EFFECTS OF OPUNTIA MEGACANTHA LEAVES EXTRACT ON RENAL ELECTROLYTE AND FLUID HANDLING IN STREPTOZOTOCIN (STZ)-DIABETIC RATS

Abstract

We previously demonstrated that Opuntia megacantha leaves' extracts can reduce blood glucose levels in diabetes mellitus. For O. megacantha leaves' extracts to have potential in the management of diabetes mellitus, it is necessary to establish its detailed effects on renal function since diabetes is associated with renal fluid and electrolyte disturbances. Therefore, the current study was designed to investigate the influence of the extracts on renal function in male diabetic Sprague-Dawley rats. Rats were made diabetic by an i.p. injection of streptozotocin (STZ, 60 mg/kg in citrate buffer). Vehicle injected animals acted as controls. Separate groups of nondiabetic and diabetic rats were orally administered O. megacantha leaves extracts (20 mg/100 g bw) or normal saline (0.1 ml.100 g⁻¹ bw) daily for 5 weeks. Urine volume and total urinary outputs of Na⁺ and K⁺ were determined from 24 h samples. O. megacantha leaves' extracts significantly (p < 0.01) increased urinary Na⁺ output in diabetic and nondiabetic rats resulting in significantly (p < 0.01) low plasma concentration by comparison with untreated animals. Treatment with the extract significantly increased FE_Na⁺ and GFR in all groups. The urinary K⁺ outputs in nondiabetic was slightly lowered, but did not reach statistically significance. O. megacantha leaves' extracts did not alter plasma aldosterone and AVP concentrations in diabetic and nondiabetic rats in nondiabetic animals. It is concluded that O. megacantha leaves extracts modulate renal water and sodium handling. The mechanisms are not clear.

• Diabetes mellitus
• Streptozotocin
• Kidney
• Opuntia megacantha

INTRODUCTION

In Zimbabwe, a wide variety of plants are used for medicinal purposes in the management of ailments that include diabetes, abdominal and chest pains [[1]]. Medicinal plants such as Aloe barbadensis, Opuntia streptacantha and Solanum verbascifolum are used to treat diabetes in Asia, South and Central America [[2]]. We have recently demonstrated that leaves extracts of O. megacantha, of the same genus as Opuntia streptacantha which has been previously established to exert hypoglycaemic effects [[2]] not only reduces blood glucose levels, but may modulate kidney function [[3]]. For O. megacantha leaves' extracts to have potential in the management of diabetes mellitus, it is necessary to establish detailed effects of the extracts on renal function since diabetes is associated with renal fluid and electrolyte disturbances. Thus, the main aim of this study was to evaluate whether O. megacantha leaves extracts can reduce the rate of decline in kidney function previously assessed as inability of the kidney to excrete Na⁺ in STZ-diabetic rats [[4]].

Some evidence from conscious diabetic rats suggests that glucose induced osmotic diuresis in the initial stages of diabetes mellitus may be associated with fluid and electrolyte depletion [[5]]. These authors reported increases in urinary electrolyte excretion after only two to three weeks of diabetes, but other studies suggest Na⁺ retention in rats five to six weeks after induction of diabetes [[4]], [[6]]. In view of these reports, treatment of diabetes mellitus should be targeted towards correction of such complications. Thus, the current study was designed to establish the effects of O. megacantha leaves' extracts on renal function in STZ-diabetic rats. We also monitored the effects of the extracts on plasma concentrations of renally active hormones, aldosterone and AVP as these may contribute to any changes in renal handling of water and electrolytes. Streptozotocin which is commonly used to induce diabetes, selectively destroys or impairs secretion of β cells of the pancreas and the systemic changes that occur after its injection are considered related to the induced diabetic state [[7]].

