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Glomerular filtration rate in early experimental diabetes
Glomerular Filtration Rate in Early ExperimentalDiabetes
Richard
C. O’Brien,
Terri J. Allen,
MBBS
BSc
Mark E. Cooper,
MBBS
Leon Bach, MBBS George
Jerums,
MD
Department of Medicine, Austin Hospital, University of Melbourne, Victoria, A us tralia
ABSTRACT A noninvasive single injection technique for the measurement of glomerularfiltration rate (GFR) using technetiumesmdiethylene triamine pentaacetic acid (DTPA) was developed for use in the rat. GFR measurements obtained by the technique correlated well with those obtained by Crsl EDTA infusion (R = 0.95, n = 7). The coefficient of variation was 8.4%. GFR was measured over 4 weeks in diabetic and control rats. GFR increased with time in both groups, with no difference between the groups; however, when corrected for body weight, diabetes was associated with an increased GFR (diabetic 13.6 f 1.7 vs. control 10.4 f 0.1 ml/min/kg p < 0.001). Insulin treated rats had higher GFRs than untreated diabetics (p < O.OS),but GFR/kg was reduced to that of nondiabetic controls. High protein intake in diabetic rats caused an increase in GFR after 1 week of diabetes, but this was not sustained by the fourth week. Genetic hypertension and angiotensin converting enzyme (ACE) inhibition with ramlpril had no effect on GFR in diabetic rats. We conclude that serial measurement of GFR in the diabetic rat is accurate and reproducible. Genetic hypertension, high protein intake, and ACE inhibition have little effect on GFR in experimental diabetes. (The Journal of Diabetic Complications 2;1:8-11, 1988.)
INTRODUCTION Glomerular filtration rate (GFR) is elevated early in the course of human diabetes,‘,* and it has been suggested that this state of “hyperfiltration” may be a pathogenic factor in the subsequent development of diabetic nephropathy.3 Increased GFR has also been demonstrated in experimental diabetes; however, this finding is variable and appears to be dependent on the degree of hyperglycemia and the duration of diabetes.4-* Conventional methods of GFR measurement are technically difficult, requiring cannulation of large vessels, and are not suitable for serial measurements. Few studies, therefore, have explored the effect of duration of diabetes on GFR, and serial studies of GFR in the same animal have not been performed. In this report we describea noninvasive single injection technique for measurement of GFR in the diabetic rat. Several factors have been shown to hasten the onset of experimental diabetic nephropathy, including high dietary protein intake and genetic hypertension.gl10 Conversely, angiotensin converting enzyme (ACE) inhibition appears to ameliorate the renal disease of both experimental and human diabetes.i1,12 We examined the possibility that these modulators of diabetic nephropathy could be acting through changes in GFR by studying the effects of variable protein intake, genetic hypertension, and ACE inhibition on GFR in the streptozotocin diabetic rat. Reprint requests: Dr. Richard C. O’Brien, Department of Medicine, Austin tiosDital. Unkersityof Melbourne, Heidelberg, 3084;
Victoria, Australia.
8
MATERIALS
AND METHODS
Male Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats weighing 180-230 g were used in this study. These animals came from our inbred stock, derived from the original strains supplied by Dr. Y.
GFRIN EXPERIMENTALDIABETES
9
distribution); and Pt = observed plasma concentration at t minutes after injection. Volume of distribution (VD) was determined in animals that had undergone total nephrectomy 3 hours previously. In controls (n = 4), untreated diabetic rats (n = 4), and diabetic rats given insulin 2 U/3 times a week (n = 4), VD was constant at 30.1 f 0.9 ml/ 100 g of body weight. Diabetic rats receiving insulin 6 U/day (n = 5) had slightly increased VD at 33.2 zj~ 0.7 ml/100 g of body weight.
