Role of kidney and liver in the renotropic activity generated in rats after uninephrectomy

Transcription

1 Nephrol Dial Transplant (1992) 7: European Dialysis and Transplant Association-European Renal Association Nephrology Dialysis Transplantation Original Article Role of kidney and liver in the renotropic activity generated in rats after uninephrectomy A. Garcia-Ocana and P. Esbrit Unidad Metabolica, Fundacion Jimenez Diaz(Centro Asociado al CSIC), Madrid, Spain Abstract. Following uninephrectomy the remnant kidney undergoes a compensatory growth apparently regulated by a humoral renotropic factor(s). Our studies were carried out to assess the role of renal and hepatic tissue in the generation of such a renotropic factor(s). The renotropic activity in extracts of rat kidney and liver obtained at different times after uninephrectomy was assayed in cortical tubules from rat kidney removed 24 h after uninephrectomy. We found that kidney extracts from 6 h and 24 h uninephrectomized rats increased [ 3 H]thymidine incorporation into tubular cell DNA, dose-dependently, compared to those from sham-operated rats. Maximal stimulation was observed at 10*-fold (6 h) or fold (24 h) extract dilution. This activity was undetected in perfused kidney extracts, or in extracts of kidneys from simultaneously uninephrectomized and partially hepatectomized rats. However, only 10-fold dilution of unperfused liver extract from 6 h or 24 h uninephrectomized rats showed significant activity. Kidney or liver extracts from 30 min uninephrectomized animals failed to display activity. Our results point to an extrarenal origin for the humoral renotropic activity generated after uninephrectomy, although the kidney seems to modulate this activity. Introduction Unilateral nephrectomy is followed by an almost immediate compensatory growth of the remnant kidney. This growth involves both hypertrophy (cell enlargement) and hyperplasia (cell division) of glomerular and tubular epithelial cells [1,2]. Present evidence suggests that a humoral growth factor(s) specific for the kidney tissue appears to regulate compensatory renal growth [3-6]. Although the nature and the origin of such a factor(s) is still unknown, several studies have suggested that this renotropic factorfs) might be elaborated or activated by the remaining kidney [7,8]. In addition, a previous study has shown that the liver could also be involved [9]. The present studies were undertaken to clarify whether the kidney and/or the liver are the source of the renotropic activity generated after unilateral nephrectomy. With this aim, we have developed a sensitive and simple assay to measure the renotropic activity of renal and liver extracts on isolated tubules from the remnant kidney of 24 h uninephrectomized rats. This system has been shown to be useful in testing the activity of a kidney growth promoter partially purified from rat plasma obtained after unilateral nephrectomy [10,11]. Key words: renotropic activity; kidney; liver; uninephrectomy Subjects and methods Surgical procedures Male Sprague-Dawley rats ( g body weight) were Correspondence and offprint requests to: A. Garcia-Ocana. Unidad used. Following an overnight fast, 42 animals were anaesthetized with ether and the left kidney excised through a Metabolica, Fundacion Jimenez Diaz. Avda. Reyes Catolicos Madrid, Spain medial laparotomy. At the time of unilateral nephrectomy

2 Role of kidney and li\er in renotropic activity in uninephrectomized rats 609 six of these animals also underwent a partial hepatectomy (2,3 of total liver mass) by the standard procedure of Higgins and Anderson [12]. Sham-operated rats (n = 28) underwent laparotomy and the kidney (n = 24) or both the kidney and the liver (n = 4) manually manipulated. These surgical procedures were always carried out between 9 a.m. and 12 noon. The animals were again anaesthetized with ether 30 rein (n=18), 6h (n = 34), or 24 h (n=18) later. For every time period studied, the liver and the kidney were removed from four to eight uninephrectomized rats and four sham-operated animals. Kidneys were also obtained from rats 6 h after simultaneous hepatectomy and either unilateral nephrectomy (n = 6) or sham operation (n = 4). At each time interval, the liver and the kidney from another group of six to eight uninephrectomized rats and four sham-operated animals were perfused with 40 ml of 0.9% NaCl (saline), carefully pumped by hand with a syringe through a cannula in the lower aorta as near