Effects of the somatostatin analogue octreotide on renal function in conscious diabetic rats

Martin Bak1,2,, Klaus Thomsen1 and Allan Flyvbjerg2

1 Institute for Basic Psychiatric Research, Department of Biological Psychiatry and 2 Institute of Experimental Clinical Research, Medical Research Laboratories, Aarhus University Hospital, Denmark



   Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Studies performed during the last decade have indicated that growth hormone (GH) and insulin-like growth factors (IGFs) may mediate the early renal changes in diabetes mellitus, i.e. hypertrophy and hyperfiltration. This and other observations have led to the suggestion that GH/IGF inhibitors, such as long-acting somatostatin analogue (e.g. octreotide and lanreotide), may be useful in order to inhibit or prevent development of long-term diabetic complications.

Methods. The present study examined the acute and chronic effects of octreotide on renal function following induction of streptozotocin (STZ)-diabetes in rats. The studies were carried out in conscious, non-fasted diabetic animals.

Results. Chronic administration of octreotide for 7 days, from onset of diabetes, prevented the decrease of effective renal vascular resistance (ERVR), and the increases in filtration fraction (FF), glomerular filtration rate (GFR), and absolute proximal tubular fluid reabsorption (APR) induced by diabetes. The renal hypertrophy was only partially prevented. In the acute study, similar changes were observed in effective renal plasma flow (ERPF) and ERVR but FF increased and GFR remained unaltered.

Conclusions. Chronic but not acute treatment with octreotide prevented the renal hyperfiltration caused by diabetes. This effect is most likely due to an increase in afferent arteriolar resistance.

Keywords: conscious; diabetes; kidney function; kidney weight; octreotide; rats



   Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The kidney hypertrophy-hyperfunction syndrome is a well-described phenomenon in human diabetes. In order to achieve a more complete understanding of the mechanisms responsible for the diabetic renal changes, many research groups are using experimental diabetic animals models. In general, consistent changes in renal volume and morphology have been reported in various models of diabetes in the past, while some controversy still appears with respect to the magnitude and time of appearance of renal functional changes in diabetes models. The variability of the published results are believed to be caused partly by methodological problems, in particular when kidney functional variables were estimated in anaesthetized animals [1,2].

During the last decades, attention has been drawn towards growth hormone (GH) and insulin-like growth factors (IGFs) as mediators of early features in diabetic kidney disease, i.e. renal hypertrophy and renal hyperfiltration [3]. In experimental diabetes lasting for days or few weeks, inhibitors of the GH/IGF axis, such as octreotide has been shown to partially [4] or completely [5] abolish the initial renal hypertrophy and in six months studies to prevent the dramatic increase in urinary albumin excretion following experimental diabetes [6]. A preventive effect of octreotide against renal hyperfiltration could not be demonstrated in diabetic rats [7]. However, as the study was carried out in uninephrectomized, anaesthetized animals, this could have masked any beneficial effect of the drug on the increased perfusion and filtration rate. Pedersen et al. [8] observed an immediate reduction in effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in control subjects and diabetic patients following octreotide i.v. In a long-term study, octreotide administration for 12 weeks induced a significant decrease in the elevated GFR [9].

The present study, performed in conscious, unstressed, and non-fasted diabetic rats, aimed at determining whether acute administration of octreotide after 2 weeks of experimental diabetes could reverse the diabetic renal hyperfiltration, and whether chronic administration of octreotide for 7 days from onset of diabetes, could prevent the diabetic renal hyperfiltration. In addition, we measured clearances of tetraethylammonium (TEA) and lithium as markers of effective renal plasma flow (ERPF) and proximal tubular fluid output (Vprox), respectively, in order to determine the mechanism by which octreotide might prevent hyperfiltration in diabetes.



   Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Specific pathogen-free adult, female Sprague–Dawley rats (Møllegaards Avlslab., Eiby, Denmark) weighing between 200 and 260 g were used. Animals were housed one per cage in a room with 12 : 12 h artificial light cycle, lights on 8 a.m. to 8 p.m., temperature 21±2°C and humidity 55±2%. The rats were fed a standard diet (R3, Lactamin, Stockholm, Sweden) containing 21% protein, 200 mmol/kg of Na+ and 200 mmol/kg of K+ for at least 2 weeks prior to initiation of the studies. Three days before the experiment, the diet was changed to a diet containing lithium chloride (10–12 mmol/kg dry weight) in order to obtain measurable plasma lithium concentrations without influencing renal function [10]. Rats had free access to food and tap water throughout the studies.