METHODS AND MATERIALS

Animals

Experiments were performed on male Sprague-Dawley rats (300–400 g bw) bred and housed in the Medical Faculty animal house, University of Zimbabwe. The rats were kept separately at room temperature in flat bottomed plastic cages that were cleaned daily and were given both food (Mouse Comproids, National Foods, Zimbabwe) and tap water ad libitum.

Induction of Diabetes

Rats were made diabetic by an intraperitonial injection of streptozotocin (STZ 60 mg/Kg) in citrate buffer, pH 6.3. Animals that exhibited glucosuria after 24 hours, tested by Combur 9 test (Boehringer, Germany), were considered diabetic. Vehicle injected animals acted as controls.

Preparation of Opuntia megacantha Leaves Extracts

Opuntia megacantha was collected around Harare and authenticated at the National Herbarium, where voucher specimens were deposited (Number 5417). Leaves were homogenized using a blender and dried for 48 hours at 40°C. 100 g of plant material was mixed with 150 mL of 80% ethanol and stirred for 4 hours and filtered. The filtrate was dried in an evaporator and a yield of 1.57% of the prepared plant material was dissolved in normal saline.

Treatment

The rats were divided into the following groups: nondiabetic control (C), treated nondiabetic (T), control diabetic (CD) and treated diabetic (TD) rats. O. megacantha leaves' extract (20 mg.100 g⁻¹ bw) was orally administered daily at 0900 h for 5 weeks. Preliminary studies showed this dose to be the most effective. Untreated groups were given normal saline (0.1 mL.100 g⁻¹ bw). Oral administration was by means of a bulbeb steel tube. Water intake, volume of urine voided and urinary contents of Na⁺ and K⁺ were measured daily. Kidney weight was determined after five weeks in control and treated nondiabetic and diabetic rats.

After 5 weeks, blood was collected by decapitation into pre-cooled fluoride tubes for glucose and in heparin tubes for Na⁺ and K⁺ measurements from all groups of rats. The plasma that was separated from the whole component of blood was stored at −70°C (Forma Scientific Inc, USA) until measurement of aldosterone and AVP.

Analytical Methods

Measurement of Electrolytes

Urine volume was determined gravimetrically. Na⁺ and K⁺ were determined by Flame Photometry (Corning model 435 Flame Photometer, Corning Limited, Halstead, U.K.). Glucose was measured using the COBAS, MIRA S (Roche, France). Osmolality was measured by freezing point depression using micro-osmometer (CamLab, Roebling, Germany).

Plasma Glucose, Osmolality, and Hormone Measurements

Plasma aldosterone concentration was measured by Coat-A-Count using a kit from Diagnostic Products Corporation, Los Angeles, U.S.A. This is a solid-phase radioimmunoassay procedure based on aldosterone-specific antibody immobilized to the wall of a polypropylene tube. The lower limit of detection was 44 fmol.L⁻¹. Inter- and intra-assay coefficients of variation were 8.1% (n = 20) and 8.3% (n = 20), respectively. AVP was determined as described by Forsling & Peysner, (1988) [[8]] using the DSL-1800 Arginine Vasopressin Radioimmunoassay kit from Diagnostic Systems Laboratories, Inc., Webster, Texas, USA. Vasopressin was extracted from plasma using Sep Pak C18 cartridges (Millipore Water Associates, Harrow, Middx, U.K.). The lower limit of detection was 0.5 fmol.L⁻¹ and intra- and interassay variations of were 7.7% (n = 12) and 11.9% (n = 12), respectively.

Clearance Measurements

Weekly glomerular filtration rate (GFR) were measured using creatinine clearance. Creatinine clearance involved 24-hour urine collection and and 0.1 mL blood collected from the tail vein simultaneously to allow calculation of creatinine clearance as a measure of GFR. The fractional excretion rates of sodium (FE_Na⁺) and potassium (FE_K⁺) were calculated.