as possible to the renal blood vessels. The perfusate was allowed to drain through a cut made on the inferior vena cava. After this manoeuvre, perfused kidney and/or liver were removed and placed in ice-cold saline. Preparation of tissue extracts A one gram portion of kidney or liver tissue was minced on ice, resuspended in 20 ml of cold saline, and homogenized in a Polytron-type homogenizer, twice for 30 s, at maximum speed. The crude homogenates were centrifuged at 2500 # for 15 min at 4 C. The supernatants (organ extracts) were aliquoted and stored at 70 C until used for assay of DNA synthesis. The protein content (mg/ml) of the extracts was: 15.6 ±2.8 and 12.2 ±3.3, for unperfused and perfused kidney, respectively (n = 9; P< 0.05); 41.4±4.7 (H = 3) and 14.2 ±2.6 (n = 7), for unperfused and perfused liver respectively (/*<0.01). Protein was measured by the method of Bradford [13]. On several occasions rat blood was collected in EDTA (final concentration: 5 mm) by cardiac puncture under ether anaesthesia, immediately before organ perfusion and removal. Blood was centrifuged at 600 g for 10 min at 4 C. Blood protein was determined in EDTA-treated blood samples and found to be ±23.0 mg/ml (n = 6). For each time interval studied, 2-3 different extracts from the kidneys or livers of 2-3 uninephrectomized or sham-operated animals were tested at least twice on different tubule assays. For every experimental time period the corresponding extracts, either perfused or unperfused, from uninephrectomized or sham-operated rats were obtained and tested in parallel on the same tubule preparation. All the assays were performed in triplicate. Kidney tubules (0.7 mg protein) were incubated at 25 C C in 1 ml of KHB containing 3 uci of [ 3 H]thymidine (85 Ci/mmol; New England Nuclear, Boston, MA) and the test additives. After 90 min of incubation, the reaction was stopped with 1 ml of ice-cold 10% trichloroacetic acid (TCA). After 5 min on ice, the reaction mixture was centrifuged at 600 g for 10 min. Then 5 ml of 5% TCA was added to the pellet and the resulting suspension was filtered through 0.45 urn filters (MFS, Dublin, CA). The filters were then washed with 5 ml of ethanol. The filter-associated radioactivity was counted in a liquid scintillation spectrometer (Rackbeta, LKB, Sweden). Statistical significance was assessed by Kruskal-Wallis test performed among activity values obtained with different dilutions of extracts, either perfused or unperfused, for every experimental time period. Student's /-test was performed between values obtained with uninephrectomized rat extracts and the corresponding sham values. All values throughout the text represent mean + SD. Results Figures 1 and 2 show the effect of 6 h and 24 h kidney extracts on DNA synthesis rate in cortical tubules from kidneys isolated 24 h after unilateral nephrectomy. Only kidney extracts from 6 h and 24 h uninephrectomized rats induced a significant increase in [ 3 H]thymidine incorporation into tubule DNA over that observed with saline control. This effect was dose-dependent, so that a lc^-fold or a lcp-fold dilution of extracts from contralateral kidneys obtained 6 h or 24 h after unilateral nephrectomy to m < Q c P uo- 120 ICO Assay of DNA synthesis Measurement of DNA synthesis was carried out in isolated cortical tubules from 24 h uninephrectomized rats, obtained as described previously [11,14]. Briefly, mm slices of kidney cortex from at least two animals were incubated in Krebs Henseleit buffer (KHB) with 0.75 mg/ml collagenase and 0.25% (w v) of bovine serum albumin, in a shaking bath at 37 C for min. Then the suspension was filtered through a mm mesh and centrifuged at 60 g for 30 s at 4C. The resulting pellet was washed and resuspended in KHB. This preparation consisted mainly of proximal tubules, assessed by optical microscopy O Dilution (-fold) Fig. 1. Incorporation of [ 3 H]thymidine into DNA of cortical tubules from 24 h uninephrectomized rat kidney in the presence of different dilutions of 6 h post-uninephrectomized rat kidney extracts:, unperfused;, unperfused with simultaneous partial hepatectomy; L C, perfused. The open symbol (O) represents the mean + SD of values corresponding to sham-operated rat kidney extracts (6 hj assayed within the dilution frame tested. Control activity corresponds to 380±49d.p.m. mg protein (100%). Points are means±sd for 2-3 extracts measured at least twice with every experimental condition. */ > <0.01, compared to the sham value.

