To evaluate the effect of astaxanthin on renal angiotensin-I converting enzyme (ACE) levels, osteopontin (OPN) and transforming growth factor β1 (TGF-β1) expressions and the extent of crystal deposition in experimentally induced calcium oxalate kidney stone disease in a male Wistar rat model.
To compare the efficacy of astaxanthin treatment with a currently used treatment strategy (citrate administration) for kidney stones.
Materials and Methods
The expression of OPN was assessed by immunohistochemistry. One step reverse transcriptase polymerase chain reaction followed by densitometry was used to assess renal OPN and TGF-β1 levels.
Renal ACE levels were quantified by an enzyme-linked immunosorbent assay method.
Crystal deposition in kidney was analysed by scanning electron microscopic (SEM)-energy-dispersive X-ray (EDX).
The renal ACE levels and the expression of OPN and TGF-β1 were upregulated in the nephrolithiasis-induced rats.
Astaxanthin treatment reduced renal ACE levels and the expression OPN and TGF-β1.
SEM-EDX analysis showed that crystal deposition was reduced in the astaxanthin-treated nephrolithiatic group.
Astaxanthin treatment was more effective than citrate administration in the regulation of renal ACE levels, OPN and TGF-β1 expressions.
Astaxanthin administration reduced renal calcium oxalate crystal deposition possibly by modulating the renal renin-angiotensin system (RAS), which reduced the expression of OPN and TGF-β1 levels.
Astaxanthin administration was more effective than citrate treatment in reducing crystal deposition and down-regulating the expression of OPN and TGF-β1.
Nephrolithiasis, characterised by mineral deposition in the nephrons, is a common clinical disorder with a high prevalence and recurrence rate. Kidney stones are predominantly composed of calcium oxalate in the form of calcium oxymonohydrate . Present day management of nephrolithiasis involves invasive procedures and drugs that are costly, have adverse side-effects and high recurrence rates. Administration of potassium citrate is found to alleviate renal stone formation and formulations containing this compound have been advocated by medical practitioners as a remedy for kidney stones . Proteins account for 1–5% of the stone matrix and osteopontin (OPN) is the most abundant protein . OPN is a 44 KDa phosphorylated glycoprotein that is thought to promote cellular adhesion through its conserved arginine-glycine-aspartic acid domain . High-molecular-weight OPN complexes are associated with pathological calcification processes, e.g. atherosclerosis, breast cancer, dental calculus, pilomatricomas and kidney stones . Previous studies have shown that OPN adheres to renal cells and enhances calcium oxalate crystal adherence and aggregation in vitro. It has also been shown that increased OPN expression in renal proximal tubular cells is modulated through reactive oxygen species generation, RAS activation and TGF-β1 expression .
The kidney is unique in having every component of the RAS with compartmentalisation in the tubular and interstitial networks. Increased nucleation of calcium oxymonohydrate crystals in the nephron lumen and the ensuing crystal cell interaction can upregulate OPN expression and increase the protein secretion by renal tubular cells through the renal RAS system . Angiotensin-I converting enzyme (ACE), the enzyme which catalyses the conversion of angiotensin I to angiotensin II, is abundant in the rat kidney and has been located in the proximal and distal tubules, the collecting ducts, and renal endothelial cells . Angiotensin II produced in the renal tissue has been found to increase the expression of TGF-β1 in vitro [9, 10]. An increased expression of TGF-β1 is also associated with many renal disease conditions including hyperoxaluria and kidney stones . There is a growing body of evidence suggesting that TGF-β1 is a potent stimulus of OPN transcription and protein expression in cultured rat renal epithelial cells . ACE thus participates in various renal functions by regulating the circulating level of angiotensin II, which in turn affects the expression of genes including OPN and TGF-β1 locally.
Oxidative stress also plays an important role in the proinflammatory effect of angiotensin II. Antioxidants and ACE inhibitors hence offer protection against renal damage arising from different pathological conditions . Astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione) is a natural antioxidant, which is attracting more attention due to its multiple effects and high antioxidant potential almost 500-times that of α-tocopherol . It is a red carotenoid pigment found in plants, algae and many marine animals, e.g. almonids, shrimps, lobsters, and crayfish. The chlorophyte alga Haematococcus pluvialis is the richest source of natural astaxanthin with a capacity to accumulate astaxanthin up to 4–5% of dry weight as per reported sources .
