The Effects of Syzygium aromaticum-Derived Oleanolic Acid on Kidney Function of Male Sprague–Dawley Rats and on Kidney and Liver Cell Lines

Abstract

Studies indicate that Syzygium spp-derived oleanolic acid (OA) enhances renal function of streptozotocin (STZ)-induced diabetic rats as evidenced by its reversal of the previously reported inability of the kidney to excrete Na⁺ in these animals. We postulated that OA influences Na⁺ excretion in the proximal tubule, the site where two-thirds of filtered NaCl is reabsorbed through a process mediated by transport proteins. Therefore, the study investigated the effects of OA on proximal tubular Na⁺ handling in male Sprague–Dawley rats using renal lithium clearance (C_Li). Renal C_Li has been used widely in animal and clinical studies to assess proximal tubular function. Sub-chronic doses of OA were administered to rats twice every third day for 5 weeks. Rats treated with deionized water served as control animals. Cytotoxicity of OA on kidney and liver cell lines was assessed by the MTT and comet assays. OA increased Na⁺ excretion of conscious male Sprague–Dawley rats from week 3 to week 5. By the end of the 5-week experimental period, OA treatment significantly reduced (p < 0.05) plasma creatinine concentration of STZ-induced diabetic rats with a concomitant elevation in glomerular filtration rate (GFR). Acute OA infusion was also associated with increases in fractional excretion of sodium (FE_Na) and lithium (FE_Li) in anesthetized rats in the absence of significant changes in GFR. The MTT assay studies demonstrated that OA increased the metabolic activity of kidney and liver cell lines. Taken together with previous observations, this study implicates the proximal tubule in OA-evoked increases in urinary Na⁺ output.

• renal function
• Syzygium aromaticum
• oleanolic acid

INTRODUCTION

Plants containing the triterpene constituent oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid, OA) have several traditional uses in folk medicines in terms of anti-inflammatory, hepatoprotective, analgesic, cardiotonic, sedative, and tonic effects.^1,2 However, increasing evidence suggests that OA possesses hypoglycemic properties and may also have beneficial effects on some processes associated with impaired kidney function in diabetes.^3,4 Indeed, OA appears to reverse the previously reported inability of the kidney to excrete Na⁺ in streptozotocin (STZ)-induced diabetic rats.⁵ These findings suggest that OA may be influencing transport processes in the proximal tubule, the site for the reabsorption of approximately two-thirds of the Na⁺ that enters the tubular fluid by glomerular filtration. Accordingly, the study investigated the effects of OA on proximal tubular Na⁺ handling using renal lithium clearance. We acknowledge that the specificity of lithium as a marker of proximal tubule sodium reabsorption is controversial; nonetheless, lithium can be considered to be a good indicator of proximal tubule function.^6–10 Available evidence suggests that some herbal extracts interfere with the concentrating and diluting mechanisms of tubular transport processes in the nephron and/or with other protein components of tubular cell membranes.^11,12 Therefore, any link between OA affecting proximal tubular Na⁺ reabsorption and secretion would require that the triterpene influences tubular transport protein components or tubular epithelial cells’ viability. To examine this possibility, this study also assessed the effects of OA on cell viability on human embryonic kidney (HEK293) and Madin–Darby bovine kidney (MDBK) cell lines. Cell culture systems have been extensively used to evaluate toxicity of drugs in specific organs.¹³ In vitro cell culture techniques that mimic the in vivo state of cell lines maintain similar levels of marker enzymes of metabolism exhibited by freshly isolated cells.¹⁴

MATERIALS AND METHODS

Drugs and Chemicals

All chemicals and drugs used for this study were of analytical grade and were purchased from standard commercial and pharmaceutical suppliers, respectively.