3 610 A. Garcia-Ocana and P. Esbrit T»ble 1. Effect of 30 min uninephrectomized rat kidney extracts on DNA synthesis in cortical tubules from 24 h uninephrectomized rat kidney Dilution (-fold) [ 3 H]-thymidine incorporation (d.p.m./mg protein) Unperfused extract Perfused extract Dilution (-fold) Fig. 2. Incorporation of [ 3 H]thymidine into DNA of cortical tubules from 24 h uninephrectomized rat kidney, in the presence of different dilutions of 24 h uninephrectomized rat kidney extracts. The number of values, control activity and symbols are as in Figure 1. */ > <0.01, compared to the sham value. respectively displayed the maximal activity, decreasing at higher concentrations. In contrast, no significant effect on tubule DNA synthesis was observed when extracts of kidneys from sham-operated rats were tested (Figures 1 and 2). Moreover no activity was detected in extracts from perfused kidneys removed 6 h or 24 h after unilateral nephrectomy, within the same dilution range which exhibited activity in unperfused kidneys (Figures 1 and 2). Plasma obtained from the same rats before organ perfusion and removal only showed activity when tested at 10% but not at dilutions matching those of the kidney extracts, and in the same assay (not shown). In addition the activity displayed by the kidney extract obtained 6 h after unilateral nephrectomy was abolished when partial hepatectomy was performed simultaneously with unilateral nephrectomy (Figure 1). Extracts from kidneys removed 30 min after unilateral nephrectomy, even unperfused, were not found to stimulate [ 3 H]thymidine incorporation into tubule DNA in a broad dilution frame (Table 1). On the other hand, we observed a slight but significant stimulatory effect on the rate of tubule DNA synthesis induced only by the highest concentration tested (10-fold dilution) of unperfused extracts from liver removed 6 h or 24 h after unilateral nephrectomy, over that of either saline control or liver extracts from sham-operated animals. This stimulation was 125±2% (6h; /><0.01), 119±7% (24 h; P<0.05); 103 ±6% (sham), compared to 100% of control (358 ±30 d.p.m./mg protein). On the contrary, perfused liver extracts from 6 h or 24 h uninephrectomized rats were not found to display activity. Moreover, neither perfused or unperfused extracts from liver removed 30 min after unilateral nephrectomy showed activity ± ±24 384±7 345 ± ± ± ± ±7 380 ±7 Values corresponding to 30 min kidney extracts from shamoperated rats, assayed within the dilution range tested, were (d.p.m./mg protein) 363 ±12 (unperfused) or 366 ±7 (perfused). Control activity was 359 ±28 d.p.m./mg protein. Activity of 10% fetal bovine serum as a positive control, was 854 ± 130 d.p.m./mg protein. Values are means ±SD for two different extracts assayed twice in triplicate. Discussion Not only the nature but also the origin of a specific regulator(s) of renal growth detected in serum or plasma of uninephrectomized animals remain unknown. Several studies have been carried out to ascertain whether the kidney itself could be involved in the generation of such a renotropic factor [7-9]. All these studies were performed with renal fragments as the target tissue for detection of the renotropic activity, and they exhibited poor sensitivity. Nevertheless, the studies showed that renal tissue, and perhaps a hepatic component, appear to play a certain role in regulating the mitogenic activity of the putative renotropin. In our studies, to assay the possible renotropic activity of kidney and liver extracts from uninephrectomized rats we used cortical tubules from rat kidney removed 24 h after unilateral nephrectomy. Previous work in our laboratory has shown that these tubules were more sensitive for measuring the renotropic activity in partially purified plasma extracts compared to those from intact or sham-operated animals [11]. By using this system, the maximal activity of unperfused kidney extracts from 6 h uninephrectomized rats on the rate of tubule DNA synthesis was induced by an extract dilution lo^fold greater than that of kidney extracts from 24 h uninephrectomized animals triggering the maximal response. Moreover, the tubule DNA synthesis-stimulating activity in 6 h or 24 h kidney extracts was found to decrease to the control level at an extract concentration higher than that which induced a maximal stimulation. These findings suggest the existence of inhibitors of renal growth in the unperfused kidney extracts, as previously reported [9,16,17], with a