Analysis of Data

Values are presented as means ± SEM. Renal function was assessed by calculating the weekly fluid voided and total urinary outputs of Na⁺ and K⁺ over the 5- week period of study. To determine the effects of the leaf extract of O. megacantha, the data were treated and presented separately for the nondiabetic and STZ-diabetic rats. Untreated animals acted as controls. The data were subjected to analysis of variance using a one-way design and Scheffes multiple comparison was used to assess any differences. A value of p < 0.05 was considered significant.

RESULTS

Table 1 compares weekly weight changes, food intake, urine volumes, urinary outputs of Na⁺ and K⁺ in untreated and treated nondiabetic and STZ-diabetic rats over a 5-week period. O. megacantha administration did not significantly influence body weight of nondiabetic rats, but reduced the rate of weight loss in diabetic rats. Food intake was significantly (p < 0.05) increased in untreated diabetic rats from week 1 by comparison with untreated nondiabetic rats. In both groups. O. megacantha leaves extract reduced daily food intake by comparison with respective controls. The leaves' extracts significantly (p < 0.01) increased water intake from week 2. Treatment with O. megacantha leaves extract reduced terminal plasma glucose levels by 21% in nondiabetic rats and by 18% in diabetic animals (Table 2). Hyperglycaemia was accompanied by no significant changes in plasma insulin concentrations.

Table 1. Comparison of Body Weight and 5 Day Measures of Food Taken, Fluid Voided, and Urinary Na⁺ and K⁺ Excreted in Untreated (C) and Treated (T) Nondiabetic(n = 8) and STZ-Diabetic Rats over 5 Weeks (n = 8 in Each Group)

Measure	Group	1	2	3	4	5
Water intake (mL.day^−1)	Nondiabetic (C)	23±1	20±1	24±1	27±1	23±1
	Nondiabetic (T)	24±1	30±1^#	30±1^#	30±1^*	30±1^#
	STZ-diabetic (CD)	77±5^*	100±3^*	113±4^*	113±5^*	121±3^*
	STZ-diabetic (TD)	85±3^*	112±3^*	120±3	138±3^*#	128±3^*
Food intake (g.day^−1)	Nondiabetic (C)	16±1	16±1	17±1	15±1	15±1
	Nondiabetic (T)	14±1	15±1	13±1^*	10±1^*	9±1^*
	STZ-diabetic (CD)	23±1^*	23±1^*	25±1^*	29±1^*	30±1^*
	STZ-diabetic (TD)	19±2^*	22±2^*	21±1^*#	23±2^*#	25±1^*#
Weight change (%)	Nondiabetic (C)	+2.2±0.3	+2.7±0.4	+5.6±0.9	+11.2±0.9	+13.1±0.7
	Nondiabetic (T)	+2.6±0.3	+2.5±0.5	+4.7±0.9	+7.8±0.9	+10.8±0.6
	STZ-diabetic (CD)	−4.9±0.7	−8.0±1.1	−10.5±1.4	−12.7±2.0	−15.7±1.2
	STZ-diabetic (TD)	−5.9±0.8	−3.9±0.5	−8.1±0.9	−11.7±0.7	−12.3±0.7
Uv(mL.day^−1)	Nondiabetic (C)	14±1	13±1	13±1	12±5	12±2
	Nondiabetic (T)	11±1	13±1	14±1	14±1	13±1
	STZ-diabetic (CD)	90±4^*	89±3^*	84±5^*	85±3^*	92±5^*
	STZ-diabetic (TD)	87±3^*	94±3^*	91±3^*	93±3^*	97±3^*
UNa^+ excreted (mmol.day^−1)	Nondiabetic (C)	22±1	23±1	22±1	19±1	22±1
	Nondiabetic (T)	18±3	30±1^*	26±1^*	36±2^*	40±1^*
	STZ-diabetic (CD)	12±1^*	9±1^*	12±1^*	9±1^*	10±1^*
	STZ-diabetic (TD)	15±1^*#	16±1^*#	15±1^*#	15±1^*#	15±1^*#
UMK^+ excreted (mmol.day^−1)	Nondiabetic (C)	18±1	17±1	18±3	23±1	18±3
	Nondiabetic (T)	20±3	17±2	18±2	19±4	19±2
	STZ-diabetic (CD)	20±1	25±3^*	36±3^*	32±2^*	37±3^*
	STZ-diabetic (TD)	23±2	23±2^*	29±4^*	28±3^*	34±2

^*p<0.01 by comparison with untreated nondiabetic animals.

