model group, and reduced in the losartan group. Conclusion Losartan has a protective effect against tubulointerstitial injury in IgAN.


Introduction
Primary IgA nephropathy (IgAN) was first described by Berger and Hinglais in 1968 and is defined by the presence of IgA-containing immune deposits in the glomerular mesangium. 1 IgAN is the most common form of primary glomerulonephritis worldwide and is the main cause of end-stage renal disease (ESRD) in patients with primary glomerular disease. 2 Idiopathic IgAN is characterized by episodic, macroscopic hematuria and often coincides with an upper respiratory tract infection; many IgAN patients exhibit slow, progressive disease, and 30% to 50% of patients develop ESRD over a 20-year period after diagnosis. 3 Currently, the treatment options are very limited. The most common medications include immunosuppressive agents if the 24-hour urinary protein level exceeds 1 g; however, long-term treatment is associated with numerous side effects, and many patients want to avoid use of immunosuppressors or have contraindications that prevent the use of this drug class. Nonimmunosuppressive options include fish oils, anticoagulants, antihypertensive agents, and tonsillectomy. In numerous studies, antihypertensive agents, particularly angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), or a combination of these, have been confirmed to reduce proteinuria and improve prognosis. [4][5][6][7][8][9][10] Park and coworkers showed that losartan treatment significantly reduced both proteinuria and urinary excretion of transforming growth factor (TGF)-b1 in IgAN patients. 6 Losartan is also widely used in clinical practice. The present study was performed to investigate the protective effect of losartan against tubulointerstitial injury in an experimental model of IgAN.

Experimental animals
Thirty 8-week-old male Sprague-Dawley rats weighing 200 to 260 g were obtained from Harbin Medical University. The animals were housed in standard conditions (22-24 C with constant humidity and a 12-hour light/12-hour dark cycle). The rats were provided standard laboratory chow and water and allowed to acclimate for 1 week before the experiment; their urine was tested to ensure that they were negative for proteinuria and had normal red blood cell counts, before they were used in this study. All procedures were approved by the animal committee of Harbin Medical University.

Animal model and groups
All rats were randomly divided into three groups (n ¼ 10 rats per group). The IgAN model was induced as previously described. 11 Briefly, rats in the model group were gavaged with 400 mg/kg bovine serum albumin (BSA, VWR, Radnor, PA, USA) every other day for 6 weeks. From the first to the ninth week, carbon tetrachloride (CCl 4 , Beijing Beihua Fine Chemicals Co., Beijing, China) was subcutaneously injected at a dose of 0.10 mL/week with castor oil (0.5 mL/ week); lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA) was also injected through the tail vein (0.05 mg/ week in the sixth and eighth weeks). Rats in the losartan group received losartan (Hangzhou MSD Pharmaceutical Co., Hangzhou, China) at a dose of 40 mg/kg/ day (concentration of 4 mg/mL) by oral gavage, beginning in the seventh week (after BSA administration was completed); losartan was administered in addition to CCl 4 and LPS treatments. At the corresponding time points, rats in the control group received phosphate-buffered saline (PBS) orally and intravenously, in volumes identical to those of BSA, CCl 4 , and LPS. Blood and urine samples were collected at various time points and stored at À80 C until analysis: specifically, blood samples were collected before the experiment and at the end of the tenth week, while urine samples were collected before the experiment and at the end of the fourth, eighth, and tenth weeks. All rats were sacrificed at the end of the tenth week of the experiment, as described in greater detail in the Histological assessment subsection below; they were perfused with ice-cold normal saline via the left ventricle for 2 minutes, and their kidneys were rapidly excised.

Analysis of biochemical indicators
Before the experiment and at the end of the first, fourth, eighth, and tenth weeks, the rats were individually housed in metabolic cages to collect 24-hour urine samples. The preservative xylene was added to the urine samples, and 10 mL of urine from each rat were centrifuged and stored in a À20 C freezer. The samples were then subjected to measurement of 24-hour urinary protein levels and red blood cell (RBC) counts using a fully automatic albumin analyzer (SIEMENS BNII, Siemens, Deerfield, IL, USA). Blood was sampled from the tail vein and immediately analyzed using an automatic biochemical analyzer (Cobas c 311, Roche, Mannheim, Germany) to measure the serum levels of blood urea nitrogen (BUN) and creatinine; the automatic biochemical analyzer was also used to assess liver function in the rats, as previously reported. 12,13 Histological assessment At the end of the tenth week, the animals were anesthetized with a 60-mg/kg intraperitoneal injection of pentobarbital sodium. As previously described, 12,14-16 the rats were perfused with ice-cold normal saline, and the kidneys were removed and decapsulated. IgA immunofluorescence labeling (FITClabeled goat anti rat IgA antibody, Santa Cruz Biotechnology, Santa Cruz, CA) was performed using 4-mm cryosections, and IgA fluorescence intensity was tested under an FLUOVIEW FV 1000 (OLYMPUS, Tokyo, Japan) confocal microscope. Right kidney tissue samples were fixed in 4% neutral formaldehyde, embedded in paraffin, and stored at 4 to 8 C prior to hematoxylin and eosin staining and immunohistochemical staining. Left kidneys were minced, frozen in liquid nitrogen, and stored at À80 C for quantitative polymerase chain reaction (qPCR) analysis. Paraffin sections (4-5 mm thick) were cut using a microtome (Thermo Scientific, Walldorf, Germany). The hematoxylin and eosin-stained sections were evaluated semiquantitatively, as previously described. 17 Briefly, kidney injury was graded from 0 to 4 as follows: 0 ¼ normal tissue; 1 ¼ changes affecting < 25% of the sample; 2 ¼ changes affecting 25% to 50% of the sample; 3 ¼ changes affecting 50% to 75% of the sample; and 4 ¼ changes affecting > 75% of the sample.

