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Effects of growth hormone and IGF-I on glomerular ultrafiltration in growth hormone-deficient rats
Regulatory Peptides, 48 (1993) 241-250
241
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01558
Effects of growth hormone and IGF-I on glomerular ultrafiltration in growth hormone-deficient rats Raimund Hirschberg Division of Nephrology and Hypertension, Harbor-UCLA Medical Center and UCLA, Los Angeles, CA (USA)
(Received 22 March 1993; revised version received20 April 1993; accepted 6 May 1993) Key words: Des(1-3)IGF-I; Glomerular hypertrophy; GFR; Single nephron glomerular filtration rate;
Glomerular ultrafiltration coefficient
Summary In growth hormone deficient states glomerular filtration rate (GFR) and renal plasma flow rate (RPF) are both reduced. Studies were performed in growth hormone deficient rats to delineate the physiologic mechanisms by which growth hormone and IGF-I contribute to the regulation of glomerular function. Growth hormone deficient dw/dw rats received, for one week, subcutaneous infusions of vehicle, des(1-3)IGF-I or were injected i.m. with recombinant human growth hormone. Subsequent renal micropuncture and clearance studies revealed a low G F R and single nephron G F R ( S N G F R ) in vehicle treated growth hormone deficient animals. Glomerular function became normal with growth hormone or IGF-I treatment, respectively. Both treatments raised S N G F R by reducing arteriolar resistance and increasing the glomerular ultrafiltration coefficient. Furthermore, the two treatments also increased the glomerular tuft volume and the kidney weight which may contribute to the rise in S N G F R and GFR. It is concluded that, (1) in growth hormone deficiency glomerular function is reduced secondary to a high renal arteriolar resistance and a low ultrafiltration coefficient. Both result from a lack in IGF-I rather than the growth hormone deficiency state per se. (2) The growth h o r m o n e - I G F - I axis may contribute to the maintenance and physiologic regulation of GFR.
Introduction Correspondence to: R. Hirschberg, Division of Nephrology and Hypertension,Harbor-UCLAMedicalCenter, 1000West Carson ST, Torrance, CA 90509, USA.
Injections of growth hormone or IGF-I raise renal plasma flow (RPF) and glomerular filtration rate (GFR) in normal rats and humans [1-15]. A single
242 intramuscular injection of growth hormone, 0.15 mg/ kg, in normal subjects induces a significant increase in RPF and GFR with a delay of several hours [2,3]. The rise in kidney function does not occur before the growth hormone-induced rise in circulating IGF-I can be demonstrated [2]. This latter finding suggests that circulating and/or renal IGF-I mediates the growth hormone-induced rise in glomerular perfusion and ultrafiltration. Indeed, subsequent studies in rats demonstrated that a short-term injection and infusion of recombinant human IGF-I (rhIGF-I) into rats acutely raises renal function [4]. In the rat rhIGF-I acutely reduces afferent and efferent arteriolar resistance in the nephron, which drives an increase in nephron blood and plasma flow [5]. As demonstrated in renal micropuncture studies this rise in nephron plasma flow contributes to the increase in single nephron glomerular filtration rate (SNGFR) during the infusion of rhIGF-I, which, in addition, is caused by an increase in the glomerular ultrafiltration coefficient, LpA [5,16]. There is preliminary evidence that the endogenous growth hormone-IGF-I axis contributes to the maintenance of adequate glomerular ultrafiltration in physiologic and pathophysiologic conditions. First, in acromegalics, both growth hormone and IGF-I serum levels are elevated [ 17-21]. Hypersomatotropic states also increase the synthesis of IGF-I in the kidney [22]. In acromegalic patients elevated serum growth hormone and IGF-I levels are associated with renal hypertrophy and glomerular hyperfiltration [7,23-29]. The elevated GFR may result from the increase in nephron and more specifically in glomerular size, but may also be caused by direct effects of growth hormone and/or IGF-I on the resistance and filtration-regulating microvasculature in the nephron. Second, certain experimental maneuvers that cause an increase in GFR are also associated with elevated IGF-I synthesis, such as feeding of high protein diets in normal rats and in animals with experimental chronic renal failure [30-32]. Third, treatment of normal rats with a synthetic growth hormone-releasing hormone antagonist which causes
a reduction in pulsatile growth hormone release and, hence, reduction in serum (and presumably renal) IGF-I levels, also decreases the glomerular filtration rate and reduces kidney wet weight and glomerular tuft volume [ 16]. GFR is reduced in growth hormone-deficiency states [3,26,27,29]. This could result from a lack of the physiologic effects of growth hormone and/or IGF-I, or from glomerular hypotrophy. If this hypothesis should materialize, it would give rise to the possibility that the growth hormone-IGF-I axis participates in the regulation and maintenance of normal glomerular function. The present study was designed to elucidate the differential effects of growth hormone and IGF-I on renal glomerular function and size using the model of the growth hormone-deficient rat.
Materials and Methods
Studies were performed in male growth hormonedeficient dw/dw rats weighing 199 + 10 (S.E.) g at the onset of the studies. Animals were purchased from Simonson, Gilroy, CA and were kindly provided by Ross Clark, Ph.D., of Genentech, South San Francisco, CA. The growth hormone deficiency trait arose spontaneously in a colony of Lewis rats and the mutation was introduced into the NIMR/AS strain and successfully maintained by crossing heterozygous animals with homozygous growth hormone deficient rats of the opposite sex [33]. Growth hormone deficient rats were randomly assigned to one of the following three groups (n = 7 each): group 1 (control) rats were anesthetized with intramuscular injections of Ketamine, 87 mg/kg, and Xylazine, 13 mg/kg, and were implanted subcutaneously with osmotic minipumps (Model 2001, Alza Corporation, Palo Alto, CA) that were filled with 50 mM acetic acid. The minipumps were primed over night in sterile 5~o glucose in water at 4°C prior to implantation. Animals in group 2 were also implanted with primed minipumps that were filled with des(13)IGF-I (Genentech Inc., South San Francisco, CA)
243 in 50 mM acetic acid. The concentration was adjusted to deliver des(1-3)IGF-I at a constant rate of 50/~g/day. Des(1-3)IGF-I is a truncated derivative of full length IGF-I that lacks the tripeptide Gly-Pro-Glu at the amino-terminus [34]. Des(1-3)IGF-I has similar actions as IGF-I but displays increased potency [35,36]. The increased potency of the truncated IGF-I does not result from increased affinity to the type I IGF-I receptors [34-37]. Rather, des(13)IGF-I has reduced affinity to IGF-binding proteins [35-37]. The third group of growth hormone deficient rats received, twice daily, intramuscular injections of recombinant human growth hormone (rhGH), 100/~g. In all three groups, treatments were given for 6 to 7 days. Animals were pair-fed a normal laboratory chow for the duration of the study and body weights were recorded daily. On day 6 or 7 animals underwent measurements of the determinants of nephron filtration by renal micropuncture using techniques that are routinely performed in this laboratory [5,16]. Briefly, rats are anesthetized with an intraperitoneal injection of Inactin (Byk-Gulden, Konstanz/Bodensee, Germany), 120 #g/kg. After tracheostomy, the left femoral artery, the left jugular vein and the bladder are cannulated with PE-50 catheters. Animals are then placed on a temperature-regulated micropuncture table. The left kidney is exposed through a mid-abdominal incision, placed in a lucite holder, surrounded with cotton that is sealed with 1 ~o agar solution and continuously bathed in saline at 37°C. The left ureter is cannulated with thinned PE-50 tubing to selectively collect urine from the left kidney. Ringer's saline containing [3H]methoxy-Inulin (NEN/Dupont, Boston, MA), 60/~Ci/ml, is infused continuously into the jugular vein at a rate of 1.8 ml/h. To replace losses of plasma that occur during this preparation [38], normal rat plasma is infused at a rate corresponding to 1 To of body weight/h for the first hour and at a rate of 0.15~o of BW/h for the reminder of the micropuncture study. Systemic blood