It has been reported that in addition to its superior antioxidant activity, astaxanthin also exhibits ACE inhibitory activity in hypertensive and obese rat models . We hypothesised that the ACE inhibitory activity of astaxanthin may influence the expression of OPN and TGF-β1 in experimentally induced nephrolithiasis. Therefore, we investigated the effect of astaxanthin treatment on the correlations of ACE levels with renal OPN and TGF-β1 expressions and calcium oxalate deposition in male Wistar rats. Oral potassium citrate intake is a common strategy for treatment of renal calculi . A comparison was made to evaluate the efficacy of astaxanthin treatment over potassium citrate administration on renal stone formation and regulation of OPN and TGF-β1 expressions.
Materials and Methods
Healthy male Wistar rats (weight 200 ± 20 g, n = 30) were housed in laboratory cages at standard conditions of humidity, temperature (25 ± 2°C) and light (12:12 h light-dark) and given access to laboratory chow and water ad libitum. After 1 week of acclimatisation, they were divided into five groups of six rats each: (1) Control rats fed on a normal diet (Control group), (2) Rats administered with 25 mg/kg body weight astaxanthin daily for 21 days (Astaxanthin control group), (3) Rats administered with 0.75% ethylene glycol daily and 0.5 μg vitamin D3 (1, 25-dihydroxycholecalciferol) on alternate days for 28 days for induction of calcium oxalate crystals (Nephrolithiatic group), (4) Nephrolithiatic rats treated with astaxanthin at 25 mg/kg body weight for 21 days (Astaxanthin-treated nephrolithiatic group), and (5) Nephrolithiatic rats treated with potassium citrate at 2 g/kg body weight for 21 days (Citrate-treated nephrolithiatic group). The dose of astaxanthin was adjusted with 1.5% astaxanthin powder from Hematococcus pluvialis microalgae. The mode of administration was oral intubation. All procedures were performed in accordance with the guidelines of Institutional Animal Ethical Committee of the School of Biosciences, Mahatma Gandhi University, Kerala, India (Approval No: B1662009/3). After the experimental period, all the rats were humanely killed and both the kidneys were isolated under RNAse-free conditions. One kidney from each rat was used for histological analyses and for renal ACE assay, and the other was used for RNA isolation and reverse transcriptase (RT)-PCR studies.
Immunohistochemical staining was used to detect OPN protein in the renal tissue of the rats. Briefly, paraffin-embedded kidney tissue samples were cut into 4-μm sections, slides prepared and incubated at 27°C for 24 h. They were deparaffinised through three changes of xylene and rehydrated. The sections were treated with citrate buffer, pH 6 at 92°C for 20 min for OPN and cooled at room temperature for 30 min. Endogenous peroxidase activity was blocked by treating the slides with 10% H2O2 for 15 min and washed in trisodium citrate buffer. Non-specific binding was blocked by 10% goat serum. The sections were then treated with the OPN antibody (Rabbit polyclonal to OPN, ab8448, Abcam, Cambridge, UK) for 1 h at room temperature. Sections were washed and incubated with goat anti-mouse Fab′2-Biotin conjugate (ab98657, Abcam) followed by streptavidin-horseradish peroxidase conjugate (Invitrogen, Life technologies, Carlsbad, CA, USA) for 30 min each. All antibodies were diluted 1:400 with trisodium citrate buffer. Sections were washed thoroughly and treated with diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. The reaction was stopped by immersing the slides in excess distilled water. Counterstaining was done with haematoxylene. Negative control sections went through the same staining process except that the primary antibody was omitted. An Olympus BX 51 microscope was used for image capturing and quantification of OPN staining pattern. Quantification of OPN immunostaining was done by counting the percentage of tubules with >50% of the cells staining positive for OPN. In all, 10 microscopic fields were examined at ×100 and the results were averaged.