Immunohistochemical staining
Paraffin-embedded kidney sections were used for immunohistochemical staining, as previously described. 18 Briefly, standard dewaxed paraffin sections were washed three times in PBS, for 3 minutes each. Then, a 0.3% H 2 O 2 treatment was applied for 10 minutes at room temperature to inhibit endogenous peroxidases. Distilled water was used to wash the sections three times for 3 minutes each; the sections were then incubated in PBS for 5 minutes. The slides were immersed in citrate buffer solution, placed in a microwave processor, heated to 95 C for 15 minutes and cooled to room temperature. After preincubation with 10% goat serum (Dako, Carpinteria, CA, USA) for 10 minutes at room temperature to block nonspecific antibody binding, the sections were incubated overnight at 4 C with primary antibodies against TGF-b1 (ab215715, 1:200, Abcam, Hong Kong) and a-smooth muscle actin (SMA) (ab32575, 1:200, Abcam). After incubation with an appropriate secondary antibody (for TGF-b1, goat anti-rabbit IgG, ab97051, Abcam; for a-SMA, goat anti-rabbit IgG, ab205718, Abcam), the sections were developed with 3,3-diaminobenzidine (Dako) to produce a brown color and counterstained with hematoxylin. The staining was observed under a DS Ri1 (Nikon, Tokyo, Japan) light microscope at a Â200 magnification. The staining intensity was quantified as follows 19 : 0 ¼ none or weak staining; 1 ¼ stained areas < 25% or weak-to-moderate staining; 2 ¼ stained area within 25% to 49% or moderate staining; 3 ¼ stained area within 50% to 75% or moderate-to-strong staining; and 4 ¼ stained areas > 75% or strong staining.

Real-time PCR analysis
qPCR was performed as previously described. 13 Total RNA was extracted from rat kidneys using TRIzol reagent (Invitrogen, Stockholm, Sweden), in accordance with the manufacturer's protocol. RNA integrity was confirmed by agarose gel electrophoresis (ChampGel-3200, Beijing, China), and the RNA concentration and purity were confirmed using the relative absorbance at 260/ 280 as measured by an ultraviolet spectrophotometer (SmartSpec TM 3000, Bio-Rad, Hercules, CA, USA). RNA samples with a ratio > 1.8 were used for qPCR analysis. Total RNA was reverse transcribed using the GoScript TM Reverse Transcription System (Promega, Madison, WI, USA), in accordance with the manufacturer's protocol; the resulting cDNA was then amplified using the GoTaq V R qPCR Master Mix (Promega) in the Stratagene Mx3000P thermocycler (Agilent, Santa Clara, CA, USA). The PCR primer sequences are listed in Table 1. The amount of each PCR product was normalized to the level of b-actin to determine the relative expression ratio of each target gene, and fold differences in gene expression levels were determined using the 2 ÀDDct method.

Statistical analysis
All data are presented as the mean AE standard deviation. Repeated measures data were performed using repeated measures analysis of variance (univariate ANOVA). Multiple samples were compared using oneway ANOVA with a random design. Ranked data were tested using the Kruskal-Wallis H test. The Mantel-Cox log-rank test was used for survival analysis. All tests were performed with SPSS Statistics, Version 17.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered significant in all statistical tests.

Expression of IgA in the control and model groups
After 10 weeks, IgA deposition was dynamically assayed by immunofluorescence to validate the model. We found that in the model group, considerable IgA deposition was observed in the mesangial area, whereas IgA deposition was minimal in the control group ( Figure 1). The results indicated that

Urinalysis and renal function assessment
After 4 weeks of the experiment, the body weight of rats in the model group was lower than that of the rats in the control and losartan groups (data not shown), and one rat died. At 10 weeks, all rats had survived in the control group, whereas two rats had died in both the model and losartan groups; the numbers of surviving rats in the three groups at each time point are shown in Figure 2a. In the model and losartan groups, the 24-hour urinary protein levels (in the model group,  (Figure 2b and c). In the tenth week of the experiment, the urinary red blood cell count in the losartan group was slightly lower than that in the model group, but the difference was not statistically significant ( Figure 2c). As shown in Figure 2d-e, significant increases in the levels of creatinine and BUN were observed in the model group, compared with the levels in the control group (P < 0.01 for both); losartan treatment abolished these differences.