Yamori in 1977. Diabetes was induced by tail vein injection of streptozotocin (STZ) in citrate buffer, 60 mg/kg for WKY rats and 45 mg/kg for SHR rats, the latter having greater sensitivity to STZ. Animals had unrestricted access to food and water. Standard diet (20% protein) was given as GR + 2 pellets (Clarke King, Melbourne, Australia), while the high protein (50%) diet contained a 36% casein supplement. Because casein contains phosphate, controls for the rats fed a high protein diet received additional dietary phosphate supplements so that urinary phosphate excretion was identical in each group. ACE inhibition was achieved with ramipril 1 mg/L in drinking water. This agent is more potent that enalapril in inhibiting plasma ACE.13 Heat treated bovine Ultralente insulin (Novo lndustri A/S, Copenhagen, Denmark), 2 U/3 times a week, was given to most groups of rats to reduce mortality. One group was untreated, and a further group received 6 U of insulin daily to achieve good glycemic control. Plasma glucose was measured by a glucoseoxidase method on an autoanalyzer (Astra-Beckman Instruments, Palo Alto, CA), and plasma renin activity was assayed by RIA.14
Measurement
Validation of GFR Method: Five diabetic and two control rats underwent carotid artery and jugular vein cannulation under general anesthesia and were allowed to recover. The following day, two clearance studies were performed in random order within 12 hours of each other, using infusion of Cr5’EDTA and the above DTPA method. Following a loading dose, Cr5’EDTA was infused into the jugular vein at 3.8 &i/hr, and after 3 hours serial samples ware taken from the carotid artery until a steady state was achieved. The calculated GFR was compared to that obtained by the DTPA technique in the same animal. Statistical analysis was performed by two-way analysis of variance (Clear Lake Research, Houston, TX).
RESULTS
of GFR:
A calibrated dose of technetiumggm reduced with stannous chloride and complexed to diethylene triamine pentaacetic acid (Sigma Chemicals, USA) was injected into the tail vein of conscious preheated rats. After 43 minutes a blood sample was taken from a different tail vein and separated in a heparinized tube. Plasma radioactivity was counted in a gamma counter (Packard Instruments, Illinois) and wascompared to a reference prepared at the time of injection. GFR was calculated according to the following equation described by Bryan etz~1.l~: Clearance
GFR measured by the DTPA technique correlated well with that measured by Cr5’EDTA infusion (R = 0.95). The method was highly reproducible, giving a mean coefficient of variation for weight-corrected GFR measured on four occasions in seven WKY rats of 8.4% (range 1.5-13.6%). There were no complications from the procedure, and measurementscould be performed repeatedly in thesame animals over a long period. The results of GFR measurements (expressed as ml/min and ml/min/kg means and SEM shown) over the first 4 weeks of diabetes in all models studied are contained in Table 1.
= V >(. In (Po/Pt)/t,
Diabetes
where V = volume of distribution; PO = theoretical plasma concentration at injection (= injected amount/volume of
TABLE
1
and GFR:
and control
GFR increased with age in both diabetic animals, with no significant difference be-
GFR, Rat Weight, and Plasma Glucose in All Groups Studied Week 0
Control (n = 8) Diabetic (D) (n = 9) D+insulin 6 u/d (n = 6) D+normal protein (n = 10) DShigh protein (n = 8) D+hypertension (n = 8) DSramipril (n = 11)
$p < 0.001.
Week 4 Plasma Glucose Weiaht (mmol/Lj (gi
GFR’
GFRAg
GFR
GFR/kg
GFR
GFR/kg
2.2 * 0.1
10.6 i 0.2
2.6 + 0.1
9.5 + 0.2
3.0 Ik0.1
10.0 f 0.3
6.7k 0.4
300
2.2 k 0.1
10.6 rt 0.2
2.7 + 0.1
11.8 f 0.4$
2.8 + 0.1
13.6 + 0.4t
36.0 + 1.7
205
1.9 + 0.1
9.0 + 0.4
3.4 + 0.1t
11.9 + 0.3$
3.4 + 0.1
10.4 + 0.1
5.2 + 1.3
327
1.9 f 0.1
10.1 zk 0.2
2.6 + 0.1
12.1 i 0.3
3.0 f 0.1
13.6 -t 0.4
32.6 f 1.5
221
2.3 k 0.1
10.5 k 0.5
3.4 + 0.17
15.5 + 0.2
3.5 * 0.2
14.0 f 0.7
26.9 i 2.9
250
2.5 k 0.1
11.6 t 0.4
2.4 f 0.2
11.1 + 0.4
3.3 * 0.1
13.8 f 0.6
29.8 + 0.4
239
1.9 f 0.1
10.0 f 0.5
2.8 IL 0.1
13.0 k 0.2
3.1 * 0.1
13.9 k 0.2
35.1 f 0.7
223
GFR isexpressed as ml/min and ml/min/kg of body weight. f p < 0.05. l
Week 4
Week 1
O’BRIEN
10
GFR
3_
ET/U.