Isolation of OA

OA was isolated from Syzygium aromaticum [(Linnaeus) Merrill & Perry] (Myrtaceae) (cloves) using a previously validated protocol in our laboratory.^3,4 Air-dried powdered flower buds of S. aromaticum were exhaustively extracted thrice at 24-h intervals sequentially with 3 L of hexane, dichloromethane, ethyl acetate, and methanol at room temperature. The solvents were removed under reduced pressure to give corresponding residues of hexane-solubles, dichloromethane-solubles, ethyl acetate-solubles (EAS), and methanol-solubles. Recrystallization of EAS which had been previously shown to contain OA ursolic acid/methyl maslinate/methyl corosolate^3,15 with ethanol yielded pure OA, whose structure was confirmed by spectroscopic analysis using 1D and 2D, ¹H and ¹³C nuclear magnetic resonance techniques. Preliminary studies indicated that the S. aromaticum-isolated OA and commercial OA were similar and hence the plant-extracted OA was used in the experiments as it costs less.

Animals

Male Sprague–Dawley rats (250–300 g body weight) bred and maintained at Biomedical Research Unit, University of KwaZulu-Natal, were used in this study. The animals had free access to standard rat chow (Meadows, Pietermaritzburg, South Africa) and water, with a 12-h light/12-h dark cycle. The experiments were performed in accordance with the institutional guidelines of the University of KwaZulu-Natal.

Experimental Design

The effects of various doses of OA on renal fluid and electrolyte handling were assessed in conscious male Sprague–Dawley rats, while the effects on proximal tubular Na⁺ handling using lithium as a marker were evaluated in anesthetized rats. In vitro effects of OA were evaluated on HEK293, MDBK, and liver (HepG2) cell lines.

Renal Fluid and Electrolyte Handling Studies

Separate groups of male Sprague–Dawley rats housed individually in Makrolon polycarbonate metabolic cages (Tecniplast, Labotec, South Africa) were treated with various doses of OA (30, 60, and 120 mg kg⁻¹, p.o.) by means of a bulbed steel tube every third day for 5 weeks at 09.00 and 15.00 (n = 7 in each group). OA was freshly dissolved in dimethyl sulfoxide (DMSO, 2 mL) and normal saline (19 mL) before use in each case. Rats treated with DMSO/saline (3 mL kg⁻¹, p.o.) acted as untreated controls. The rats were given both food and water ad libitum. Urine volume and urinary concentrations of creatinine, urea, Na⁺, K⁺, and Cl⁻ were determined every third day for 5 weeks at 09.00. Mean arterial blood pressure (MAP) was monitored every third consecutive day for 5 weeks at 09.00 using non-invasive tail cuff method with photoelectric sensors (IITC Model 31 Computerized Blood Pressure Monitor, Life Sciences, Woodland Hills, CA, USA). The unit works with IITC hardware system to measure blood pressure and heart rate in conscious rats. The animals had been warmed in an enclosed chamber (IITC Model 303sc Animal Test Chamber IITC Life Sciences) for 30 min at ±30°C before taking BP readings. At the end of the 5-week experimental period, blood samples were also collected from all groups of animals by cardiac puncture into individual pre-cooled heparinized containers. Separated plasma was analyzed for Na⁺, K⁺, creatinine, and urea concentrations.

Determination of Proximal Tubule Na⁺ Handling

Male Sprague–Dawley rats were fed standard rodent chow supplemented with lithium chloride (12 mmol kg⁻¹ dry weight) for 48 h prior to experimentation in order to raise plasma lithium to measurable concentrations without affecting renal sodium or water excretion.¹⁶ The rats were prepared for acute renal clearance studies and MAP using a modified procedure that has been previously described.³ Briefly, the rats were anesthetized by intraperitoneal injection of inactin [5-ethyl-5-(1′-methylpropyl)-2-thiobarbiturate, 0.11 g kg⁻¹ body weight] (Sigma Aldrich, St. Louis, MO, USA) and tracheotomy was performed. The right jugular vein was cannulated to allow a continuous intravenous infusion of saline. A minimal abdominal incision was made, and a urinary bladder catheter was inserted for the collection of urine samples. The body temperature was maintained at 37 ± 1°C with a heated table throughout the experimental period. Immediately following the cannulation of the right jugular vein, a priming dose of creatinine (3 μg in 0.3 mL of 0.077 M NaCl) was given i.v. and the animals were placed on a continuous i.v. infusion of 0.077 M NaCl containing creatinine (0.15 μg mL⁻¹) at 9 mL h⁻¹ (Harvard Syringe Infusion Pump 22, Harvard Apparatus, Holliston, MA, USA). After a 3 h 30 min equilibration period, urine samples were taken every 30 min over the 4 h post-equilibration period of 1 h control, 1 h 30 min treatment, and 1 h 30 min recovery periods; blood samples were taken once per hour for the measurement of electrolyte and clearance marker concentrations. Animals were then divided into two groups that received either vehicle (0.077 M NaCl) or OA in 0.077 M NaCl at 90 μg h⁻¹ for 1.5 h (treatment period). The infusate was then switched back to the vehicle for a further 1 h 30 min. Proximal tubular Na⁺ handling was assessed by the determination of endogenous lithium in plasma and urine and by the fractional excretion (FE) of lithium and sodium. For MAP measurements, a catheter was implanted in the left carotid artery for continuous recording of arterial blood pressure at 30 min intervals via a pressure transducer (Statham MLT 0380, Ad Instruments, Bella Vista, NSW, Australia), compatible with PowerLab System ML410/W (Bella Vista, NSW, Australia) and withdrawal of blood samples (0.3 mL). Plasma and urine samples were stored at 4°C until assay of creatinine, urea, and electrolytes was undertaken.