One week before induction of diabetes, the animals were anaesthetized with halothane/N2O. Using semi-aseptic surgical techniques, sterile Tygon catheters (Norton Performance Plastics, Arkon, OH, USA) were advanced into the abdominal aorta and the inferior vena cava via the femoral vessels. A sterile chronic suprapubic catheter was implanted into the bladder. All catheters were produced and fixed, with minor modifications, as described previously [1]. After instrumentation, the rats were infused with saline s.c. (5 ml) and given a long acting analgesic, buprenorphinum (Temgesic, Reckitt & Colman, Hull, UK) s.c. They were housed individually, and after a recovery period of 5–6 days, they were accustomed to restriction by daily training sessions in restraining cages. The duration of each daily session was gradually increased from 1 to 3 h a day.

The rats were randomly allocated into diabetic and control groups. Diabetes was induced by i.v. injection of streptozotocin (STZ, 40 mg/kg body weight (BW)) in acidic 154 mmol/l NaCl (pH 4.5) following 12 h of food deprivation. Eighteen hours after STZ administration, and daily thereafter, the animals were weighed, urine analysis were performed for glucose and ketones using Neostix 4TM (Ames Limited, Stoke Poges, Slough, UK) and tail vein blood glucose was determined by Haemoglucotest 1–44 and Reflolux II reflectance meter (Boehringer-Mannheim, Mannheim, Germany). After administration of STZ, hyperglycemic rats with blood glucose levels above 25 mmol/l were treated daily with a very long-acting heat-treated insulin (Ultralente Insulin, Novo Nordisk A/S, Bagsvaerd, Denmark) to obtain a blood glucose level of 20–25 mmol/l. Day 0 was defined as the day following STZ administration.

The renal effects of octreotide (Sandostatin, Sandoz Pharma AG, Basle, Switzerland) in experimental diabetes were studied in two separate renal clearance studies. In one study, octreotide was given acutely after 14 days of onset of experimental diabetes, and in the other study octreotide was given chronically beginning at day 0. In the latter study, a somewhat shorter period of diabetes of 7 days was chosen in order to avoid experienced problems with blocked catheters. We have previously shown that renal functional changes are present within this time period of experimental diabetes [11]. In each study, the rats were randomly allocated into treatment with octreotide or vehicle. In the acute study, after a vehicle period, octreotide was given as a single bolus of 10 µg i.v., followed by an i.v. infusion rate of 10 µg/h during the rest of the clearance study. In the chronic study, octreotide was given in a dose of 100 µg s.c. twice daily. In previous studies from our group we have shown that the octreotide doses used in the present study maintain high diurnal serum octreotide concentration (i.e. above 1000 ng/l) [5], abolish the rise in endogenous kidney IGF-I levels [5] and normalize the diabetes-associated increase in urinary albumin excretion [6].

Clearance experiments were carried out on day 14 in the acute study and on day 7 in the chronic study.

Renal clearance protocol
The experiments were carried out between 8 a.m. and 1 p.m. The rats were transferred to a restraining cage and connected to infusion pumps via the vein catheter and to a blood pressure transducer via the arterial catheter. Throughout the experiment a half isotone saline (77 mM NaCl) was infused at a rate of 70 µl/min in order to maintain a urine flow necessary for accurate bladder emptying. A rate of 30 µl/min was used during acute octreotide infusion since it is known to induce anti-dioresis. [14C]Tetraethylammonium bromide (0.83 µCi/ml, New England Nuclear, Boston, MA, USA) together with [3H]inulin (2.5 µCi/ml, Amersham, Rainham, UK) and LiCl (13 mmol/l) were infused (10 µl/min) together with the saline as markers of ERPF, GFR, and Vprox, respectively.