pressure is recorded from the femoral artery continuously with a pressure transducer and amplifying recorder. 1 h is allowed for equilibration of the inulin in the extracellular space. Thereafter, the determinants of glomerular ultrafiltration are measured: tubular pressures are measured in early proximal tubules with a 1-3 #m sharpened pipette that is filled with 1.8 M saline and connected to a servo-nulling pressure system (Model 5 A, Instrumentation for Physiology & Medicine, Inc., San Diego, CA). Early proximal tubules are identified by injecting a small amount of lightly stained Ringer's saline into tubules and are defined as proximal tubule loops that are followed by a further three or more surface loops. Tubular pressure is measured in > 5 early loops that are randomly selected from all quadrants of the accessible kidney surface. Measurements of efferent arteriolar pressure are obtained from accessible star vessels. Five or more exactly timed (3 min) collections of early proximal tubule fluid are obtained with oil-filled 9-11/~m sharpened pipettes to determine the single nephron G F R (SNGFR). Efferent arteriolar blood collections are taken from star vessels with 11-13 #m sharpened glass pipettes. The glomerular capillary hydrostatic pressure, PG, cannot be measured directly in dw/dw rats because these animals rarely display surface glomeruli that are accessible to micropuncture. Thus, P c was estimated from stop-flow pressure measurements as previously described [39]. At the beginning and end of the period of micropuncture, arterial blood (200 #1) was collected from the femoral artery for the measurement of hematocrit, serum inulin, albumin and total protein concentrations. Urine is collected from the left and right kidney, respectively, in pre-weight microcentrifuge tubes for the determination of whole kidney GFR. Afferent total protein concentrations are measured in arterial serum samples with the Lowry assay [40]. Afferent and efferent serum albumin concentrations are measured with a micro-albumin ELISA assay and the efferent total protein concentration is calcu-
244
lated from the afferent albumin/protein ratio and the efferent serum albumin levels as previously described [5]. [3H]Inulin concentrations in tubular fluid, urine and serum are measured in a liquid scintillation counter. From these primary measurements, the GFR, SNGFR, nephron filtration fraction (SNFF), single nephron plasma and blood flow rates (SNPF, SNBF), afferent and efferent arteriolar resistances ( R A , R E ) , glomerular capillary pressure (PG) and the glomerular ultrafiltration coefficient, LpA, are all calculated as previously described [5,16,39]. After termination of the micropuncture measurements an arterial blood sample is obtained to determine the serum IGF-I levels. To separate IGF-I from the binding proteins, serum samples are acidified with 1 M HCI and separated by H P L C as previously described from this laboratory [16]. The two fractions containing the IGF-I are dried in a Speed-Vac concentrator, taken up in assay buffer and the IGF-I concentrations are measured with a non-equilibrium RIA [16,41] using a specific polyclonal antibody, kindly donated by the Hormone and Pituitary Program, NIH, Baltimore, MD. The left kidney is obtained, fixed in alcoholic Bouin's solution and thin sections are stained with the PAS method. The glomerular volume is estimated from cross-sectional areas ofglomerular tufts ( > 100/ kidney), that were measured with a computerized
digitizing plate [16]. Data are expressed as m e a n + S . E . M . Statistical evaluations were performed with the analysis of variance and the Neuman-Keuls muir±comparison test.
Results
Due to the pair-feeding protocol the food intake was maintained similar in the three groups of rats, averaging 15.6+0.3 g/day in the control rats, 15.5 + 0.2 g/day in group 2 animals receiving truncated IGF-I, and 15.9 + 0.2 g/day in the rats in group 3 that were treated with growth hormone. Despite of the similar food intakes, the daily gain in body weight was different. Both the IGF-I (1.7+0.3 g/day, P < 0 . 0 5 ) and the growth hormone-treated rats (2.8 + 0.3 g/day, P < 0 . 0 5 ) gained significantly more weight each day than the control animals that were treated with vehicle (0.4 + 0.1 g/day). The mean arterial pressure (MAP) was slightly but significantly lower (P<0.05) in the rats in group 2 that were treated with IGF-I as compared to the growth hormone-treated or the control rats (Table I). The hematocrit which is mainly an estimate for the animal's fluid status during the micropuncture procedures was similar in the three groups (Table I). The serum IGF-I levels in the vehicle controls (Table I)
TABLE I Physiologic data in growth hormone-deficient vehicle treated, d e s ( 1 - 3 ) I G F - I or growth h o r m o n e treated d w / d w rats
G r o u p 1, n = 7 d w / d w , vehicle G r o u p 2, n = 7 d w / d w , des(1-3)IGF-I G o u p 3, n = 7 d w / d w , growth h o r m o n e
MAP (mmHg)
HCT ( °~o )
IGF-I (ng/ml)
GFR L (ml/min)
GFR R (ml/min)
129 _+2 115" _+2 126 +3
46 + 1 44 + 1 45 _+ 1
176 + 11 323* + 60 496* + 73
0.63 + 0.07 1.09" + 0.11 1.01" _+0.06
0.84 + 0.09 1.44" + 0.21 1.41" + 0.12
* P < 0 . 0 5 vs. vehicle control; data are m e a n + S.E.M. M A P = m e a n arterial blood pressure; GFRL,R = glomerular filtration rate of the left and right kidney, respectively.
245
were lower than usually found in normal rats in this laboratory [5,16]. Both treatment with des(13)IGF-I and growth hormone increased the steadystate serum IGF-I levels about 2-fold and almost 3-fold in the group 1 and group 2 rats, respectively, (Table I). Although values tended to be greater in the growth hormone treated animals compared to the rats receiving IGF-I (Table I), this trend did not achieve statistical significance due to the large standard errors in the two groups. The left and right whole kidney G F R was each lower in the growth hormone deficient rats that received only vehicle infusion (Table I), compared to values that are usually obtained in normal rats in this laboratory [4,5,16]. Both, treatment with des(13)IGF-I or growth hormone raise G F R greatly, by about 60~o (Table I). In all three groups the G F R of the right kidney tended to be greater than the G F R of the left kidney. This is commonly observed in micropuncture studies and may result from physiological differences and from the manipulation that is performed on the left kidney. Nephron plasma (and blood) flow rates and nephron G F R tend to be low in growth hormone deficient control rats (Table II). In contrast, in groups 2 and 3, SNPF and S N G F R were increased into the normal range by the treatments with des(1-3)IGF-I or
growth hormone, respectively, that were given for about 1 week (Table II). There were no significant differences in the nephron filtration fraction (SNFF) among all three groups (Table II). The proximal tubule hydrostatic pressure (P-r) was slightly higher in the group 3 animals which were treated with growth hormone compared to the other two groups (Table II). This difference, although significant (P<0.05), was small and the physiologic importance may be challenged. The afferent as well as the efferent arteriolar resistance were reduced in groups 2 and 3 compared to the values obtained in the vehicle control rats (Table II). This finding indicates that arteriolar dilation was achieved by treatment with des(13)IGF-I as well as with growth hormone. The glomerular capillary pressure, Po, was significantly lower in the animals that received des(13)IGF-I (P<0.05) and tended to be lower in the growth hormone treated growth hormone-deficient animals compared to the vehicle controls (P: n.s.). The significant decrease in Po in the des(1-3)IGFI-treated rats resulted from a decrease in the RA/R E ratio in these latter rats compared to the group 3 animals that received growth hormone. Furthermore, the group 2 rats also had a lower systemic blood pressure than the other two groups (Table I) and the afferent arteriolar resistance was somewhat greater,
TABLE II Determinants of glomular ultrafiltration in growth hormone-deficient controls receiving vehicle infusion and in rats receiving des(1-3)IGF-I or growth hormone for 1 week SNGFR (nl/min) Group 1, n = 7 dw/dw, vehicle Group 2, n = 7 dw/dw, des(1-3)IGF-I Group 3, n = 7 dw/dw, growth hormone
SNPF (nl/min)
SNFF
PA RE (10 9 dyn s c m - 5)
P'r (mmHg)
PG (mmHg)
LpA (nl/s/mmHg)
21 + 1
85 _+6 149+11"
39+4*
180+ 10"
45.3 + 4.4 23.1" + 2.5 20.6* _+ 1.0
10.6 + 0.3 11.4 + 0.4 11.9" _+0.7
47 + 1
36_+3*
0.26 + 0.02 0.24 + 0.01 0.22 + 0.02
0.046 + 0.004 0.127" + 0.036 0.125" + 0.019
20.6 + 2.1 9.7* _+0.8 8.2* + 0.7
40+2* 44+_2
* P < 0 . 0 5 vs. vehicle control; data are mean + S.E.M. SNGFR, S N F F = single nephron GFR, single nephron plasma flow, single nephron filtration fraction; RAm = afferent and efferent arteriolar resistance, PT.G = tubular and glomerular hydrostatic pressure; LpA = glomerular ultrafiltration coefficient.