Analytic Procedures

Urine volume was determined gravimetrically. Na⁺ and K⁺ concentrations were determined by ion activity using the Beckman Coulter Counter (Synchron CX3 Clinical Systems, Fullerton, CA, USA). Urea and creatinine analyses were performed using the Beckman Coulter instrument. Creatinine estimation employed the reaction of creatinine and sodium picrate to form creatinine picrate. Urea estimation employed the hydrolytic degradation of urea in the presence of urease. The methods used reagent kits from Beckman Coulter, Ireland, Inc., and the concentrations were measured using Beckman Coulter (Synchron CX3 Clinical Systems).

Li⁺ was measured by flame emission spectrometry (Optima 2100 DV, Perkin Elmer, Shelton, CT, USA). The instrument was calibrated with Li⁺ working standards (0.16, 0.32, 0.64, 1.28, and 2.56 mg L⁻¹) made from Li⁺ stock standard solution (5 mg L⁻¹). Manually diluted (1:20) pool samples were then aspirated into the air/acetylene flame where neutral Li⁺ ions absorb light emitted from the Li⁺ hollow cathode lamp at 670.8 nm.¹⁷ The standard solutions and samples were diluted in deionized water to suppress the interfering substances. FE of Na⁺ and Li⁺ was determined simultaneously.

Calculations

Lithium clearance (C_Li) was used as a marker for the output of Na⁺ from the proximal tubules.⁸ Renal clearances (C) and FE were calculated with the standard formulae where C = U × V/P and FE = 100 × C/GFR, where U is the urinary concentration, V is the urine flow rate, and P is the plasma concentration. FE_{Na distal} was calculated as C_Na/C_Li. GFR, the glomerular filtration rate, as assessed by creatinine clearance (C_Cr) was calculated using the standard formulae from measurements of the plasma and urinary concentrations of creatinine and urine flow rate in the fifth week.

Cell Viability Studies

Cell culture

The HEK293 cell line was obtained from the University of the Witwatersrand Medical School. HepG2 cells and MDBK cells were purchased from Highveld Biological Pty Ltd., Kelvin, South Africa. All cell lines were initially propagated in 25 cm² flasks (Bibby Sterilin, Stone, Staffordshire, UK), at 37°C in EMEM (Gibco BRL, Inchinnan, Scotland) (5 mL) containing 10% (v/v) fetal bovine serum, 20 mM HEPES, 10 mM NaHCO₃, 100 U mL⁻¹ penicillin G, and 100 μg mL⁻¹ streptomycin sulfate (Whittaker Bioproducts, Walkersville, MD, USA) at pH 7.5. Cells were divided 1:3 every 3–4 days and stored in a biofreezer (−80°C) in complete medium containing 10% DMSO.

MTT assay

The sensitivity of cells to the activity of OA was determined using a standard spectrophotometric 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.¹⁸ Cells were trypsinized and seeded into 48-well plates (Bibby Sterilin) at a seeding density of 1.8 × 10⁴ cells/well and incubated for 24 h to permit attachment and growth of cells to semi-confluency.