GFR I Kg
mUmin
mllmin
2-
o
Diabetic+High
+ -)
Diabetic n=lO DiabetictHypettension
a
Diabetic+Ramipril
,.
F’rotein n=8 n=8
1
n=l
0
1I 0
2 4 Time (weeks)
w-
6
Diabetic+High
-
Diabetic 1~10 Diaktic+Hypertension
Protein n=R
*
Diabetic+Ramipril
!
n=l
n=8
I 8
0
8
2
4
6
8
Time (wccks~
tween the two groups. However, untreated diabetic animals had reduced growth, and when corrected for body weight, GFR in this group was significantly higher than in controls at all times after the induction of diabetes (p < 0.001). Insulin (6 U/day) caused a significant rise in GFR after 2 weeks of diabetes (p < 0.05). Weightcorrected GFR was reduced by insulin to that of nondiabetic controls after 4 weeks.
Effect on GFR of Factors Known
o
+
FIG 1 The effects of modulators of diabetic nephropathy on GFR in diabetic rats.
to Modulate
Diabetic
Nephropathy:
Diabetic rats fed a high protein diet had a significant (p < 0.01) early increase in GFR compared to their diabetic controls; however, this effect disappeared by the fourth week of diabetes (Figure 1). Despite adequate suppression of ACE as documented by a 69% rise in plasma renin activity in ramipril treated animals (ramipri12.74 f 0.4 vs. control 1.62 f 0.3 ng/ml/hr p
DISCUSSION DTPA is handled by the kidney in a manner similar to inulin, and single injection techniques using this compound are widely used for the evaluation of GFR in humans.16 This study demonstrates that a comparable method is accurate and reproducible when applied to the diabetic rat. The method allows serial studies of GFR in the same animal, thereby improving validity of measurements obtained, as well as reducing animal use. When measured by infusion techniques, the reported range of GFR is 1.68-3.68 ml/min in control rats, and 2.04-4.26 ml/min in diabetic rats.4-8 Our GFR findings for both groups fall within those ranges. Some authors have reported a decrease in GFR in severely hyperglycemic animals, compared to controls.6a8 We found no difference between these groups, and a possible explanation is that severely hyperglycemic animals tolerate surgery poorly, thus producing an artifactually low GFR when measured by the infusion technique or during micropuncture. Low dose insulin therapy with moderate hyperglycemia has been shown to variably increase GFR in experimental diabetes.4-8 We found that maintenance of good glycemic control normalized GFR after 4 weeks of treatment;
however GFR was increased at 1 week of diabetes when compared to nondiabetic controls. It is possible that variations in the duration of diabetes have produced the differing results seen in previous experiments, and clearly further longitudinal studies are required. High dietary protein intake has been shown to accelerate the onset of proteinuria and glomerulosclerosis in diabetic rats, and it has been suggested that hemodynamic mechanisms, particularly increased intraglomerular pressure and increased renal plasma flow leading to increased GFR, are responsible.g In our experiments GFR was increased in the high protein group only in the first week of diabetes, suggesting that the intrarenal hemodynamic effects of diabetes may override those of high protein intake once the disease is established. Alternatively, it is possible that the hyperphagia reported in diabetic animals17 may increase protein intake in animals on a “normal” diet. A further increase in protein consumption may therefore produce no additional effect. Changes in intrarenal hemodynamics, particularly lowering of intraglomerular pressure, are postulated as the mechanism by which ACE inhibitors may ameliorate diabetic nephropathy. Reduction of intraglomerular pressure will by itself lead to a fall in GFR. Our finding of no change in overall GFR in the ramipril treated group suggests that the inhibition of ACE has caused other hemodynamic changes, such as an increase in renal plasma flow, so that GFR is maintained. Genetic hypertension was not associated with any increase in GFR in diabetic animals, and it is therefore likely that the significantly accelerated diabetic nephropathy reported in this modello is due to factors other than alteration of GFR. Weconcludethat, with the possibleexception of insulin, factors known to modulate the development of diabetic nephropathy do not appearto substantially influence GFR in the first few weeks of experimental diabetes. This does not support the concept that changes in total GFR participate in the pathogenesis of experimental diabetic nephropathy.