246 TABLE III Indicators of renal and glomerular hypertrophyin control and treated growth hormone deficient rats
Group 1, n = 7 dw.dw, vehicle Group 2, n = 7 dw/dw, des(1-3)GF-I Group 3, n = 7 dw/dw, growth hormone
Glomeruli/kidney (n)
LKW (g)
LKW/BW (g/100 g)
Glomerular tuft volume (104 #m3)
29,644 _+2100
0.76 _+0.01 0.91" + 0.03 0.90* + 0.02
0.372 _+0.006 0.467** + 0.14 0.379 + 0.019
28.6 + 1.5 34.6* + 2.0 33.7* + 1.8
29,894 + 1761 27,453 _+1518
* P<0.05 vs. vehicle control; * P<0.05 vs. group 3; data are mean + S.E.M. LKW = left kidney wet weight; BW = body weight.
although not significantly, compared to the group 3 rats. The reduced systemic blood pressure and the slightly higher afferent arteriolar resistance allowed for a lesser transmission of blood pressure into the glomerular capillary in the group 2 rats that were treated with the truncated IGF-I. The glomerular ultrafiltration coefficient, LpA, was increased about 3-fold in groups 2 and 3 ( P < 0.05 for each comparison) compared to the vehicle-treated controls (Table II). There was no difference in LpA between the des(1-3)IGF-I treated rats and the animals that received growth hormone. The number of glomeruli that contribute to the whole kidney G F R was estimated from the ratio G F R / S N G F R . Results indicate that in all three groups a similar number of functional glomeruli was present (Table III). The propensity of I G F - I and growth hormone to induce renal and nephron growth was estimated from the kidney wet weight, the ratio of the left kidney wet weight divided by the body weight (LKW/BW) and the histomorphometric estimation of the glomerular tuft volume (Table III). In both groups of rats that received treatment with either growth hormone or I G F - I the kidney wet weight and the glomerular tuft volume were significantly greater than in the growth hormone-deficient controls (P<0.05 for each comparison). However, the specific kidney weight, i.e., LKW/BW, was greatest
in the rats receiving des(1-3)IGF-I as compared to the control as well as the growth hormone treated animals (Table III).
Discussion In the present study moderate dosages of both des(1-3)IGF-I and recombinant human growth hormone increase glomerular perfusion and ultrafiltration rates as well as increase renal and nephron size in growth hormone-deficient rats. In fact, the growth hormone deficiency state presents with elevated afferent and efferent arteriolar resistance, a normal PG and a reduced LpA. The increased arteriolar resistance allows only for a lesser-than-normal nephron blood flow. Since the glomerular capillary pressure, Po, as well as the ultrafiltration coefficient, LpA, are low, a reduced glomerular ultrafiltration rate results. Indeed, growth hormone deficiency in man presents with reduced G F R and R P F [3,26,27,29,42]. This laboratory has previously studied the baseline renal function in growth hormone-deficient subjects and demonstrated a lower-than-normal G F R and RPF and elevated renal vascular resistance [3,42]. The present studies confirm these earlier findings and, furthermore, delineate the pathophysiologic mechanisms that cause these changes in renal function in
247 growth hormone deficiency; the reduced G F R and RPF in hyposomatotropic states are caused by arteriolar vasoconstriction in the nephron and a reduced LpA. Both of these abnormalities are corrected with the administration of either the truncated IGF-I or growth hormone, both of which render the RPF and G F R normal. Similarly, in growth hormone-deficient humans, RPF and G F R are raised towards normal with a single injection of growth hormone [3]. Furthermore, in growth hormonedeficient subjects, other maneuvers such as an infusion of amino acids acutely raise RPF and GFR, indicating that the glomerular perfusion and filtration are not only reduced due to a lower kidney size, rather than determined functionally [42]. Several lines of evidence suggest that the growth hormone-induced rise in G F R is mediated through IGF-I. First, in normal human subjects, growth hormone raises G F R and RPF and lowers renal vascular resistance; all of these changes are induced in the same direction by administration of IGF-I [ 2,8,10,12,14]. Second, the growth hormone-induced rise in renal function is delayed and occurs only several hours after a single injection of rhGH, at a time, when IGF-I serum levels (and presumably renal tissue IGF-I concentrations) have also risen [2]. Third, as shown in the present study, treatment of growth hormone-deficient rats with either truncated IGF-I or growth hormone, each reduces renal arteriolar resistance and elevates nephron filtration and perfusion. Both treatments affect the same determinants of glomerular ultrafiltration, namely arteriolar resistance and LpA, but not P6. Fourth, the normalization of glomerular function in growth hormone deficient rats requires only the correction of the IGF-I deficiency without concomitant administration of growth hormone, as shown in group 2 in the present study. Although not measured, it is reasonable to assume that the growth hormone secretion in these latter rats has remained low during the week of des(1-3)IGF-I treatment. The glomerular ultrafiltration coefficient, LpA, is the product of the pressure-dependent permeability
of the glomerular ultrafiltration barrier, Lp, and the surface area A that is available for ultrafiltration in the glomerulus. With presently available methods it is experimentally impossible to measure each of these terms independently in vivo, and it remains unknown, whether Lp, A or both are affected by IGF-I. Theoretically, both may be increased by IGF-I. However, it is the most likely scenario that IGF-I increases the surface area which could result from mesangial relaxation. Indeed, mesangial cells display specific IGF-I receptors and several effects of IGF-I have been demonstrated in cultured rat mesangial cells [43,44]. Angiotensin II, for instance, which has been shown to reduce LpA in vivo [45,46] also contracts cultured mesangial cells in vitro [47], but direct effects of IGF-I to relax mesangial cells are yet to be demonstrated experimentally. IGF-I increases glomerular function, in part, by lowering arteriolar resistance as demonstrated in the present as well as in previous studies [5,16]. The vasodilatory effects of IGF-I are observed within several minutes of an intravenous infusion of IGF-I in rats [4]. This effect of IGF-I may be mediated, in part, by vasodilatory prostaglandins, such as prostacyclin [4,14,48]. Administration of indomethacin, a potent cyclooxygenase inhibitor, prevents [4] or reduces [48] the IGF-I-induced acute increase in RPF and G F R in the rat. However, vasodilating cyclooxygenase products may have only a permissive effect, rather than being a true mediator. Recent evidence suggests an important role for E D R F / N O to mediate the IGF-I-induced reduction in arteriolar resistance and increase in renal blood flow [48]. Haylor and coworkers infused acutely IGF-I into normal rats and measured the renal blood flow with an electromagnetic flow probe and calculated the renal vascular resistance [48]. Infusion of IGF-I concomitant with administration of the nitric oxide synthase inhibitor L-NAME completely prevented the rise in RBF [48]. In the present study both IGF-I and growth hormone induce renal and glomerular growth. Previous data suggest, that IGF-I increases kidney weight and
248 nephron size primarily by hypertrophy, rather than hyperplasia [49]. Indeed, G u l e r and associates demo n s t r a t e d that I G F - I induces renal growth in excess o f b o d y growth and growth o f other organs [50]. Furthermore, (renal) I G F - I has been associated with the induction of renal c o m p e n s a t o r y growth in a number o f experimental disease states [ 5 1 - 5 5 ] . The glomerular h y p e r t r o p h y m a y have contributed to the I G F - I - or growth h o r m o n e - i n d u c e d rise in glomerular function that was d e m o n s t r a t e d in the growth h o r m o n e deficient rats in the present experiments. However, it is unlikely that the increase in glomerular size is solely responsible for the normalization o f n e p h r o n function in these studies, since we have previously d e m o n s t r a t e d that the functional effects occur quickly, within minutes of the onset of systemic I G F - I infusions in n o r m a l rats [4]. Possibly, the increase in n e p h r o n volume contributed to a rise in the ultrafiltration surface area and, thus, m a y have helped to increase L p A . In summary, growth h o r m o n e administration corrects the reduced glomerular function and size in growth h o r m o n e deficient rats and raises I G F - I synthesis and serum levels. The functional and growth effects of growth h o r m o n e on the nephron can also be induced by administration of m o d e s t dosages o f d e s ( 1 - 3 ) I G F - I , suggesting that the lower glomerular function and size in growth h o r m o n e deficiency results primarily from a lack o f I G F - I . Moreover, the findings in the present study also suggest that growth h o r m o n e a n d / o r I G F - I contribute to the regulation o f glomerular function.