The samples for RNA isolation and RT-PCR were collected under RNAse-free conditions and stored in RNAlater® solution (Sigma-Aldrich). RNA isolation was done using TRI reagent (Sigma-Aldrich) as per the manufacturers' instructions. The quality of the RNA was analysed and quantification was done using a NanoDrop®1000 Ultraviolet (UV)-Vis Spectrophotometer (Thermo Fisher Scientific, USA) at 260 nm. The primers were for OPN, TGF-β1 and β-actin were taken from literature [6, 18]. Conventional RT-PCR was carried out as follows, 1 μg RNA was used to synthesise cDNA using the One Step Primescript RT-PCR kit (TaKaRa Bio Inc. Shiga, Japan). The reaction mixtures consisted of RT-PCR buffer, Taq polymerase, Primescript RT enzyme, RNAase-free distilled water, forward and reverse primers and RNA sample. The sense and antisense rat OPN primers were: 5′-CTG GCA GTG GTT TGC CTT TGC C-3′ and 5′-CGT CAG ATT CAT CCG AGT TCA C-3′. The β- actin gene was used as the internal standard for normalisation. The sense and antisense primers of the β-actin gene were: 5′-ATG CCA TCC TGC GTC TGG ACC TGG C-3′ and 5′-AGC ATT TGC GGT GCA CGA TGG AGG G-3′. Denaturation was carried out at 94°C for 30 s, annealing at 60°C for30 s and extension at 72°C for 30 s. In all, 30 cycles of amplification were done and the final extension was done at 72°C for 10 min .
The TGF-β1 primers were: sense 5′-GGACTACTACGCCAAAGAAG-3′ (base pairs [bp] 715–734) and antisense 5′-TCAAAAGACAGCCACTCAGG-3′. The PCR product length was 294 bp. The reaction was done at 94°C for 30 s (denaturation), 54°C for 1 min (annealing), 72°C for 1 min (first extension) and 72°C for 7 min (final extension), for 35 cycles (Rotor-Gene Q, Qiagen). The template concentration and the cycle number were optimized to ensure linearity of response and to avoid saturation of the reaction .
The PCR products were resolved on 1.5% agarose gel and the bands were identified based on product size using a 100 bp DNA ladder. The band for OPN was identified at 395 bp. The gel was viewed using a UV transilluminator. The images were recorded using Syngene gel documentation system and GeneSnap software (Version 6.03, SynGene, USA). The band densities of the scanned images were quantified by densitometric analysis using Quantity One® 1-D image analysis software of Bio-Rad Image Analysis Systems (CA, USA). They were normalised to the levels obtained for the β-actin gene by finding the ratio of the value of the OPN and TGF-β1 gene to that of β-actin.
The kidneys were washed in ice cold PBS (0.02 mol/L, pH 7.2) and homogenised using a Teflon homogeniser, subjected to two freeze-thaw cycles and centrifuged at 1500g for 15 min. The ACE activities were analysed in kidney tissue homogenate using commercially available Rat Angiotensin I Converting Enzyme ELISA kit (E02A0498, Shanghai Blue Gene Biotech. Co. Ltd., China). Carl Pearson's correlation was calculated for the renal ACE activity with TGF-β1 and OPN expressions in the kidney.
The tissue sections of 4–5 mm block size for scanning electron microscopic (SEM) analysis were processed as per standard protocol, dried using a critical point drier and gold coated (E-1010 Ion splutter unit, Hitachi, Japan). They were analysed using S-2400 SEM (Hitachi, Japan) at ×800. The elemental spectra of crystals within the specimen and the corresponding calcium maps were performed by energy-dispersive X-ray (EDX) analysis using EDAX (Ametek Instruments India Pvt. Ltd).
The results were presented as the mean (sd). One-way anova followed by Tukey's post hoc multiple comparison test was used for comparison among the groups. SPSS/PC+ version 18 (SPSS Inc. Chicago, Illinois, USA) was used and a P < 0.05 was considered to indicate statistical significance.
The immunohistochemical staining results showed that OPN protein expression was weak in the renal tissues of the control groups (Fig. 1A,B). The renal cortex of the nephrolithiatic group showed high frequency and intensity of staining for OPN in the proximal tubules, distal tubules and the collecting ducts. The intensity of staining was higher associated with crystal deposition particularly in the proximal tubules. Crystals were positive for OPN (Fig. 1C). The astaxanthin-treated nephrolithiatic group showed only a faint expression of OPN protein, which was hardly detectable (Fig. 1D). In the citrate-treated nephrolithiatic rat kidneys, OPN expression was similar to that of the nephrolithiatic group (Fig. 1E). Quantitative analysis of OPN staining showed a significant reduction in the astaxanthin-treated nephrolithiatic group when compared with the nephrolithiatic and the citrate-treated nephrolithiatic groups (Fig. 1F).