ACKNOWLEDGMENTS The authors thank the staff of the Austin Hospital Department of Nuclear Medicine for their help. Dr. O’Brien is supported by a National Research Council Scholarship.
Health and Medical
GFRIN EXPERIMENTALDIABETES
REFERENCES 1. Ditzel J, Schwartz M: Abnormally increased glomerular filtration rate in short-term insulin-treated diabetic subjects. Diabetes 16:264-7, 1967. 2. Mogensen CE: Kidney function and glomerular permeability to macromolecules in early juvenile diabetes. Stand J C/in Lab invest 28:91-100, 1971. CE: Early glomerular hyperfiltration in insulin 3. Mogensen dependent diabetics and late nephropathy. Stand J C/in Lab Invest 46:201, 1986. J, Steven K, Parving H-H: 4. Jensen PK, Sandahl Christiansen Renal function in streptozotocin diabetic rats. Diabetologia 21:409-14, 1981. 5. Jensen PK, Sandahl Christiansen J, Steven K, Parving H-H: Strict metabolic control and renal function in the streptozotocin diabetic rat. Kidney Infernational 31:47-51, 1987. TH, Troy JL, Brenner BM: Glomerular haemo6. Hostetter dynamics in experimental diabetes mellitus. Kidney International 19:410-15, 1981. 7. Carney L, Wong NLM, Dirks JH: Acute effects of streptozotocin diabetes on rat renal function J Lab C/in Med93:950-61, 1979. LD. Davidman M, Keane WF: Determinants of 8. Michels glomerularfiltration and plasma flow in experimental diabetic rats. J Lab C/in Med 98:869-84, 1981. 9. Zatz R, Meyer TW, Rennke HG, Brenner BM: Predominance of haemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc Nat/ Acad Sci USA 82:5963-7, 1985.
11
10 Cooper ME, Allen TJ, Doyle AE: Accelerated progression of diabetic nephropathy in the spontaneously hypertensive streptozotocin diabetic rat. Clin Exp Pharmacol Physiol 13:655-62, 1986. 11 Zatz R, Rentz B. Brenner BM, et al: Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J C/in Invest 77:1925-30, 1986. 12 Marre M, Leblanc H, Suarez L, Guyenne T-T, Menard J, Passa P: Converting enzyme inhibition and kidney function in normotensive diabetic patients with persistent microalbuminuria. Br Med J 294:1448-52, 1987. K, Lang RE: 13 Unger T, Moursi M, Ganten D, Hermann Antihypertensive action of the converting enzyme inhibitor perindopril (S9490-3) in spontaneously hypertensive rats: Comparison with enalapril (MK 421) and Ramipril (HOE498). J Cardiovasc Pharmacol8:276-85, 1988. 14 Mendelsohn FAO, Hutchinson J, Johnston Cl: A review of plasma renin measurements and their clinical significance. Aust NZ J Med 1:86-93, 1971. 15 Bryan CW, Jarchow RC, Maher JF: Measurement of glomerular filtration rate in small animals without urine collection. J Lab C/in Med 80:845-56, 1972. of renal function with 16. Dubovsky EV, Russel CD: Quantitation glomerular and tubular agents. Sem Nucl Med XII 4:308-29, 1982. 17. Hebden RA, Gardiner SM, Bennet T, MacDonald IA: The influence of streptozotocin-induced diabetes mellitus on fluid and electrolyte handling in rats. C/in Sci 70:111-17, 1986.