Acknowledgements These studies were m a d e possibly by a grant from the A m e r i c a n H e a r t Association, Dallas, T X (No. 901218). The author would like to express his gratitude to the H o r m o n e and Pituitary Program, N I H , Baltimore, M D , for providing specific a n t i - I G F - I antibody for R I A ; and Ross Clark, Ph.D., for allowing to use the growth h o r m o n e deficient rats, as well as
G e n e n t e c h Inc., South San F r a n c i s c o , CA, for the d o n a t i o n o f r h G H and d e s ( 1 - 3 ) I G F - I . The author also appreciates the provision of the Digital Vax c o m p u t e r system and the Clinfo software m a d e available to these studies by the Clinical R e s e a r c h Center at H a r b o r - U C L A M e d i c a l Center through N I H grant G C R C R R 00425. Parts o f this m a n u s c r i p t were previously presented in abstract form (J. Am. Soc. Nephrol., 2 (1991) 681).
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249 11 Ritz, E., TOnshoff, B., Worgall, S., Kovacs, G. and Mehls, O., Influecne of growth hormone and insulin-like growth factor I on kidney function and kidney growth, Pediatr. Nephrol., 5 (1991) 509-512. 12 Haffner, D., Ritz, E., Mehls, O., Rosman, J., Blum, W., Heinrich, U. and H~lbinger, A., Growth hormone-induced rise in glomerular filtration rate is not obliterated by angiotensinconverting enzyme inhibitors, Nephron, 55 (1990) 63-68. 13 Haffner, D., Zacharewicz, S., Mehls, O., Heinrich, U. and Ritz, E., The acute effect of growth hormone on GFR is obliterated in chronic renal failure, Clin. Nephrol., 32 (1989) 266269. 14 T0nshoff, B., Nowack, R., Kurilenko, S., Blum, W., Seyberth, H., Mehls, O. and Ritz, E., Growth hormone-induced glomerular hyperfiltration is dependent on vasodilating prostanoids, Am. J. Kidney Dis., 21 (1993) 145-151. 15 Baumann, U., Eisenhauer, T. and Hartmann, H., Increase of glomerular filtration rate and renal plasma flow by insulin-like growth factor I during euglycaemic clamping in anaesthetized rats, Eur. J. Clin. Invest., 22 (1992) 204-209. 16 Hirschberg, R. and Kopple, J., The growth hormone-insulinlike growth factor I axis and renal glomerular function, J. Am. Soc. Nephrol., 2 (1992) 1417-1422. 17 Ezzat, S., Snyder, P., Young, W., Boyajy, L., Newman, C., Klibanski, A., Molitch, M., Boyd, A., Sheeler, C. and Cook, D., Octreotide treatment of acromegaly. A randomized, multicenter study, Ann. Intern. Med., 112 (1992), 711-718. 18 Vance, M. and Harris, A., Long-term treatment of 189 acromegalic patients with somatostatin analog octreotide. Resuits of the International Multicenter Acromegaly Study Group, Arch. Intern. Med., 151 (1991) 1573-1578. 19 Holly, J., Cotterill, A., Jennett, R., Shears, D., al-Othman, S., Chard, T. and Wass, J., Inter-relations between growth hormone, insulin, insulin-like growth factor I (IGF-I), IGFbinding protein- 1 (IGFBP- 1) and sex hormone binding globulin in acromegaly, Clin. Endocrinol., 34 (1991) 275-280. 20 Mehltretter, G., Heinz, S., Schopohl, J., Von Werder, K. and M~iller, O., Long-term treatment with SMS 201-995 in resistant acromegaly: Effectiveness of high doses and continuous subcutaneous infusion, Klin. Wochenschr., 69 (1991) 83-90. 21 Salmeda, P., Juustila, H., Pyhtinen, J., Jokinen, K., Alavaikko, M. and Ruokonen, A., Effective clinical response to long term octreotide treatment with reduced serum concentrations of growth hormone, insulin-like growth factor-I and the aminoterminal peptide of type III procollagen in acromegaly, J. Clin. Endocrinol. Metab., 70 (1990), 1193-1201. 22 Miller, S., Rotwein, P., Bortz, J., Bechtel, P., Hansen, V., Rogers, S. and Hammerman, M., Renal expression of IGF-I in hypersomatotropic states, Am. J. Physiol., 259 (1990) F251-F257. 23 O'Shea, M. and Layish, D., Growth hormone and the kidney:
24
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A case presentation and review of the literature, J. Am. Soc. Nephrol., 3 (1992) 157-161. Dullaart, R., Meijer, S., Marbach, P. and Sluiter, W., Effect of a somatostatin analogue, octreotide, on renal hemodynamics and albuminuria in acromegalic patients, Eur. J. Clin. Invest., 22 (1992) 494-502. Ikkos, D., Ljunggren, H. and Luft, R., Glomerular filtration rate and renal plasma flow in acromegaly, Acta Endocrinol. (Copenh.), 21 (1956), 226-236. Falkheden, T., Renal function following hypophysectomy in man, Acta Endocrinol. (Copenh.), 42 (1963) 571-590. Falkheden, T. and Wickbom, I., Renal function and kidney size following hypophysectomy in man, Acta Endocrinol. (Copenh.), 48 (1965) 348-354. Gershberg, H., Heinemann, H. and Stumpf, H., Renal function studies and autopsy report in a patient with gigantism and acromegaly, J. Clin. Endocrinol. Metab., 17 (1957) 377-385. Falkheden, T. and SjOgren, B., Extracellular fluid volume and renal function in pituitary insufficiency and acromegaly, Acta Endocrinol. (Copenh.), 46 (1964) 80-88. Hirschberg, R. and Kopple, J., Response of insulin-like growth factor I and renal hemodynamics to a high- and low-protein diet in the rat, J. Am. Soc. Nephrol., 1 (1991) 1034-1040. El Nahas, A., Le Carpentier, J., Bassett, A. and Hill, D., Dietary protein and insulin-like growth factor-I content following unilateral nephrectomy, Kidney Int., 36, Suppl. 27 (1989), S15-S19. Lowe, W., Adamo, M., Werner, H., Roberts, C. and LeRoith, D., Regulation by fasting of rat insulin-like growth factor I and its receptor: Effects on gene expression and binding, J. Clin. Invest., 84 (1989) 619-626. Charlton, H., Clark, R., Robinson, I., Porter-Goff, A., Cox, B., Bugnon, C. and Bloch, B., Growth hormone-deficient dwarfism in the rat: A new mutation, J. Endocrinol., 119 (1988) 51-58. Bagley C., May, B., Szabo, L., McNamara, P., Ross, M., Francis, G., Ballard, F. and Wallace, J., A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide, Biochem. J., 259 (1989) 665-671. Clark, R. and Mortensen, D., Recombinant human DESIGF-I is more potent than IGF-I in promoting growth and inducing hypoglycemia in the rat, Abstract, 72nd Annual Meeting, The Endocrine Society, Atlanta, GA, June, 1990. Ballard, F., Francis, G., Bagley, C., Szabo, L. and Wallace, J., Effects of insulin-like growth factors on protein metabolism: Why are some molecular variants more potent?, Biochem. Soc. Symp., 55 (1989) 91-104. Ross, M., Francis, G., Szabo, L., Wallace, J. and Ballard, F., Insulin-like growth factor (IGF)-binding proteins inhibit the biological activities of IGF-I and IGF-2 but not des-(1-3)IGF-1, Biochem. J., 258 (1989) 267-272.