Thereafter, the medium (0.5 mL) containing the cells was changed and various doses of OA (0, 5, 10, 20, 40, and 80 μmol L⁻¹) were added to the wells and cells incubated at 37°C for 24, 48 and 72 h. After each incubation period, the medium was removed and 200 μL of MTT in phosphate-buffered saline (5 mg mL⁻¹) was added to the wells. The cells were incubated for 4 h to allow for the formation of blue formazan crystals. The MTT solution was then replaced with DMSO (200 μL/well) and absorbance was measured at 570 nm in a UV–visible spectrophotometer (Thermoscientific Biomate, Cambridge, UK). The percentage cell viability was then calculated as follows: [A₅₇₀ treated cells – background]/[A₅₇₀ control cells – background] × 100.

Single-cell gel electrophoresis (comet) assay

The single-cell gel electrophoresis (comet) assay is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells.^19,20 HEK293 and MDBK cells were seeded into 24-well plates (Bibby Sterilin) and incubated for 24 h to permit attachment and growth to semi-confluency. Thereafter, the cells were prepared as for MTT studies, but only two doses of OA (20 and 80 μmol L⁻¹) were used. Preliminary studies indicated that all doses of OA (5–80 mmol L⁻¹) used in the study did not induce any damage on the DNA; hence, comet assay studies were conducted with median and highest concentrations, that is, 20 and 80 μmol L⁻¹, respectively. Frosted microscope slides were each covered with 400 μl of 1% normal melting point agarose in Ca²⁺- and Mg²⁺-free Kenny’s solution (0.4 M NaCl, 9 mM KCl, 0.7 mM K₂HPO₄, and 2 mM NaHCO₃) and a coverslip was placed over the agarose, which was then allowed to solidify. The coverslips were removed and approximately 1.8 × 10⁴ cells were added to 175 μL of 0.5% low melting point agarose in Kenny’s solution. The last layer of 200 μL of 1% normal melting point agarose was then added. The coverslips were removed, and the slides were placed in ice cold, freshly prepared lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10) for 1 h. Thereafter, the slides were subjected to electrophoresis for 35 min at 25 V (0.83 V cm⁻¹) in a BioRad horizontal gel electrophoresis unit (BioRad, Richmond, CA, USA) containing chilled electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13.5). Slides were washed with three changes of neutralization buffer (0.4 M Tris, pH 7.5), each for 5 min, to remove alkali and detergents. Slides were then stained with ethidium bromide (50 μL of a 20 μg mL⁻¹ solution) and a coverslip was placed over the agarose. Slides were viewed using an Olympus inverted fluorescent microscope with a CC12 fluorescent camera (excitation filter of 450–490 nm and a barrier filter of 520 nm) (Wirsam Scientific & Precision Eq. LTD, Johannesburg, South Africa) which allowed for computerized image analysis for determination of DNA tails, linearly related to the frequency of DNA strand breaks.²¹

Statistical analysis

Data are presented as the mean ± standard error of mean (SEM). Statistical comparison of the differences between the control and experimental groups was performed with GraphPad InStat Software (version 5.00, GraphPad Software, San Diego, CA, USA), using one-way analysis of variance, followed by Tukey–Kramer multiple comparison test. A value of p < 0.05 was considered significant.

RESULTS

Structure of OA

The data from ¹H- and ¹³C-NMR (1D and 2D) spectroscopic analyses of the white powder obtained after recrystallization of the EAS with ethanol were comparable with literature data.^3,22

Renal Fluid and Electrolyte Excretion

Figures 1 and 2 compare the effects of OA on urinary Na⁺ outputs in groups of anesthetized and conscious rats with their respective control animals. Acute infusion (1 h 30 min) of OA (90 μg h⁻¹) in anesthetized animals increased urinary Na⁺ excretion rate from the pre-treatment value of 713 to 847 μmol h⁻¹ by the end of the treatment period (Figure 1). On the other hand, sub-chronic administration (5 weeks) of various doses of OA (30, 60, and 120 mg kg⁻¹, p.o.) twice every third day significantly (p < 0.05) increased urinary Na⁺ outputs in a dose-independent manner from week 2 to week 5 (Figure 2). However, OA treatment did not change the urine flow and K⁺ and Cl⁻ excretion rates of both conscious and anesthetized animals (data not shown).