^*#p<0.01 by comparison with treated sSTZ-animals.

Table 2. Changes in Kidney Weight, Plasma Concentrations of Glucose, Insulin, Na⁺, K⁺, Aldosterone AVP, and Osmolality in Untreated and Treated Nondiabetic and Diabetic Rats After 5 Weeks (n = 8 in All Groups)

Measure	Nondiabetic (C)	Nondiabetic (T)	STZ-Diabetic (CD)	STZ-Diabetic (TD)
Kidney weight (g.100 g^−1 bwt)	0.43±0.15	0.31±0.11	0.92±0.13^*	0.89±0.07^*
Glucose (mmol.L^−1)	7.7±0.2	6.1±0.1^*	36.4±0.3^*	29.7±0.7^*#
Insulin (ng.mL^−1)	13.6±0.6	12.6±0.7	1.3±0.1^*	1.5±0.2^*
Na^+ (mmol.L^−1)	137±1	134±1^*	142±1.0^*	133±1^*#
K^+ (mmol.L^−1)	4.9±0.3	4.5±0.2	3.6±0.5^*	3.7±0.3^*
Aldosterone (mmol.^−1)	1.3±0.3	1.6±0.3	1.3±0.1	1.8±0.4
AVP (fmol. L^−1)	5.8±0.6	6.1±0.9	1.1±0.3^*	1.3±0.2^*
Osmolality (mOsm.Kg^−1H2O)	289±5	271±6	139±9^*	327±7^*

Renal Effects of O. megacantha Leaves Extract

Glycosuria and polyuria were observed from the first week in STZ-diabetic rats. O. megacantha leaves' extracts did not significantly affect the weekly volume of urine in nondiabetic and diabetic rats despite increased water intake (Table 1). STZ-diabetic rats were associated with a decline in mean weekly urinary outputs of Na⁺ that persisted up to week 5. Plasma concentrations of Na⁺ were significantly (p < 0.01) higher in untreated STZ-diabetic animals by comparison with nondiabetic control rats (Table 2). O. megacantha leaves' extracts significantly (p < 0.01) increased urinary Na⁺ excretion in diabetic and nondiabetic rats resulting in significantly (p < 0.01) low plasma concentration by comparison with untreated animals. The treatment significantly (p < 0.05) increased FE_Na⁺ from weeks 3 to 5 in nondiabetic rats and and weeks 1 to 4 in diabetic animals (Figure 1C). The urinary output of K⁺ in STZ-diabetic rats was significantly elevated resulting in low plasma concentrations by comparison with nondiabetic rats (Table 1). Administration of O. megacantha leaves extracts caused significant reduction in urinary K⁺ outputs in STZ-diabetic rats. The urinary K⁺ excretion in nondiabetic was slightly lowered, but did not reach statistical significance. Administration of leaves' extracts increased FE_K⁺ rates from week 3 in nondiabetic rats, but no significant effects were observed in STZ-diabetic rats (Figure 1B).

Figure 1. GFR (A), FeK⁺(B) and Fe_Na⁺ (C) rates in nondiabetic and STZ-diabetic control rats and nondiabetic and STZ-diabetic rats administered O. megacantha leaves' extract for 5 weeks vertical bars indicate S.E. of means.