RT-PCR was used to detect expression of OPN and TGF-β1 in the rat kidneys. Analysis of OPN using total RNA showed distinct bands at 395 bp in the nephrolithiatic group and citrate-treated nephrolithiatic group. In both the control groups and the astaxanthin-treated nephrolithiatic group no clear bands were seen (Fig. 2A). The corresponding band intensities of β-actin are shown in Fig. 2B. Quantitative analysis of the OPN expression by densitometry expressed as the ratio of OPN band intensity relative to that of β-actin indicated that the nephrolithiatic group had relatively high expression. The citrate-treated nephrolithiatic rat tissue also had a high OPN expression similar to the nephrolithiatic group whereas the controls and the astaxanthin-treated nephrolithiatic groups showed very little OPN expression (Fig. 2C).
The TGF-β1 protein was concentrated as a distinct band at 294 bp in the nephrolithiatic group and citrate-treated nephrolithiatic group. In both the control groups and the astaxanthin-treated nephrolithiatic group only faint bands were seen (Fig. 3A). Figure 3B gives the β-actin expression in the corresponding samples. The quantitative analysis of TGF-β1 expression was consistent with the OPN expression, with the nephrolithiatic group and citrate-treated nephrolithiatic group exhibiting highest concentrations in the mentioned order and the control and the astaxanthin-treated nephrolithiatic groups the least (Fig. 3C).
Renal ACE values were significantly higher in the nephrolithiatic group vs the control groups. The ACE levels decreased in the astaxanthin-treated nephrolithiatic group (Table 1). Correlation of the renal ACE activity with OPN expression showed that was a positive correlation of r = 0.946. Correlation of renal ACE with TGF-β1 was also strongly positive (r = 0.845). The results are expressed graphically in Fig. 4A and B, respectively.
Table 1. Renal ACE values
Mean (sd) renal ACE levels, ng/mL
Values represent mean ±SD for six observations. Statistical analysis was carried out by one-way anova followed by post hoc multiple comparison test. A P < 0.05 was considered to indicate statistical significance.
*†‡ⱡ represent comparisons with control, astaxanthin control, nephrolithiatic- and astaxanthin-treated nephrolithiatic groups, respectively.
The SEM-EDX analysis of the kidney showed crystal deposition in the cortical region particularly in the proximal convoluted tubules of the nephrolithiatic group. Most crystals were ‘plate’ shaped confirming their chemical composition as calcium oxymonohydrate. Elemental analysis revealed high amounts of calcium in the nephrolithiatic group (Fig. 5A). The amount of calcium was significantly reduced in the astaxanthin-treated nephrolithiatic group vs the nephrolithiatic group (Fig. 5B). Crystal formation was absent in this group. The citrate-treated nephrolithiatic group showed a reduction in the calcium deposition and the crystals were less regular (Fig. 5C).
Renal epithelial cells respond to calcium oxymonohydrate crystals characteristically by increasing the expression of specific genes that encode transcriptional activators, regulators of extracellular matrix, growth factors and by the production of pro- and anti-inflammatory molecules, e.g. OPN and monocyte chemotactic protein-1 that are also modulators of biomineralisation processes . OPN plays a major role in the adhesion function of calcium oxalate crystals by forming a major component of extracellular matrix proteins . In the present study, there was an increase in OPN protein in the renal tissue and in proximity with the calcium oxymonohydrate crystals in the nephrolithiatic group. Supersaturation, nucleation, growth, aggregation and retention in the renal tubules are the main events leading to clinical nephrolithiasis. It has been previously reported that immobilised OPN is seen to increase crystal aggregation and OPN adhering to the surface of collagen granules can cause an increase in calcium oxalate crystal adherence and aggregation . Thus, it is clear that any molecule that can modulate renal OPN expression can be expected to regulate the aggregation and retention of calcium oxalate crystals in the tissue. In the present study, OPN protein and mRNA levels were reduced in the nephrolithiatic rats when administered with astaxanthin at a dose of the 25 mg/kg body weight.