4 Role of kidney and liver in renotiopic activity in uninephrectornized rats 611 different potency and/or clearance rate than that of the renotropic stimulators) in these extracts. Our results are also consistent with the presence in the uninephrectomized rat kidney extracts of an apparently short-lived renal growth-promoting activity, decreasing at 24 h after unilateral nephrectomy, as recently reported in uninephrectomized mice [15]. We have recently observed a similar pattern of response in renal cells with a partially purified preparation of a renotropic activity present in 24 h uninephrectomized rat plasma [10,11]. The relationship between the latter and the activity of kidney extracts described in this report is unknown at present. Interestingly, extracts of perfused kidneys from 6 h or 24 h uninephrectomized rats did not show any detectable tubule DNA synthesis-stimulating activity. This finding suggests a paramount role of blood components in this activity. However, rat plasma obtained immediately before organ perfusion and removal, assayed in parallel with kidney extracts, only displayed significant activity at 10%. The latter concentration is fold higher than that corresponding to the 24 h unperfused kidney extract dilution exhibiting the maximal activity, based on an estimated blood concentration in unperfused kidney extracts equivalent to about 3 mg protein, or 15 ul blood per millilitre of extract (see Subjects and methods). To our knowledge only one previous report has shown some renotropic activity, although in the range of 10% over control, in 24 h uninephrectomized rat plasma at such a low concentration [4]. Our results though suggest that the interaction between circulating substances and the renal tissue is what is responsible for the activity of unperfused kidney extracts. This is consistent with the previously reported effect of unpurified plasma or sera on the activity of kidney (possibly unperfused, although it was not stated) extracts from uninephrectomized rats to stimulate DNA synthesis in renal cortical fragments [7,8]. In this regard the possible role of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), which appear to accumulate in the growing kidney within 24 h of unilateral nephrectomy [18,19], in the activity of our kidney extracts, cannot be ruled out from the present data. However, doses of recombinant human IGF-I (2 ng/ml) or EGF (5 ng/ml) displaying maximal activity (^30% over control) in our tubule assay, did not modify the maximum effect of kidney extracts (not shown). Furthermore, tubule DNA synthesis-stimulating activity was not detected in kidney extracts, even unperfused, from 30 min uninephrectomized rats. Although we did not test these extracts at dilutions less than 100-fo!d, this is a much higher concentration than that of 6 h kidney extract exhibiting the maximal activity. Thus our results strongly suggest that the activity of 30 min kidney extracts, if any, would be much lower than that of 6 h kidney extracts. Recent reports have provided evidence that 30 min after unilateral nephrectomy, rat plasma maximally stimulated the production of inositol-l,4,5-triphosphate, 1,2-diacylglycerol and cyclic GMP in renal cortical slices. In addition, this stimulation was no longer detected in 6 h uninephrectomized rat plasma [20,21]. Results from these reports suggest that an increased formation of intracellular messengers might regulate the early phase of compensatory kidney growth triggered by one or more circulating renotropic factors. Assuming that the activities reported therein and the tubule DNA synthesis-promoting activity in kidney extracts reported here are caused by the same renotropic factor(s), those results and our data taken together suggest an extrarenal origin for this factor. On the other hand, partial hepatectomy performed on simultaneously uninephrectomized rats was found to abolish the stimulation triggered by unperfused 6 h kidney extracts