Figure 1. Comparison between the effects of OA infusion in anesthetized rats with control animals on GFR (A), MAP (B), and Na⁺ excretion rate (C). OA was administered at 90 μg h⁻¹ for 1 h 30 min during the treatment period. Values are presented as means for each 30-min collection; vertical bars indicate SEM of means (n = 6) in each group.

Note: *p < 0.05 in comparison with control animals.

Figure 2. Comparison between the effects of OA treatment twice every third day for 5 weeks with control animals on MAP (A) and Na⁺ excretion rates (B) in conscious rats. Values are presented as means for weekly collection; vertical bars indicate SEM of means (n = 6) in each group.

Note: *p < 0.05 in comparison with control animals.

MAP and GFR Measurements

The GFR of control and OA-infused animals remained steady over the 4 h experimental period and did not statistically differ during the treatment (Figure 1). OA administration for 1 h 30 min to anesthetized rats, however, significantly (p < 0.05) reduced MAP from a pre-treatment value of 105 to 92 mm Hg at the end of the treatment period (n = 6) without influencing the GFR (Figure 1). The MAP of conscious animals was significantly (p < 0.05) decreased by all doses of OA from week 2 until the end of the 5-week experimental period (Figure 2). OA also decreased significantly (p < 0.05) the plasma creatinine concentration of conscious animals with a concomitant increase in GFR by the end of the 5-week experimental period, but plasma urea concentration was not altered (Table 1). Kidney mass was not altered by sub-chronic OA treatment (Table 1).

Table 1. Terminal plasma biochemical parameters of control and rats administered OA twice every third consecutive day for 5 weeks (n = 6 in all groups).

Parameter	Control	30 mg kg^−1 body weight	60 mg kg^−1 body weight	120 mg kg^−1 body weight
Na^+ (mmol L^−1)	142 ± 1	141 ± 2	140 ± 2	142 ± 1
K^+ (mmol L^−1)	4.25 ± 0.25	4.14 ± 0.21	4.03 ± 0.22	4.17 ± 0.20
Cl^− (mmol L^−1)	104 ± 1	102 ± 1	103 ± 1	101 ± 2
Urea (mmol L^−1)	7.5 ± 0.6	8.1 ± 0.3	8.5 ± 0.5	8.4 ± 0.2
Creatinine (μmol L^−1)	27 ± 1	20 ± 3*	21 ± 2*	20 ± 3*
GFR (mL min^−1)	2.58 ± 0.16	3.04 ± 0.16*	3.51 ± 0.17*	3.73 ± 0.17*
Kidney weights (g 100 g^−1)	0.43 ± 0.20	0.47 ± 0.06	0.48 ± 0.05	0.47 ± 0.01

Notes: *p < 0.05 in comparison with respective control animals. **p < 0.05 in comparison with respective non-diabetic animals.

Renal Clearance Measurements

Lithium clearance (C_Li) was used as a marker for proximal tubular sodium clearance.²³ The lithium doses used resulted in plasma lithium concentrations between 0.2 and 0.3 mmol L⁻¹ with no difference between the groups. FE_Li and FE_Na did not differ between the control and the experimental groups prior to the infusion of OA (Figure 3). The infusion of OA for 1 h 30 min significantly increased FE_Li, FE_{Na prox}, and FE_{Na dist} in comparison with control animals at the corresponding time (Figure 3). However, OA infusion slightly reduced the FE_{Na distal} in comparison with FE_{Na prox} to levels that did not achieve statistical significance at the corresponding time period. In all cases, the FE_Na was not accompanied by any changes in FE_K and FE_Cl (data not shown).

Figure 3. Comparison between the effects of OA infusion in anesthetized rats with control animals on FE_{Na prox}, FE_{Na dist} (A) and FE_Li (B). OA was administered at 90 μg h⁻¹ for 1 h 30 min during the treatment period. Values are presented as means, and vertical bars indicate SEM (n = 6 in each group).