250 38 Maddox, D., Price, D. and Rector, F., Effects of surgery on plasma volume and salt and water excretion in rats, Am. J. Physiol., 233 (1977) F600-F606. 39 Blantz, R. and Tucker, B., Measurements of glomerular dynamics. In M. Martinez-Maldonado (Ed.), Methods in Pharmacology, Vol. 4B, Plenum Publishing Corporation, Boston, 1978, pp. 141-163. 40 Lowry, O., Rosebrough, N., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 41 Furlanetto, R., Underwood, L., Van Wyk, J. and D'Ercole, A., Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay, J. Clin. Invest., 60 (1977) 648-657. 42 Hirschberg, R. and Kopple, J., Role of growth hormone in the amino-acid induced acute rise in renal function in man, Kidney Int., 32 (1987) 382-387. 43 Doi, T., Striker, L., Elliot, S., Conti, F. and Striker, G., Insulinlike growth factor-I is a progression factor for human mesangial cells, Am. J. Pathol., 134 (1989) 395-404. 44 Conti, F., Striker, L., Lesniak, M., MacKay, K., Roth, J. and Striker, G., Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells, Endocrinology, 122 (1988) 2788-2795. 45 Thomson, S., Tucker, B., Gabbai, F. and Blantz, R., Functional effects of glomerular hyemodynamics of short-term cyclosporine in male rats, J. Clin. Invest., 83 (1989) 960-969. 46 Schor, N., Ichikawa, I. and Brenner, B., Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat, Kidney Int., 20 (1981) 442-251. 47 Ausiello, D., Kriesberg, J., Roy, C. and Karnovsky, M., Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin, J. Clin. Invest., 65 (1980) 754-760.
48 Haylor, J., Singh, I. and E1 Nahas, A., Nitric oxide synthesis inhibitor prevents vasodilation by insulin-like growth factor I, Kidney Int., 39 (1991) 333-335. 49 Hirschberg, R. and Nast, C., Glomerular hypertrophy and segmental glomerular sclerosis are not linked in subtotally nephrectomized rats, Abstract, J. Am. Soc. Nephrol., 3 (1992), 739. 50 Guler H., Zapf, J., Scheiwiller, E. and Froesch, E., Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats, Proc. Natl. Acad. Sci. USA, 85 (1988) 4889-4893. 51 Marshall, S., Flyvbjerg, A., Frystyk, J., Korsgaard, L. and Orskov, H., Renal insulin-like growth factor I and growth hormone receptor binding in experimental diabetes and after unilateral nephrectomy in the rat, Diabetologia, 34 (1991) 632639. 52 Fagin, A. and Melmed, S., Relative increase in insulin-like growth factor I messanger ribonucleic acid levels in compensatory renal hypertrophy, Endocrinology, 120 (1987) 718-724. 53 Flyvbjerg, A., Orskov, H., Nyborg, K., Frystyk, J., Marshall, S., Bornfeldt, K., Arnqvist, H. and Jorgensen, K., Kidney IGF-I accumulation occurs in four different conditions with rapid initial kidney growth in rats, In E. Spencer (Edit), Modern Concepts of insulin-like gorwth factors, Elsevier, New York, 1991, pp. 207-217. 54 Mulroney, S., Haramati, A., Werner, H., Boncy, C., Roberts, C. and LeRoith, D., Altered expression of insulin-like growth factor-I (IGF-I) and IGF receptor genes after unilateral nephrectomy in immature rats, Endocrinology, 130 (1992) 249356. 55 Mulroney, S., Haramati, A., Roberts, C. and LeRoith, D., Renal IGF-I mRNA levels are inhanced following unilateral nephrectomy in immature but not adult rats, Endocrinology, 128 (1991) 2660-2662.
241
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01558
Effects of growth hormone and IGF-I on glomerular ultrafiltration in growth hormone-deficient rats Raimund Hirschberg Division of Nephrology and Hypertension, Harbor-UCLA Medical Center and UCLA, Los Angeles, CA (USA)
(Received 22 March 1993; revised version received20 April 1993; accepted 6 May 1993) Key words: Des(1-3)IGF-I; Glomerular hypertrophy; GFR; Single nephron glomerular filtration rate;
Glomerular ultrafiltration coefficient
Summary In growth hormone deficient states glomerular filtration rate (GFR) and renal plasma flow rate (RPF) are both reduced. Studies were performed in growth hormone deficient rats to delineate the physiologic mechanisms by which growth hormone and IGF-I contribute to the regulation of glomerular function. Growth hormone deficient dw/dw rats received, for one week, subcutaneous infusions of vehicle, des(1-3)IGF-I or were injected i.m. with recombinant human growth hormone. Subsequent renal micropuncture and clearance studies revealed a low G F R and single nephron G F R ( S N G F R ) in vehicle treated growth hormone deficient animals. Glomerular function became normal with growth hormone or IGF-I treatment, respectively. Both treatments raised S N G F R by reducing arteriolar resistance and increasing the glomerular ultrafiltration coefficient. Furthermore, the two treatments also increased the glomerular tuft volume and the kidney weight which may contribute to the rise in S N G F R and GFR. It is concluded that, (1) in growth hormone deficiency glomerular function is reduced secondary to a high renal arteriolar resistance and a low ultrafiltration coefficient. Both result from a lack in IGF-I rather than the growth hormone deficiency state per se. (2) The growth h o r m o n e - I G F - I axis may contribute to the maintenance and physiologic regulation of GFR.
Introduction Correspondence to: R. Hirschberg, Division of Nephrology and Hypertension,Harbor-UCLAMedicalCenter, 1000West Carson ST, Torrance, CA 90509, USA.