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Richard
C. O’Brien,
Terri J. Allen,
MBBS
BSc
Mark E. Cooper,
MBBS
Leon Bach, MBBS George
Jerums,
MD
Department of Medicine, Austin Hospital, University of Melbourne, Victoria, A us tralia
ABSTRACT A noninvasive single injection technique for the measurement of glomerularfiltration rate (GFR) using technetiumesmdiethylene triamine pentaacetic acid (DTPA) was developed for use in the rat. GFR measurements obtained by the technique correlated well with those obtained by Crsl EDTA infusion (R = 0.95, n = 7). The coefficient of variation was 8.4%. GFR was measured over 4 weeks in diabetic and control rats. GFR increased with time in both groups, with no difference between the groups; however, when corrected for body weight, diabetes was associated with an increased GFR (diabetic 13.6 f 1.7 vs. control 10.4 f 0.1 ml/min/kg p < 0.001). Insulin treated rats had higher GFRs than untreated diabetics (p < O.OS),but GFR/kg was reduced to that of nondiabetic controls. High protein intake in diabetic rats caused an increase in GFR after 1 week of diabetes, but this was not sustained by the fourth week. Genetic hypertension and angiotensin converting enzyme (ACE) inhibition with ramlpril had no effect on GFR in diabetic rats. We conclude that serial measurement of GFR in the diabetic rat is accurate and reproducible. Genetic hypertension, high protein intake, and ACE inhibition have little effect on GFR in experimental diabetes. (The Journal of Diabetic Complications 2;1:8-11, 1988.)
INTRODUCTION Glomerular filtration rate (GFR) is elevated early in the course of human diabetes,‘,* and it has been suggested that this state of “hyperfiltration” may be a pathogenic factor in the subsequent development of diabetic nephropathy.3 Increased GFR has also been demonstrated in experimental diabetes; however, this finding is variable and appears to be dependent on the degree of hyperglycemia and the duration of diabetes.4-* Conventional methods of GFR measurement are technically difficult, requiring cannulation of large vessels, and are not suitable for serial measurements. Few studies, therefore, have explored the effect of duration of diabetes on GFR, and serial studies of GFR in the same animal have not been performed. In this report we describea noninvasive single injection technique for measurement of GFR in the diabetic rat. Several factors have been shown to hasten the onset of experimental diabetic nephropathy, including high dietary protein intake and genetic hypertension.gl10 Conversely, angiotensin converting enzyme (ACE) inhibition appears to ameliorate the renal disease of both experimental and human diabetes.i1,12 We examined the possibility that these modulators of diabetic nephropathy could be acting through changes in GFR by studying the effects of variable protein intake, genetic hypertension, and ACE inhibition on GFR in the streptozotocin diabetic rat. Reprint requests: Dr. Richard C. O’Brien, Department of Medicine, Austin tiosDital. Unkersityof Melbourne, Heidelberg, 3084;
Victoria, Australia.
8
MATERIALS
AND METHODS
Male Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats weighing 180-230 g were used in this study. These animals came from our inbred stock, derived from the original strains supplied by Dr. Y.
GFRIN EXPERIMENTALDIABETES
9
distribution); and Pt = observed plasma concentration at t minutes after injection. Volume of distribution (VD) was determined in animals that had undergone total nephrectomy 3 hours previously. In controls (n = 4), untreated diabetic rats (n = 4), and diabetic rats given insulin 2 U/3 times a week (n = 4), VD was constant at 30.1 f 0.9 ml/ 100 g of body weight. Diabetic rats receiving insulin 6 U/day (n = 5) had slightly increased VD at 33.2 zj~ 0.7 ml/100 g of body weight.