Note: *p < 0.05 in comparison with control animals.

Cell Culture Studies

MTT assay

Figure 4 shows the percentage cell viability of HepG2, HEK293, and MDBK cell lines. The viability and/or metabolic activity of HepG2 cells increased significantly (p < 0.05) after 24- and 48-h exposure periods when compared to the control. However, the cell viability and/or metabolic activity decreased significantly (p < 0.05) after the 72-h exposure period in comparison to 24- and 48-h exposure periods, but remained above that of the control. OA treatment significantly increased (p < 0.05) the viability and/or metabolic activity of HEK293 and MDBK in a dose-dependent manner after 24-h exposure in comparison to both the control and the longer exposure periods. However, there was a slight decrease in the viability and/or metabolic activity of both kidney cell lines after 48- and 72-h exposure, which did not reach statistical significance when compared to the control.

Figure 4. The effects of OA on the viability and/or metabolic activity of HepG2 (A), HEK293 (B), and MDBK (C) cells in vitro after exposure to various concentrations of OA for 24, 48, and 72 h. Values are presented as means, and vertical bars indicate SEM (n = 6).

Notes: *p < 0.05 in comparison with control. ♦p < 0.05 in comparison with 24 and 48 h.

DNA integrity

Figure 5 shows the effects of OA on DNA integrity of HEK293 and MDBK cell lines, respectively, as assessed by the comet assay. Figure 5A shows no apparent DNA damage of control cells that were only suspended in the growth medium whilst Figure 5B indicates DNA damage as indicated by the comet tails in positive control cells treated with hydrogen peroxide (H₂O₂). H₂O₂ is considered to be an important reactive oxidant species and causes cell death via either apoptosis or necrosis.²⁴ HEK293 cells remained intact following exposure for 24 and 72 h to OA at concentrations of 20 and 80 μmol L⁻¹, indicating that OA does not cause DNA damage in kidney cell lines. Similar results were obtained for the MDBK cells (results not shown).

Figure 5. The effects of OA on DNA integrity of HEK293 cells in vitro after exposure to various concentrations of OA for 24 and 72 h. Control for 24 h (A), H₂O₂ — 50 μmol L⁻¹ for 24 h (B), OA — 20 μmol L⁻¹ for 24 h (C) and 72 h (D), and 80 μmol L⁻¹ for 24 h (E) and 72 h (F).

DISCUSSION

The results of the present study apart from confirming our previous observations of the natriuretic effects of OA in rats³ suggest that this increased urinary Na⁺ output is in part mediated via the inhibition of proximal tubular Na⁺ reabsorption by the triterpene. Renal C_Li, which has been widely used in animal studies and clinical investigations to assess proximal tubular function in the mammalian kidney,^6,7,25–28 was used to estimate changes in proximal tubule Na⁺ reabsorption. The technique is based on the assumptions that Li⁺ is reabsorbed in the proximal tubules to the same degree as Na⁺.²⁹ Thus, a change in FE_Li estimates Na⁺ amount leaving the proximal tubule if GFR is unchanged. However, this is a controversial technique as there is evidence that lithium can be reabsorbed by the distal nephron under certain conditions such as Na⁺ and K⁺ depletion.¹⁰ It is reasonable to assume that C_Li is a valid marker of proximal tubule function in this study, since the animals were Na⁺ and K⁺ replete. Therefore, any coincidental proximal tubular action of OA was associated with predominant secretion of Na⁺.