Injections of growth hormone or IGF-I raise renal plasma flow (RPF) and glomerular filtration rate (GFR) in normal rats and humans [1-15]. A single
242 intramuscular injection of growth hormone, 0.15 mg/ kg, in normal subjects induces a significant increase in RPF and GFR with a delay of several hours [2,3]. The rise in kidney function does not occur before the growth hormone-induced rise in circulating IGF-I can be demonstrated [2]. This latter finding suggests that circulating and/or renal IGF-I mediates the growth hormone-induced rise in glomerular perfusion and ultrafiltration. Indeed, subsequent studies in rats demonstrated that a short-term injection and infusion of recombinant human IGF-I (rhIGF-I) into rats acutely raises renal function [4]. In the rat rhIGF-I acutely reduces afferent and efferent arteriolar resistance in the nephron, which drives an increase in nephron blood and plasma flow [5]. As demonstrated in renal micropuncture studies this rise in nephron plasma flow contributes to the increase in single nephron glomerular filtration rate (SNGFR) during the infusion of rhIGF-I, which, in addition, is caused by an increase in the glomerular ultrafiltration coefficient, LpA [5,16]. There is preliminary evidence that the endogenous growth hormone-IGF-I axis contributes to the maintenance of adequate glomerular ultrafiltration in physiologic and pathophysiologic conditions. First, in acromegalics, both growth hormone and IGF-I serum levels are elevated [ 17-21]. Hypersomatotropic states also increase the synthesis of IGF-I in the kidney [22]. In acromegalic patients elevated serum growth hormone and IGF-I levels are associated with renal hypertrophy and glomerular hyperfiltration [7,23-29]. The elevated GFR may result from the increase in nephron and more specifically in glomerular size, but may also be caused by direct effects of growth hormone and/or IGF-I on the resistance and filtration-regulating microvasculature in the nephron. Second, certain experimental maneuvers that cause an increase in GFR are also associated with elevated IGF-I synthesis, such as feeding of high protein diets in normal rats and in animals with experimental chronic renal failure [30-32]. Third, treatment of normal rats with a synthetic growth hormone-releasing hormone antagonist which causes
a reduction in pulsatile growth hormone release and, hence, reduction in serum (and presumably renal) IGF-I levels, also decreases the glomerular filtration rate and reduces kidney wet weight and glomerular tuft volume [ 16]. GFR is reduced in growth hormone-deficiency states [3,26,27,29]. This could result from a lack of the physiologic effects of growth hormone and/or IGF-I, or from glomerular hypotrophy. If this hypothesis should materialize, it would give rise to the possibility that the growth hormone-IGF-I axis participates in the regulation and maintenance of normal glomerular function. The present study was designed to elucidate the differential effects of growth hormone and IGF-I on renal glomerular function and size using the model of the growth hormone-deficient rat.
Materials and Methods
Studies were performed in male growth hormonedeficient dw/dw rats weighing 199 + 10 (S.E.) g at the onset of the studies. Animals were purchased from Simonson, Gilroy, CA and were kindly provided by Ross Clark, Ph.D., of Genentech, South San Francisco, CA. The growth hormone deficiency trait arose spontaneously in a colony of Lewis rats and the mutation was introduced into the NIMR/AS strain and successfully maintained by crossing heterozygous animals with homozygous growth hormone deficient rats of the opposite sex [33]. Growth hormone deficient rats were randomly assigned to one of the following three groups (n = 7 each): group 1 (control) rats were anesthetized with intramuscular injections of Ketamine, 87 mg/kg, and Xylazine, 13 mg/kg, and were implanted subcutaneously with osmotic minipumps (Model 2001, Alza Corporation, Palo Alto, CA) that were filled with 50 mM acetic acid. The minipumps were primed over night in sterile 5~o glucose in water at 4°C prior to implantation. Animals in group 2 were also implanted with primed minipumps that were filled with des(13)IGF-I (Genentech Inc., South San Francisco, CA)
243 in 50 mM acetic acid. The concentration was adjusted to deliver des(1-3)IGF-I at a constant rate of 50/~g/day. Des(1-3)IGF-I is a truncated derivative of full length IGF-I that lacks the tripeptide Gly-Pro-Glu at the amino-terminus [34]. Des(1-3)IGF-I has similar actions as IGF-I but displays increased potency [35,36]. The increased potency of the truncated IGF-I does not result from increased affinity to the type I IGF-I receptors [34-37]. Rather, des(13)IGF-I has reduced affinity to IGF-binding proteins [35-37]. The third group of growth hormone deficient rats received, twice daily, intramuscular injections of recombinant human growth hormone (rhGH), 100/~g. In all three groups, treatments were given for 6 to 7 days. Animals were pair-fed a normal laboratory chow for the duration of the study and body weights were recorded daily. On day 6 or 7 animals underwent measurements of the determinants of nephron filtration by renal micropuncture using techniques that are routinely performed in this laboratory [5,16]. Briefly, rats are anesthetized with an intraperitoneal injection of Inactin (Byk-Gulden, Konstanz/Bodensee, Germany), 120 #g/kg. After tracheostomy, the left femoral artery, the left jugular vein and the bladder are cannulated with PE-50 catheters. Animals are then placed on a temperature-regulated micropuncture table. The left kidney is exposed through a mid-abdominal incision, placed in a lucite holder, surrounded with cotton that is sealed with 1 ~o agar solution and continuously bathed in saline at 37°C. The left ureter is cannulated with thinned PE-50 tubing to selectively collect urine from the left kidney. Ringer's saline containing [3H]methoxy-Inulin (NEN/Dupont, Boston, MA), 60/~Ci/ml, is infused continuously into the jugular vein at a rate of 1.8 ml/h. To replace losses of plasma that occur during this preparation [38], normal rat plasma is infused at a rate corresponding to 1 To of body weight/h for the first hour and at a rate of 0.15~o of BW/h for the reminder of the micropuncture study. Systemic blood
pressure is recorded from the femoral artery continuously with a pressure transducer and amplifying recorder. 1 h is allowed for equilibration of the inulin in the extracellular space. Thereafter, the determinants of glomerular ultrafiltration are measured: tubular pressures are measured in early proximal tubules with a 1-3 #m sharpened pipette that is filled with 1.8 M saline and connected to a servo-nulling pressure system (Model 5 A, Instrumentation for Physiology & Medicine, Inc., San Diego, CA). Early proximal tubules are identified by injecting a small amount of lightly stained Ringer's saline into tubules and are defined as proximal tubule loops that are followed by a further three or more surface loops. Tubular pressure is measured in > 5 early loops that are randomly selected from all quadrants of the accessible kidney surface. Measurements of efferent arteriolar pressure are obtained from accessible star vessels. Five or more exactly timed (3 min) collections of early proximal tubule fluid are obtained with oil-filled 9-11/~m sharpened pipettes to determine the single nephron G F R (SNGFR). Efferent arteriolar blood collections are taken from star vessels with 11-13 #m sharpened glass pipettes. The glomerular capillary hydrostatic pressure, PG, cannot be measured directly in dw/dw rats because these animals rarely display surface glomeruli that are accessible to micropuncture. Thus, P c was estimated from stop-flow pressure measurements as previously described [39]. At the beginning and end of the period of micropuncture, arterial blood (200 #1) was collected from the femoral artery for the measurement of hematocrit, serum inulin, albumin and total protein concentrations. Urine is collected from the left and right kidney, respectively, in pre-weight microcentrifuge tubes for the determination of whole kidney GFR. Afferent total protein concentrations are measured in arterial serum samples with the Lowry assay [40]. Afferent and efferent serum albumin concentrations are measured with a micro-albumin ELISA assay and the efferent total protein concentration is calcu-
244
lated from the afferent albumin/protein ratio and the efferent serum albumin levels as previously described [5]. [3H]Inulin concentrations in tubular fluid, urine and serum are measured in a liquid scintillation counter. From these primary measurements, the GFR, SNGFR, nephron filtration fraction (SNFF), single nephron plasma and blood flow rates (SNPF, SNBF), afferent and efferent arteriolar resistances ( R A , R E ) , glomerular capillary pressure (PG) and the glomerular ultrafiltration coefficient, LpA, are all calculated as previously described [5,16,39]. After termination of the micropuncture measurements an arterial blood sample is obtained to determine the serum IGF-I levels. To separate IGF-I from the binding proteins, serum samples are acidified with 1 M HCI and separated by H P L C as previously described from this laboratory [16]. The two fractions containing the IGF-I are dried in a Speed-Vac concentrator, taken up in assay buffer and the IGF-I concentrations are measured with a non-equilibrium RIA [16,41] using a specific polyclonal antibody, kindly donated by the Hormone and Pituitary Program, NIH, Baltimore, MD. The left kidney is obtained, fixed in alcoholic Bouin's solution and thin sections are stained with the PAS method. The glomerular volume is estimated from cross-sectional areas ofglomerular tufts ( > 100/ kidney), that were measured with a computerized
digitizing plate [16]. Data are expressed as m e a n + S . E . M . Statistical evaluations were performed with the analysis of variance and the Neuman-Keuls muir±comparison test.