Yamori in 1977. Diabetes was induced by tail vein injection of streptozotocin (STZ) in citrate buffer, 60 mg/kg for WKY rats and 45 mg/kg for SHR rats, the latter having greater sensitivity to STZ. Animals had unrestricted access to food and water. Standard diet (20% protein) was given as GR + 2 pellets (Clarke King, Melbourne, Australia), while the high protein (50%) diet contained a 36% casein supplement. Because casein contains phosphate, controls for the rats fed a high protein diet received additional dietary phosphate supplements so that urinary phosphate excretion was identical in each group. ACE inhibition was achieved with ramipril 1 mg/L in drinking water. This agent is more potent that enalapril in inhibiting plasma ACE.13 Heat treated bovine Ultralente insulin (Novo lndustri A/S, Copenhagen, Denmark), 2 U/3 times a week, was given to most groups of rats to reduce mortality. One group was untreated, and a further group received 6 U of insulin daily to achieve good glycemic control. Plasma glucose was measured by a glucoseoxidase method on an autoanalyzer (Astra-Beckman Instruments, Palo Alto, CA), and plasma renin activity was assayed by RIA.14
Measurement
Validation of GFR Method: Five diabetic and two control rats underwent carotid artery and jugular vein cannulation under general anesthesia and were allowed to recover. The following day, two clearance studies were performed in random order within 12 hours of each other, using infusion of Cr5’EDTA and the above DTPA method. Following a loading dose, Cr5’EDTA was infused into the jugular vein at 3.8 &i/hr, and after 3 hours serial samples ware taken from the carotid artery until a steady state was achieved. The calculated GFR was compared to that obtained by the DTPA technique in the same animal. Statistical analysis was performed by two-way analysis of variance (Clear Lake Research, Houston, TX).
RESULTS
of GFR:
A calibrated dose of technetiumggm reduced with stannous chloride and complexed to diethylene triamine pentaacetic acid (Sigma Chemicals, USA) was injected into the tail vein of conscious preheated rats. After 43 minutes a blood sample was taken from a different tail vein and separated in a heparinized tube. Plasma radioactivity was counted in a gamma counter (Packard Instruments, Illinois) and wascompared to a reference prepared at the time of injection. GFR was calculated according to the following equation described by Bryan etz~1.l~: Clearance
GFR measured by the DTPA technique correlated well with that measured by Cr5’EDTA infusion (R = 0.95). The method was highly reproducible, giving a mean coefficient of variation for weight-corrected GFR measured on four occasions in seven WKY rats of 8.4% (range 1.5-13.6%). There were no complications from the procedure, and measurementscould be performed repeatedly in thesame animals over a long period. The results of GFR measurements (expressed as ml/min and ml/min/kg means and SEM shown) over the first 4 weeks of diabetes in all models studied are contained in Table 1.
= V >(. In (Po/Pt)/t,
Diabetes
where V = volume of distribution; PO = theoretical plasma concentration at injection (= injected amount/volume of
TABLE
1
and GFR:
and control
GFR increased with age in both diabetic animals, with no significant difference be-
GFR, Rat Weight, and Plasma Glucose in All Groups Studied Week 0
Control (n = 8) Diabetic (D) (n = 9) D+insulin 6 u/d (n = 6) D+normal protein (n = 10) DShigh protein (n = 8) D+hypertension (n = 8) DSramipril (n = 11)
$p < 0.001.
Week 4 Plasma Glucose Weiaht (mmol/Lj (gi
GFR’
GFRAg
GFR
GFR/kg
GFR
GFR/kg
2.2 * 0.1
10.6 i 0.2
2.6 + 0.1
9.5 + 0.2
3.0 Ik0.1
10.0 f 0.3
6.7k 0.4
300
2.2 k 0.1
10.6 rt 0.2
2.7 + 0.1
11.8 f 0.4$
2.8 + 0.1
13.6 + 0.4t
36.0 + 1.7
205
1.9 + 0.1
9.0 + 0.4
3.4 + 0.1t
11.9 + 0.3$
3.4 + 0.1
10.4 + 0.1
5.2 + 1.3
327
1.9 f 0.1
10.1 zk 0.2
2.6 + 0.1
12.1 i 0.3
3.0 f 0.1
13.6 -t 0.4
32.6 f 1.5
221
2.3 k 0.1
10.5 k 0.5
3.4 + 0.17
15.5 + 0.2
3.5 * 0.2
14.0 f 0.7
26.9 i 2.9
250
2.5 k 0.1
11.6 t 0.4
2.4 f 0.2
11.1 + 0.4
3.3 * 0.1
13.8 f 0.6
29.8 + 0.4
239
1.9 f 0.1
10.0 f 0.5
2.8 IL 0.1
13.0 k 0.2
3.1 * 0.1
13.9 k 0.2
35.1 f 0.7
223
GFR isexpressed as ml/min and ml/min/kg of body weight. f p < 0.05. l
Week 4
Week 1
O’BRIEN
10
GFR
3_
ET/U.