Comparison of liver function
CCl 4 is hepatotoxic; thus, in this study, we also tested the liver function in each group of rats. As depicted in Figure 3a and b, the alanine aminotransferase and aspartate aminotransferase levels were slightly higher in the model and losartan groups at 10 weeks, but were not significantly different from the levels in the control group. The albumin (ALB) level visibly decreased over time in the model group, but this difference was not statistically significant; however, after losartan treatment, the ALB level was similar to that of the control group (Figure 3c).

Assessment of renal pathological injury
After 10 weeks of treatment, the light microscopy data (Figure 4a) showed that the structures of the glomeruli, renal tubules, and interstitium were normal in the control group. In the model group, the    glomeruli exhibited proliferating mesangial cells with an increased mesangial matrix, a widened mesangial region, vacuolization of some renal tubular epithelial cells, and infiltration of the tubulointerstitial area by numerous inflammatory cells. These alterations were significantly diminished after losartan treatment. The tubulointerstitial injury index of each group after 10 weeks was scored: the model and losartan groups showed greater injury than the control group (P < 0.05 for both), while the losartan group showed less injury than the model group (P < 0.05) (Figure 4b).
Protein expression of TGF-b1 and a-SMA in renal tissue As expected, the protein expression levels of TGF-b1 and a-SMA at 10 weeks were significantly higher in the model group than in the control group (P < 0.05 for both), based on immunohistochemical analysis of renal tissue. After losartan treatment, the protein expression levels of TGF-b1 and a-SMA were lower than those in the model group (P < 0.05 for both) (Figure 5a-d).
Gene expression of TGF-b1 and MCP-1 in renal tissue The mRNA expression levels of TGF-b1 and MCP-1 at 10 weeks were significantly higher in the kidneys of model rats than in the kidneys of control rats. In addition, losartan treatment reduced the mRNA expression levels of TGF-b1 and MCP-1, relative to those in the model group (P < 0.05 for both) (Figure 6a and b).

Discussion
Primary glomerulonephritis remains the main cause of chronic kidney disease in China, and IgAN is the most frequent histopathologic lesion, comprising 42.6% of primary glomerular diseases. 20 IgAN is also well recognized as the most prevalent glomerular disease worldwide 21 and is characterized by the accumulation of IgA deposits, predominantly in the glomerular mesangium, accompanied by mesangial proliferation. 22 A significant proportion of patients follow a progressive course and eventually develop ESRD. 23 Notably, ACEIs and ARBs are more effective nonimmunosuppressive treatment options for IgAN than alternative antihypertensive drugs or other agents, including fish oils and anticoagulants. 24 In this study, we examined the effect of losartan on IgAN. There are many methods available for development of an IgAN animal model, including the use of ddY mice with high serum IgA levels, administration of immune complexes to induce IgAN, and injection of CCl 4 to cause liver injury through a reduction of IgA in mesangial IgA deposits. However, each method has limitations: ddY mice exhibit a high degree of variability in the age of onset and severity of the disease, and injection of CCl 4 can cause severe liver injury and increased mortality. 25,26 Therefore, in the present study, we reduced the dose of CCl 4 injected into the rats; we also injected CCl 4 by a subcutaneous route, rather than an intraperitoneal injection. After 10 weeks of modeling, we found that liver function was not obviously damaged in the rats, but there were significant depositions of IgA in the glomeruli. Thus, we successfully generated an IgA model, although two rats died during the course of this experiment. Urinary protein levels and red blood cell counts increased over time in the model group. The administration of losartan for 1 week significantly reduced the 24-hour urinary protein level (measured at week 8 in the experiment), but did not influence the urinary red blood cell count; this finding is potentially because ARB therapy does not affect active lesions. 27 However, steroids and immunosuppressive agents can repair active lesions without influencing chronic lesions or regions affected by glomerular hypertension and hyperfiltration. 28 In the model group, the rats exhibited gradual increases in serum BUN and creatinine levels, compared with rats in the control group; losartan therapy lowered both the serum BUN and creatinine levels, while reducing the renal pathological injury, as confirmed by hematoxylin and eosin staining. ARB therapy has a renoprotective effect through reduction of glomerular hypertension and hyperfiltration, as well as by lowering the expression levels of fibrogenic and inflammatory factors, 29 including TGF-b1, a-SMA and MCP-1; effects on these factors were confirmed by immunohistochemistry and qPCR analyses in the present study. However, additional studies are needed to elucidate the potential mechanisms of ARB therapy-dependent improvement in renal function in our model of IgAN.
In conclusion, the findings in the current study indicate that the administration of BSA, LPS, and CCl 4 can successfully induce the onset of IgAN in a rat model. Losartan can reduce the 24-hour urinary protein, serum BUN, and serum creatinine levels, without influencing liver function or increasing the level of serum ALB. Notably, losartan ameliorated injury to the glomeruli and kidney tubules. The results of this study demonstrate that prevention of the release of inflammatory and fibrogenic factors contributes to losartan-dependent attenuation of renal injury.