Results
Due to the pair-feeding protocol the food intake was maintained similar in the three groups of rats, averaging 15.6+0.3 g/day in the control rats, 15.5 + 0.2 g/day in group 2 animals receiving truncated IGF-I, and 15.9 + 0.2 g/day in the rats in group 3 that were treated with growth hormone. Despite of the similar food intakes, the daily gain in body weight was different. Both the IGF-I (1.7+0.3 g/day, P < 0 . 0 5 ) and the growth hormone-treated rats (2.8 + 0.3 g/day, P < 0 . 0 5 ) gained significantly more weight each day than the control animals that were treated with vehicle (0.4 + 0.1 g/day). The mean arterial pressure (MAP) was slightly but significantly lower (P<0.05) in the rats in group 2 that were treated with IGF-I as compared to the growth hormone-treated or the control rats (Table I). The hematocrit which is mainly an estimate for the animal's fluid status during the micropuncture procedures was similar in the three groups (Table I). The serum IGF-I levels in the vehicle controls (Table I)
TABLE I Physiologic data in growth hormone-deficient vehicle treated, d e s ( 1 - 3 ) I G F - I or growth h o r m o n e treated d w / d w rats
G r o u p 1, n = 7 d w / d w , vehicle G r o u p 2, n = 7 d w / d w , des(1-3)IGF-I G o u p 3, n = 7 d w / d w , growth h o r m o n e
MAP (mmHg)
HCT ( °~o )
IGF-I (ng/ml)
GFR L (ml/min)
GFR R (ml/min)
129 _+2 115" _+2 126 +3
46 + 1 44 + 1 45 _+ 1
176 + 11 323* + 60 496* + 73
0.63 + 0.07 1.09" + 0.11 1.01" _+0.06
0.84 + 0.09 1.44" + 0.21 1.41" + 0.12
* P < 0 . 0 5 vs. vehicle control; data are m e a n + S.E.M. M A P = m e a n arterial blood pressure; GFRL,R = glomerular filtration rate of the left and right kidney, respectively.
245
were lower than usually found in normal rats in this laboratory [5,16]. Both treatment with des(13)IGF-I and growth hormone increased the steadystate serum IGF-I levels about 2-fold and almost 3-fold in the group 1 and group 2 rats, respectively, (Table I). Although values tended to be greater in the growth hormone treated animals compared to the rats receiving IGF-I (Table I), this trend did not achieve statistical significance due to the large standard errors in the two groups. The left and right whole kidney G F R was each lower in the growth hormone deficient rats that received only vehicle infusion (Table I), compared to values that are usually obtained in normal rats in this laboratory [4,5,16]. Both, treatment with des(13)IGF-I or growth hormone raise G F R greatly, by about 60~o (Table I). In all three groups the G F R of the right kidney tended to be greater than the G F R of the left kidney. This is commonly observed in micropuncture studies and may result from physiological differences and from the manipulation that is performed on the left kidney. Nephron plasma (and blood) flow rates and nephron G F R tend to be low in growth hormone deficient control rats (Table II). In contrast, in groups 2 and 3, SNPF and S N G F R were increased into the normal range by the treatments with des(1-3)IGF-I or
growth hormone, respectively, that were given for about 1 week (Table II). There were no significant differences in the nephron filtration fraction (SNFF) among all three groups (Table II). The proximal tubule hydrostatic pressure (P-r) was slightly higher in the group 3 animals which were treated with growth hormone compared to the other two groups (Table II). This difference, although significant (P<0.05), was small and the physiologic importance may be challenged. The afferent as well as the efferent arteriolar resistance were reduced in groups 2 and 3 compared to the values obtained in the vehicle control rats (Table II). This finding indicates that arteriolar dilation was achieved by treatment with des(13)IGF-I as well as with growth hormone. The glomerular capillary pressure, Po, was significantly lower in the animals that received des(13)IGF-I (P<0.05) and tended to be lower in the growth hormone treated growth hormone-deficient animals compared to the vehicle controls (P: n.s.). The significant decrease in Po in the des(1-3)IGFI-treated rats resulted from a decrease in the RA/R E ratio in these latter rats compared to the group 3 animals that received growth hormone. Furthermore, the group 2 rats also had a lower systemic blood pressure than the other two groups (Table I) and the afferent arteriolar resistance was somewhat greater,
TABLE II Determinants of glomular ultrafiltration in growth hormone-deficient controls receiving vehicle infusion and in rats receiving des(1-3)IGF-I or growth hormone for 1 week SNGFR (nl/min) Group 1, n = 7 dw/dw, vehicle Group 2, n = 7 dw/dw, des(1-3)IGF-I Group 3, n = 7 dw/dw, growth hormone
SNPF (nl/min)
SNFF
PA RE (10 9 dyn s c m - 5)
P'r (mmHg)
PG (mmHg)
LpA (nl/s/mmHg)
21 + 1
85 _+6 149+11"
39+4*
180+ 10"
45.3 + 4.4 23.1" + 2.5 20.6* _+ 1.0
10.6 + 0.3 11.4 + 0.4 11.9" _+0.7
47 + 1
36_+3*
0.26 + 0.02 0.24 + 0.01 0.22 + 0.02
0.046 + 0.004 0.127" + 0.036 0.125" + 0.019
20.6 + 2.1 9.7* _+0.8 8.2* + 0.7
40+2* 44+_2
* P < 0 . 0 5 vs. vehicle control; data are mean + S.E.M. SNGFR, S N F F = single nephron GFR, single nephron plasma flow, single nephron filtration fraction; RAm = afferent and efferent arteriolar resistance, PT.G = tubular and glomerular hydrostatic pressure; LpA = glomerular ultrafiltration coefficient.
246 TABLE III Indicators of renal and glomerular hypertrophyin control and treated growth hormone deficient rats
Group 1, n = 7 dw.dw, vehicle Group 2, n = 7 dw/dw, des(1-3)GF-I Group 3, n = 7 dw/dw, growth hormone
Glomeruli/kidney (n)
LKW (g)
LKW/BW (g/100 g)
Glomerular tuft volume (104 #m3)
29,644 _+2100
0.76 _+0.01 0.91" + 0.03 0.90* + 0.02
0.372 _+0.006 0.467** + 0.14 0.379 + 0.019
28.6 + 1.5 34.6* + 2.0 33.7* + 1.8
29,894 + 1761 27,453 _+1518
* P<0.05 vs. vehicle control; * P<0.05 vs. group 3; data are mean + S.E.M. LKW = left kidney wet weight; BW = body weight.
although not significantly, compared to the group 3 rats. The reduced systemic blood pressure and the slightly higher afferent arteriolar resistance allowed for a lesser transmission of blood pressure into the glomerular capillary in the group 2 rats that were treated with the truncated IGF-I. The glomerular ultrafiltration coefficient, LpA, was increased about 3-fold in groups 2 and 3 ( P < 0.05 for each comparison) compared to the vehicle-treated controls (Table II). There was no difference in LpA between the des(1-3)IGF-I treated rats and the animals that received growth hormone. The number of glomeruli that contribute to the whole kidney G F R was estimated from the ratio G F R / S N G F R . Results indicate that in all three groups a similar number of functional glomeruli was present (Table III). The propensity of I G F - I and growth hormone to induce renal and nephron growth was estimated from the kidney wet weight, the ratio of the left kidney wet weight divided by the body weight (LKW/BW) and the histomorphometric estimation of the glomerular tuft volume (Table III). In both groups of rats that received treatment with either growth hormone or I G F - I the kidney wet weight and the glomerular tuft volume were significantly greater than in the growth hormone-deficient controls (P<0.05 for each comparison). However, the specific kidney weight, i.e., LKW/BW, was greatest
in the rats receiving des(1-3)IGF-I as compared to the control as well as the growth hormone treated animals (Table III).