GFR I Kg
mUmin
mllmin
2-
o
Diabetic+High
+ -)
Diabetic n=lO DiabetictHypettension
a
Diabetic+Ramipril
,.
F’rotein n=8 n=8
1
n=l
0
1I 0
2 4 Time (weeks)
w-
6
Diabetic+High
-
Diabetic 1~10 Diaktic+Hypertension
Protein n=R
*
Diabetic+Ramipril
!
n=l
n=8
I 8
0
8
2
4
6
8
Time (wccks~
tween the two groups. However, untreated diabetic animals had reduced growth, and when corrected for body weight, GFR in this group was significantly higher than in controls at all times after the induction of diabetes (p < 0.001). Insulin (6 U/day) caused a significant rise in GFR after 2 weeks of diabetes (p < 0.05). Weightcorrected GFR was reduced by insulin to that of nondiabetic controls after 4 weeks.
Effect on GFR of Factors Known
o
+
FIG 1 The effects of modulators of diabetic nephropathy on GFR in diabetic rats.
to Modulate
Diabetic
Nephropathy:
Diabetic rats fed a high protein diet had a significant (p < 0.01) early increase in GFR compared to their diabetic controls; however, this effect disappeared by the fourth week of diabetes (Figure 1). Despite adequate suppression of ACE as documented by a 69% rise in plasma renin activity in ramipril treated animals (ramipri12.74 f 0.4 vs. control 1.62 f 0.3 ng/ml/hr p
DISCUSSION DTPA is handled by the kidney in a manner similar to inulin, and single injection techniques using this compound are widely used for the evaluation of GFR in humans.16 This study demonstrates that a comparable method is accurate and reproducible when applied to the diabetic rat. The method allows serial studies of GFR in the same animal, thereby improving validity of measurements obtained, as well as reducing animal use. When measured by infusion techniques, the reported range of GFR is 1.68-3.68 ml/min in control rats, and 2.04-4.26 ml/min in diabetic rats.4-8 Our GFR findings for both groups fall within those ranges. Some authors have reported a decrease in GFR in severely hyperglycemic animals, compared to controls.6a8 We found no difference between these groups, and a possible explanation is that severely hyperglycemic animals tolerate surgery poorly, thus producing an artifactually low GFR when measured by the infusion technique or during micropuncture. Low dose insulin therapy with moderate hyperglycemia has been shown to variably increase GFR in experimental diabetes.4-8 We found that maintenance of good glycemic control normalized GFR after 4 weeks of treatment;
however GFR was increased at 1 week of diabetes when compared to nondiabetic controls. It is possible that variations in the duration of diabetes have produced the differing results seen in previous experiments, and clearly further longitudinal studies are required. High dietary protein intake has been shown to accelerate the onset of proteinuria and glomerulosclerosis in diabetic rats, and it has been suggested that hemodynamic mechanisms, particularly increased intraglomerular pressure and increased renal plasma flow leading to increased GFR, are responsible.g In our experiments GFR was increased in the high protein group only in the first week of diabetes, suggesting that the intrarenal hemodynamic effects of diabetes may override those of high protein intake once the disease is established. Alternatively, it is possible that the hyperphagia reported in diabetic animals17 may increase protein intake in animals on a “normal” diet. A further increase in protein consumption may therefore produce no additional effect. Changes in intrarenal hemodynamics, particularly lowering of intraglomerular pressure, are postulated as the mechanism by which ACE inhibitors may ameliorate diabetic nephropathy. Reduction of intraglomerular pressure will by itself lead to a fall in GFR. Our finding of no change in overall GFR in the ramipril treated group suggests that the inhibition of ACE has caused other hemodynamic changes, such as an increase in renal plasma flow, so that GFR is maintained. Genetic hypertension was not associated with any increase in GFR in diabetic animals, and it is therefore likely that the significantly accelerated diabetic nephropathy reported in this modello is due to factors other than alteration of GFR. Weconcludethat, with the possibleexception of insulin, factors known to modulate the development of diabetic nephropathy do not appearto substantially influence GFR in the first few weeks of experimental diabetes. This does not support the concept that changes in total GFR participate in the pathogenesis of experimental diabetic nephropathy.