The infusion was initiated by a 15 min interval during which a bolus of the different markers was given at a rate of four times (40 µl/min) the continuous infusion velocity. This was followed by an equilibration interval of 90 min. In the acute study, the equilibration interval was followed by urine collection in three vehicle periods of 20 min. Octreotide infusion was then started, and after a new equilibration interval of 30 min, urine was again collected in three experimental periods of 20 min. In the chronic study, the initial equilibration interval was followed by urine collection in three periods of 20 min. Blood samples (200 µl) were drawn from the arterial catheter after 105, 165, and 255 min in the acute study and after 105 and 165 min in the chronic study. Blood replacement with donor blood was given after each blood sample. Mean arterial blood pressure was recorded continuously using a Uniflow (Baxter, Irvine, CA, USA) transducer connected to a pre-amplifier and PC registration. Following the vehicle periods of the acute study and during the full length of the infusion experiment in the chronic study, net losses of water and sodium were replaced by a PC-controlled servo-system described earlier taking into account the basic infusion rate of fluid and sodium [12]. After cessation of the chronic experiment, the left kidney was weighed after perfusion fixation at 115 mmHg in vivo with a 4% formalin solution [13].

Approximately 30% of the rats were excluded due to occlusion of one or more of the various catheters in the acute study, and a somewhat lower percentage (about 20%) were excluded in the chronic study. Two rats in the acute study and four rats in the chronic study were excluded because of bladder emptying errors.

Analysis
Urine volume was determined by gravimetry. Li+ concentration was determined in plasma and urine by flame emission photometry and atomic absorption spectrophotometry, respectively. [14C]Tetraethylammonium bromide and [3H]inulin in plasma and urine were determined by dual label liquid scintillation counting (Wallac model 1409, Helsinki, Finland). Sample (15 µl) and 285 µl of water were mixed with 2.5 ml of scintillation liquid (Ultima Gold, Packard Instruments, Meriden, USA). Calculation of d.p.m. was performed by automatic efficiency control.

Calculations
Renal clearances (C) were calculated by the standard formula:


where U is the urine concentration, V is the urine flow rate and P is the plasma concentration. In previous studies the renal extraction fraction of TEA has been shown to be approximately 90% and the validity of TEA as an estimate of ERPF has been documented [14,15]. With the concentration of TEA used in this study, TEA is without effects on efferent renal sympathetic nerve activity in rats [16]. By use of CTea, CIn and CLi the following parameters were calculated:


In all calculations, it was assumed that the renal venous pressure was 5 mmHg throughout the experiment.

Data presentation and statistics
All values are presented as mean±SEM. Overall statistical analysis were performed by one way ANOVA (between groups) or one way ANOVA for repeated measurements (within groups). Individual comparisons within (vehicle vs octreotide in the acute study) or between groups (control vs diabetes in the acute and the chronic study) were performed by subsequent use of Student's t-test for paired and unpaired data, respectively. Differences were considered statistically significant at the 0.05 level.



   Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Acute effect of octreotide in diabetic rats
As shown in Table 1Go, diabetic animals were characterized by growth retardation, hyperglycaemia, and reduced mean arterial blood pressure (MAP). The haematocrit values were similar in diabetic and control rats. Octreotide treatment had no effect on body weight, MAP, haematocrit, or blood glucose. The plasma lithium concentration was about 0.20 mM in all groups (Table 1Go).


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Table 1. Body weight, MAP, haematocrit, and blood glucose in control rats and in rats diabetic for 14 days before and during acute i.v. octreotide infusion: data are given as mean±SEM

 
The renal variables from the study with acute octreotide administration are presented in Table 2Go. Compared with control rats, the diabetic rats showed increased GFR, FF, and APR. Treatment with octreotide led to an increase in ERVR and FF whereas ERPF decreased. Octreotide did not affect the GFR. In the control group, similar haemodynamic effects were seen. Concerning the proximal tubule, Vprox, as measured by lithium clearance, and FELi was reduced by octreotide in the control rats but not in the diabetic rats. In the distal nephron, the fractional reabsorptive escape of sodium (CNa/Vprox) and sodium clearance (CNa) were increased by octreotide in the diabetic group whereas urine flow rate (V) and the fractional distal escape of water (V/Vprox) were reduced in both groups.


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Table 2. Renal clearance data in control rats and in rats diabetic for 14 days before and during acute i.v. octreotide infusion: data are given as mean±SEM

 

Chronic effect of octreotide in diabetic rats
As shown in Table 3Go, changes in growths, blood glucose and MAP were similar to those described for the acute study. The renal changes in the diabetic animals were also similar to those seen in the acute study (Table 4Go). In the diabetic rats, octreotide led to an increase of ERVR and a decrease of ERPF and GFR while FF was unaltered and Vprox was reduced (P<0.05). No such effects were seen in the control group in response to octreotide. No essential effects were seen in the distal nephron in any of the groups.