Discussion In the present study moderate dosages of both des(1-3)IGF-I and recombinant human growth hormone increase glomerular perfusion and ultrafiltration rates as well as increase renal and nephron size in growth hormone-deficient rats. In fact, the growth hormone deficiency state presents with elevated afferent and efferent arteriolar resistance, a normal PG and a reduced LpA. The increased arteriolar resistance allows only for a lesser-than-normal nephron blood flow. Since the glomerular capillary pressure, Po, as well as the ultrafiltration coefficient, LpA, are low, a reduced glomerular ultrafiltration rate results. Indeed, growth hormone deficiency in man presents with reduced G F R and R P F [3,26,27,29,42]. This laboratory has previously studied the baseline renal function in growth hormone-deficient subjects and demonstrated a lower-than-normal G F R and RPF and elevated renal vascular resistance [3,42]. The present studies confirm these earlier findings and, furthermore, delineate the pathophysiologic mechanisms that cause these changes in renal function in
247 growth hormone deficiency; the reduced G F R and RPF in hyposomatotropic states are caused by arteriolar vasoconstriction in the nephron and a reduced LpA. Both of these abnormalities are corrected with the administration of either the truncated IGF-I or growth hormone, both of which render the RPF and G F R normal. Similarly, in growth hormone-deficient humans, RPF and G F R are raised towards normal with a single injection of growth hormone [3]. Furthermore, in growth hormonedeficient subjects, other maneuvers such as an infusion of amino acids acutely raise RPF and GFR, indicating that the glomerular perfusion and filtration are not only reduced due to a lower kidney size, rather than determined functionally [42]. Several lines of evidence suggest that the growth hormone-induced rise in G F R is mediated through IGF-I. First, in normal human subjects, growth hormone raises G F R and RPF and lowers renal vascular resistance; all of these changes are induced in the same direction by administration of IGF-I [ 2,8,10,12,14]. Second, the growth hormone-induced rise in renal function is delayed and occurs only several hours after a single injection of rhGH, at a time, when IGF-I serum levels (and presumably renal tissue IGF-I concentrations) have also risen [2]. Third, as shown in the present study, treatment of growth hormone-deficient rats with either truncated IGF-I or growth hormone, each reduces renal arteriolar resistance and elevates nephron filtration and perfusion. Both treatments affect the same determinants of glomerular ultrafiltration, namely arteriolar resistance and LpA, but not P6. Fourth, the normalization of glomerular function in growth hormone deficient rats requires only the correction of the IGF-I deficiency without concomitant administration of growth hormone, as shown in group 2 in the present study. Although not measured, it is reasonable to assume that the growth hormone secretion in these latter rats has remained low during the week of des(1-3)IGF-I treatment. The glomerular ultrafiltration coefficient, LpA, is the product of the pressure-dependent permeability
of the glomerular ultrafiltration barrier, Lp, and the surface area A that is available for ultrafiltration in the glomerulus. With presently available methods it is experimentally impossible to measure each of these terms independently in vivo, and it remains unknown, whether Lp, A or both are affected by IGF-I. Theoretically, both may be increased by IGF-I. However, it is the most likely scenario that IGF-I increases the surface area which could result from mesangial relaxation. Indeed, mesangial cells display specific IGF-I receptors and several effects of IGF-I have been demonstrated in cultured rat mesangial cells [43,44]. Angiotensin II, for instance, which has been shown to reduce LpA in vivo [45,46] also contracts cultured mesangial cells in vitro [47], but direct effects of IGF-I to relax mesangial cells are yet to be demonstrated experimentally. IGF-I increases glomerular function, in part, by lowering arteriolar resistance as demonstrated in the present as well as in previous studies [5,16]. The vasodilatory effects of IGF-I are observed within several minutes of an intravenous infusion of IGF-I in rats [4]. This effect of IGF-I may be mediated, in part, by vasodilatory prostaglandins, such as prostacyclin [4,14,48]. Administration of indomethacin, a potent cyclooxygenase inhibitor, prevents [4] or reduces [48] the IGF-I-induced acute increase in RPF and G F R in the rat. However, vasodilating cyclooxygenase products may have only a permissive effect, rather than being a true mediator. Recent evidence suggests an important role for E D R F / N O to mediate the IGF-I-induced reduction in arteriolar resistance and increase in renal blood flow [48]. Haylor and coworkers infused acutely IGF-I into normal rats and measured the renal blood flow with an electromagnetic flow probe and calculated the renal vascular resistance [48]. Infusion of IGF-I concomitant with administration of the nitric oxide synthase inhibitor L-NAME completely prevented the rise in RBF [48]. In the present study both IGF-I and growth hormone induce renal and glomerular growth. Previous data suggest, that IGF-I increases kidney weight and
248 nephron size primarily by hypertrophy, rather than hyperplasia [49]. Indeed, G u l e r and associates demo n s t r a t e d that I G F - I induces renal growth in excess o f b o d y growth and growth o f other organs [50]. Furthermore, (renal) I G F - I has been associated with the induction of renal c o m p e n s a t o r y growth in a number o f experimental disease states [ 5 1 - 5 5 ] . The glomerular h y p e r t r o p h y m a y have contributed to the I G F - I - or growth h o r m o n e - i n d u c e d rise in glomerular function that was d e m o n s t r a t e d in the growth h o r m o n e deficient rats in the present experiments. However, it is unlikely that the increase in glomerular size is solely responsible for the normalization o f n e p h r o n function in these studies, since we have previously d e m o n s t r a t e d that the functional effects occur quickly, within minutes of the onset of systemic I G F - I infusions in n o r m a l rats [4]. Possibly, the increase in n e p h r o n volume contributed to a rise in the ultrafiltration surface area and, thus, m a y have helped to increase L p A . In summary, growth h o r m o n e administration corrects the reduced glomerular function and size in growth h o r m o n e deficient rats and raises I G F - I synthesis and serum levels. The functional and growth effects of growth h o r m o n e on the nephron can also be induced by administration of m o d e s t dosages o f d e s ( 1 - 3 ) I G F - I , suggesting that the lower glomerular function and size in growth h o r m o n e deficiency results primarily from a lack o f I G F - I . Moreover, the findings in the present study also suggest that growth h o r m o n e a n d / o r I G F - I contribute to the regulation o f glomerular function.
Acknowledgements These studies were m a d e possibly by a grant from the A m e r i c a n H e a r t Association, Dallas, T X (No. 901218). The author would like to express his gratitude to the H o r m o n e and Pituitary Program, N I H , Baltimore, M D , for providing specific a n t i - I G F - I antibody for R I A ; and Ross Clark, Ph.D., for allowing to use the growth h o r m o n e deficient rats, as well as
G e n e n t e c h Inc., South San F r a n c i s c o , CA, for the d o n a t i o n o f r h G H and d e s ( 1 - 3 ) I G F - I . The author also appreciates the provision of the Digital Vax c o m p u t e r system and the Clinfo software m a d e available to these studies by the Clinical R e s e a r c h Center at H a r b o r - U C L A M e d i c a l Center through N I H grant G C R C R R 00425. Parts o f this m a n u s c r i p t were previously presented in abstract form (J. Am. Soc. Nephrol., 2 (1991) 681).
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