ACKNOWLEDGMENTS The authors thank the staff of the Austin Hospital Department of Nuclear Medicine for their help. Dr. O’Brien is supported by a National Research Council Scholarship.
Health and Medical
GFRIN EXPERIMENTALDIABETES
REFERENCES 1. Ditzel J, Schwartz M: Abnormally increased glomerular filtration rate in short-term insulin-treated diabetic subjects. Diabetes 16:264-7, 1967. 2. Mogensen CE: Kidney function and glomerular permeability to macromolecules in early juvenile diabetes. Stand J C/in Lab invest 28:91-100, 1971. CE: Early glomerular hyperfiltration in insulin 3. Mogensen dependent diabetics and late nephropathy. Stand J C/in Lab Invest 46:201, 1986. J, Steven K, Parving H-H: 4. Jensen PK, Sandahl Christiansen Renal function in streptozotocin diabetic rats. Diabetologia 21:409-14, 1981. 5. Jensen PK, Sandahl Christiansen J, Steven K, Parving H-H: Strict metabolic control and renal function in the streptozotocin diabetic rat. Kidney Infernational 31:47-51, 1987. TH, Troy JL, Brenner BM: Glomerular haemo6. Hostetter dynamics in experimental diabetes mellitus. Kidney International 19:410-15, 1981. 7. Carney L, Wong NLM, Dirks JH: Acute effects of streptozotocin diabetes on rat renal function J Lab C/in Med93:950-61, 1979. LD. Davidman M, Keane WF: Determinants of 8. Michels glomerularfiltration and plasma flow in experimental diabetic rats. J Lab C/in Med 98:869-84, 1981. 9. Zatz R, Meyer TW, Rennke HG, Brenner BM: Predominance of haemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc Nat/ Acad Sci USA 82:5963-7, 1985.
11
10 Cooper ME, Allen TJ, Doyle AE: Accelerated progression of diabetic nephropathy in the spontaneously hypertensive streptozotocin diabetic rat. Clin Exp Pharmacol Physiol 13:655-62, 1986. 11 Zatz R, Rentz B. Brenner BM, et al: Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J C/in Invest 77:1925-30, 1986. 12 Marre M, Leblanc H, Suarez L, Guyenne T-T, Menard J, Passa P: Converting enzyme inhibition and kidney function in normotensive diabetic patients with persistent microalbuminuria. Br Med J 294:1448-52, 1987. K, Lang RE: 13 Unger T, Moursi M, Ganten D, Hermann Antihypertensive action of the converting enzyme inhibitor perindopril (S9490-3) in spontaneously hypertensive rats: Comparison with enalapril (MK 421) and Ramipril (HOE498). J Cardiovasc Pharmacol8:276-85, 1988. 14 Mendelsohn FAO, Hutchinson J, Johnston Cl: A review of plasma renin measurements and their clinical significance. Aust NZ J Med 1:86-93, 1971. 15 Bryan CW, Jarchow RC, Maher JF: Measurement of glomerular filtration rate in small animals without urine collection. J Lab C/in Med 80:845-56, 1972. of renal function with 16. Dubovsky EV, Russel CD: Quantitation glomerular and tubular agents. Sem Nucl Med XII 4:308-29, 1982. 17. Hebden RA, Gardiner SM, Bennet T, MacDonald IA: The influence of streptozotocin-induced diabetes mellitus on fluid and electrolyte handling in rats. C/in Sci 70:111-17, 1986.
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