GFR and Plasma Aldosterone and AVP Concentrations

After 5 weeks of O. megacantha leaves' extracts treatment plasma concentrations of aldosterone and AVP in nondiabetic and diabetic rats were not significantly altered by comparison with respective controls (Table 2). GFR values approximated 4 mL.min⁻¹ in untreated nondiabetic rats throughout the study period (Figure 1A). Treatment with the extract significantly increased GFR from weeks 2 in nondiabetc rats and from week 3 in STZ-diabetic animals. However, GFR values in diabetic rats were significantly low by comparison with nondiabetic animals.

DISCUSSION

This study determined the effects of O. megacantha leaves' extracts on renal electrolyte retention in STZ-diabetic rats with the hope of providing further evidence for possible use of the extract in the management of diabetes mellitus. After 5 weeks of O. megacantha administration, the inability of the kidney to excrete Na⁺ in STZ-diabetis mellitus was reversed suggesting that the extracts protects renal function in diabetes mellitus. This suggests that O. megacantha leaves' extracts may play a role in protecting renal function in diabetes mellitus. We have previously reported hypoglycaemic effects of oral administration of O. megacantha leaves extracts in nondiabetic and STZ-diabetic rats [[9]]. The glucose metabolic control and improvement of kidney function associated with O. megacantha administration are decisive intermediary endpoints likely to lead to the postment of diabetic renal disease.

O. megacantha leaves extracts administration daily for 5 weeks increased the weekly urinary outputs of Na⁺ urinary with a concomitant reduction of plasma concentrations of the cation in nondiabetic and diabetic rats. It would appear that the extract did not influence the plasma concentrations of hormones known to influence renal electrolyte excretion, aldosterone and AVP to elicit renal electrolyte handling. In the current study plasma concentrations of these hormones in treated rats did not significantly differ from values of respective control animals (Table 2). Variations in the activity of sodium retaining hormones such as as aldosterone have been excluded in involvement to explain disturbances of body sodium in diabetes [[10]]. The absence of increases of plasma AVP levels in diabetic rats despite further increases in plasma osmolality following O. megacantha adminstration is perhaps surprising and suggests involvement of disturbances of other factors in fluid and electrolyte regulation. Perhaps, low plasma Na⁺ concentrations suppressed the secretion of AVP. Indeed, studies have shown that the sensitivity of AVP response to osmotic stimuli is significantly decreased in diabetes mellitus [[11]]. These authors reported that plasma concentrations of Na⁺ influenced plasma AVP levels rather than plasma osmolality in diabetes mellitus. Such changes would enable the kidney to increase renal excretion of electrolytes and water. The mechanisms involved cannot be explained by the present study but may be due to renal haemodynamics responses to the extract as evidenced by an increase in mean weekly GFR in treated rats. Indeed, O. megacantha leaves extracts increased GFR in both nondiabetic and diabetic rats. Since, a decrease in GFR auguments the degree of sodium retention [[12]] and macroalbuminuria [[13]], intervention trials in insulin-dependent diabetes mellitus should adopt reversing a decline in GFR as an outcome measure. The results of the current study suggest that the leaves extract of O. megacantha increased renal sodium loss as shown by reduction in plasma Na⁺ concentration in diabetic and non-diabetic rats (Table 2). The reduced levels of plasma sodium could be a result of O. megacantha leaves' extracts induced increases in GFR and Fe_Na+, reducing tubular sodium reabsorption. Therefore, we cannot exclude the possibility that the leaves's extract could at least play a permissive role in lowering body sodium.

In summary, extracts of O. megacantha leaves increase renal Na⁺ loss and reduce decline in GFR in STZ-diabetic rats by mechanisms that are not clear. Although the effects of the extracts on renal fluid, sodium and potassium have been studied, renal handling of other ions such as magnesium, calcium and phosphate warrant investigations. The effects of the extract on other complications of diabetes mellitus need studying.

ACKNOWLEDGMENT

This study was supported by research grant number YYHO1O/3781 from the Research Board, University of Zimbabwe.