In the present study, there was an elevated renal level of ACE in the nephrolithiatic group. ACE elevation plays a crucial role in the progression of renal disease, primarily by increasing the intraglomerular pressure followed by an increase in renal angiotensin II production. Angiotensin II contributes to the progression of renal injury and angiotensin II dependent transcriptional and post-transcriptional mechanisms enhance TGF-β1 expression . The elevation in TGF-β1 levels in turn leads to an over expression of collagen fibres and subsequently interstitial fibrosis and renal dysfunction [22, 23]. In the present study, there was also enhanced TGF-β1 expression in the nephrolithiasis-induced rats possibly linked to the elevated ACE levels. Furthermore, angiotensin II and TGF-β1 together upregulate the expression of OPN in immortalised rat renal proximal tubular cells . It is well documented that the interruption of the RAS is an effective strategy to ameliorate renal crystal burdening in patients with hyperoxaluria . The present data indicates that administration of astaxanthin reduces renal ACE levels to the normal range. A similar observation was made in hypertensive rat models where astaxanthin reduced ACE activity and circulating levels of angiotensin II .
It has been suggested that ACE inhibitors can block angiotensin II formation by increasing efferent arteriolar pressure leading to a reduction in intraglomerular pressure mitigating renal injury. In the present study, renal ACE levels and the expressions of OPN and TGF-β1 were significantly decreased with astaxanthin treatment in the astaxanthin-treated nephrolithiatic group. Consistent with this observation the SEM-EDX analysis revealed that there were no crystal depositions in the tubular lumens of the astaxanthin-treated nephrolithiatic group, indicating the ability of astaxanthin to disrupt crystal formation. Citrate therapy has been used for a long time in the clinical management of kidney stones [7, 17]. In the present study, we found that the renal tissue of citrate-treated rats had high calcium content with some stray crystal deposits as opposed to the astaxanthin-treated group, which had a normal histology and calcium levels. The renal ACE levels and the relative expressions of OPN and TGF-β1 in the kidney were also high despite the administration of citrate. These results suggest that astaxanthin can be considered as a better antilithiatic agent than potassium citrate.
It could be regarded that the reported effects of astaxanthin in the present study possibly stem from its unique chemical properties based on its molecular structure with two carbonyl groups, two hydroxyl groups, and eleven conjugated ethylenic double bonds. The presence of the hydroxyl and keto moieties on each ionone ring explains its ability to be esterified and a higher antioxidant activity and a more polar nature than other carotenoids [24, 25]. Within the cell, astaxanthin is found to effectively scavenge lipid radicals, destroy peroxides and protect fatty acids and membranes from oxidative damage . Previous studies reported that astaxanthin showed significant antioxidant activity in the kidney . Based on the present observations, it can be assumed that the protective effect of astaxanthin may be associated with its well established antioxidant effect and its ability to modulate the renal RAS system. In addition, the nephroprotective effect of ACE modulators is also mediated through changes in TGF-β1 expression . Thus the protective mechanism of astaxanthin plausibly results from its modulatory effect on renal ACE levels, which in turn down regulates the expressions of OPN and TGF-β1, and thereby reduces crystal deposition and associated renal damage.
It can be concluded from our present investigation that astaxanthin can effectively reduce crystal deposition in the renal tubules in experimentally induced nephrolithiasis. Astaxanthin possibly exerts this effect through an ACE modulatory effect, which successively coordinates the expression of OPN and TGF-β1 in the kidneys of the rats. Moreover, treatment with astaxanthin was found to be more effective than the potassium citrate therapy used in the clinical management of kidney stones. Hence astaxanthin offers promising potential as an antilithiatic agent. Further research is warranted to elucidate the molecular mechanism of the ACE modulatory activity of astaxanthin.
The authors are grateful to Radhakrishnan R, LMMD DIVISION, RGCB, Trivandrum, Kerala, India for providing facility and guidance in RT-PCR studies and Sreekumar, SCTIMST, Trivandrum, Kerala, India for the SEM-EDX analysis.