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Table 3. Body weight, MAP, haematocrit and blood glucose in control rats and in diabetic rats treated s.c. with octreotide for 7 days from onset of diabetes. Data are given as mean±SEM

 

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Table 4. Renal clearance data in control rats and in diabetic rats treated s.c. with octreotide for 7 days from onset of diabetes. Data are given as mean±SEM

 



   Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we examined whether acute administration of octreotide after 2 weeks of experimental diabetes could reverse the diabetic renal hyperfiltration, and whether chronic administration of octreotide for 7 days from diabetes onset could prevent the diabetic renal hyperfiltration. In both studies, the diabetic rats showed the well-known hyperfiltration, whereas ERPF was unaltered. In diabetic rats, octreotide given chronically from onset of diabetes prevented the increase in GFR. Octreotide also led to an increase in ERVR and a decrease in ERPF in these rats. As FF showed a tendency to a decrease rather than an increase, the changes in ERVR and ERPF suggests that octreotide causes afferent arteriolar vasoconstriction. In the acute study, similar changes were observed in ERPF and ERVR but FF increased and GFR remained unaltered. This suggests that additional efferent arteriolar vasoconstriction may occur in the acute condition.

In control rats, acute octreotide administration caused an increase in ERVR and a decrease in ERPF without any change in FF, suggesting that the effect of octreotide on the afferent arteriole is not specific for the diabetic state. However, as the effect seems to vanish during chronic treatment, the control rats appear less sensitive to octreotide than diabetic rats in the chronic setting.

Clinical studies confirm that somatostatin and its analogues may affect renal perfusion and filtration rate. Vora et al. [17] and Pedersen et al. [8], using i.v. native somatostatin or octreotide, respectively, observed an immediate reduction in RPF and GFR in control subjects and diabetic patients. In a long-term study, octreotide administration for 12 weeks induced a significant decrease in the elevated GFR [9].

At the level of proximal tubule, we found that chronic treatment with octreotide led to a decrease in Vprox in diabetic rats, which support the notion that octreotide led to afferent arteriolar vasoconstriction in these animals. In the distal nephron, the acute natriuresis observed in the diabetic rats and the acute anti-diuresis observed in both diabetic rats and control rats were not maintained in the chronic study. This finding is expected, as, in the chronic setting, sodium and water excretion must match the intake. A decrease of the urine flow rate after acute octreotide has also been observed in patients [8,17].

In the control rats given octreotide acutely, the urine flow rate showed a tendency to be higher than the fluid infusion rate although the latter was high in order to match the infusion rate of the diabetic rats. An excessive urine flow rate is seen regularly in our studies and is probably a consequence of the experimental design, which involves a positive water balance during the first hour of equilibration followed by a negative balance in the following hours. When the rats are prevented from excreting the excess of fluid by servo control of the water balance, the urine flow rate increases even more as also observed in the present chronic study. A urine flow rate exceeding the infusion rate is not seen in the diabetic animals because they are not in a positive water balance during the first hours of the study.

It was concluded that chronic but not acute treatment with octreotide prevented the renal hyperfiltration caused by diabetes. This effect is most likely due to an increase in afferent arteriolar resistance.



   Acknowledgments
 
This study was supported by grants from the Danish Health Research Council (grant no. 9700592), the Novo Foundation, the Johanne and Aage Louis-Hansen Memorial Foundation, the Nordic Insulin Foundation, the Eva and Henry Frænkels Memorial Foundation, the Beckett Foundation, Arvid Nilssons Foundation, Mogens Svare Mogensens Foundation, Bernhard and Marie Kleins Grant for Research in Diabetes and the Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration, Danish Health Research Council (grant no. 9600822). We are grateful to Mrs Else Tusch, Jette Birk and Ninna Rosenqvist for excellent technical assistance.



   Notes
 
Correspondence and offprint requests to: Martin Bak, MD, Department of Biological Psychiatry, Institute for Basic Psychiatric Research, Aarhus University Hospital, Skovagervej, DK-8240 Risskov, Denmark. Email: martinbak{at}dadlnet.dk Back



   References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 23. 2.01
Revision received 3. 5.01.



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