Specifically, OA infusion increased FE_Na and FE_Li, likely reflecting an inhibition of proximal tubular epithelial Na⁺ reabsorption. Indeed, the pronounced increase in urinary Na⁺ excretion rate evoked by OA infusion in the absence of significant changes in GFR suggests that OA inhibits proximal tubular reabsorption. The proximal tubule reabsorbs approximately two-thirds of the NaCl that enters the tubular fluid by glomerular filtration³⁰ mediated by transport proteins.^31–33 The proximal tubule Na⁺ transporters on which OA exerts inhibitory action could not be identified by the current observations, nor is it clear whether OA has a direct effect on epithelial transport or works through an intermediary. Na⁺ is mainly reabsorbed across the proximal tubular cell in a process mediated by proximal tubule apical NHE3 followed by uphill active extrusion via basolateral Na⁺/K⁺ ATPase.^34,35 The literature on natriuretic hormones’ actions in the proximal tubule, however, shows that transport inhibition is effected via parallel independent inhibition of apical NHE3 and basolateral Na⁺/K⁺ ATPase.³⁶ We, therefore, speculate that OA inhibited apical NHE3 and basolateral Na⁺/K⁺ ATPase accounting for Na⁺ excretion since NHE3 is a major Na⁺ transport pathway in the renal proximal tubule.³¹ Therefore, the reduction of FE_{Na dist} by OA to values that were below FE_{Na prox}, but statistically insignificant may be of biological importance compensating for changes in distal tubule sodium handling.

The GFR of anesthetized rats remained constant and the plasma concentration of electrolytes was unchanged in anesthetized rats following OA infusion, while sub-chronic treatment of conscious animals elevated the GFR with a concomitant reduction in plasma creatinine concentration. The current study cannot explain this discrepancy, but this may be attributed to the differences in the experimental protocols. In the anesthetized rats, GFR was assessed during the 1 h 30 min OA infusion period, while the GFR of conscious rats was assessed at the end of 5 weeks following oral administration of OA twice every third day. The OA-evoked increases in urinary Na⁺ excretion in conjunction with reduced plasma creatinine concentration and an elevated GFR over the 5-week experimental period suggest that these changes were in part mediated via enhanced renal plasma flow. The blood pressure lowering properties and amelioration of kidney function by OA confirm previous observations in STZ-induced diabetic rats.³⁷

The results further suggest that OA-evoked increase in Na⁺ excretion was not due to toxicity of this compound as shown by the MTT and comet assays. The MTT assay indicated that cell viability was significantly increased after 24 h, but long periods of exposure (48 and 72 h) of kidney cell lines to OA led to a progressive decrease in cell viability. The increased cell viability above that of the control could be as a result of a stimulatory effect on the cells by the OA, leading to increased cell proliferation in a time- and dosage-independent manner. Similar findings were obtained by other researchers.^38,39 We speculate that the decrease in cell viability with longer exposure periods could be partly attributed to the depletion of nutrients in the culture medium, which was not changed for the duration of the experimental period. This interpretation is supported by the observation that the control cells exhibited the same trend, indicating that the decrease was not totally drug induced. This was supported by the results of the comet assay where the treated cells lacked noticeable DNA tails which are clearly observed for the positive control. The comet assay is a simple and rapid technique to measure DNA damage or repair and genotoxicity^19,20 in individual cells. Undamaged DNA retains a highly organized association with matrix proteins in the nucleus, while this organization is disrupted in damaged cells. The concentrations of OA used in the current study (5–80 μmol L⁻¹) compared with those previously used in other in vitro studies for the triterpene and crude extracts containing such compounds.^40–42

Kidney structure is affected in diabetes due to glomerular hypertrophy caused by accumulation of extracellular matrix proteins and injury of the epithelial cells.^43,44 We have, however, reported that extracts of Persea americana Mill (Lauraceae) (“Avocado”) decrease the viability of kidney cell lines in vitro.⁴⁵ Furthermore, aristolochic acid isolated from Aristolochia fangchi was shown to induce cellular injury and apoptosis in proximal tubular (LLC-PK1) epithelial cell lines.^46–49 The results described in this study introduce the first in vivo evidence that the previously reported OA-evoked increases in urinary Na⁺ output are in part mediated via inhibition of Na⁺ reabsorption and increases in proximal tubular Na⁺ secretion. The study provides the first in vitro evidence that OA does not exhibit toxicity in kidney and liver cell lines and implicates the proximal tubule in the natriuretic effects of OA.

ACKNOWLEDGMENTS

This study was partly funded by the University of KwaZulu-Natal, Research Division. The authors are grateful to the Biomedical Research Unit, University of KwaZulu-Natal, for the supply of animals.

Declaration of interest: The authors declare that there is no interest that could be perceived as prejudicing the impartiality of the research reported.