on tubule DNA synthesis. This could be accounted for by a possible effect of partial hepatectomy directly on a renotropic substance secreted by the liver, or indirectly through a putative metabolic insufficiency affecting the synthesis of such a renotropin. In this regard a previous report has shown that partial hepatectomy of a lesser degree than that performed by us also impaired compensatory renal growth in rats [9]. These findings suggest that the liver could play a key role in the formation (or the activation) of renotropic factors. Nevertheless, only unperfused 6 h and 24 h liver extracts at the maximal concentration tested, greater than that of corresponding kidney extracts displaying the maximal activity, significantly increased tubule DNA synthesis. Although this again suggests a stimulatory effect by a blood component(s) present in the unperfused liver extract, activity should have been detected at a higher extract dilution, since blood represents 70% of the protein content of unperfused hepatic tissue, a higher value than that for kidney (see Subjects and methods). Other investigators have recently shown in rabbit liver homogenates growth inhibitory and growth stimulatory activities of renal cortical cells, detected on the 3rd day after unilateral nephrectomy [22]. Although we did not test the activity of extracts from livers removed beyond 24 h after unilateral nephrectomy, the renotropic activity of rat liver extracts could be manifested earlier than in rabbit livers, as described for the circulating renotropic factor(s) in both species [5,23]. Tne presence of different regulators of kidney growth in unpurified liver extracts makes it difficult to draw conclusions from activity data obtained with these extracts. Our results, by using a sensitive and simple assay

5 612 A. Garcia-Ocafla and P. Esbnt to detect a renotropic activity, strongly suggest that the circulating renotropic factor(s) generated after unilateral nephrectomy has an extrarenal origin, although the kidney might play a somewhat important role in the regulation of its activity. Whether the liver is the site of formation of this factor(s) awaits further studies using purified extracts, which are in progress. Acknowledgements. This work was supported in part by a grant from Direction General de Investigation Cientifica y Tecnica (DGICYTPM88-OO13-CO2-O2) of Spain. A.G-O. is a fellow of Conchita Rabago Foundation. References 1. Fine L. The biology of renal hypertrophy. Kidney Int 1986; 29: Wesson LG. Compensatory growth and other growth responses of the kidney. Nephron 1989; 51: Austin H, Goldin H, Preuss HG. Humoral regulation of renal growth. Nephron 1981; 27: Dicker SE, Morris CA. Presence of a renotrophic factor in plasma of unilaterally nephrectomized rats. J Phvsiol 1980; 299: Yun GC, Areas J, Yamamoto N, Preuss HG. Renotropic stimulation in rat kidney cell culture. Life Sci 1988; 42: De Miguel F, Manzano F, Esbrit P. Specificity of the renotropic activity in the plasma of uninephrectomized rats carrying non-renal tumours. Med Sci Res 1989; 17: Dicker SE, Morris CA. Origin of the humoral factor responsible for compensatory renal hypertrophy. J Physiol 1980; 301: Preuss HG, Goldin H. A renotropic system in rats J Clin Invest 1976; 57: Dicker SE, Morris CA, Shipolini R. Regulation of compensatory kidney hypertrophy by its own products. J Physiol 1977; 269: Manzano F, Esbnt P, Garcia-Ocafia A, Garcia-Caflero R, Jimenez-Clavero MA. Partial purification and characterisation of a renal growth factor from plasma of uninephrectomised rats. Nephrol Dud Transplant 1989; 4: Esbrit P, Garria-Ocana A, Garcia-Canero R, Manzano F, Jimenez-Clavero MA. Biological properties of a renotropic protein present in plasma of uninephrectomized rats. Renal Physiol Biochem 1991; 14: Higgins GM, Anderson RM. Restoration of the liver of the white rats following partial surgical removal. Arch Palhol 1931; Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: Manzano F, Esbnt P, Lopez-Novoa JM. Lipid methylation, hormone action and compensatory hypertrophy in renal cortical tubules. Clin Physiol Biochem 1989; 7: Hise MK, Chacko KV: Potent-intrinsic growth promoting factors in mouse kidney following nephron loss (Abstract) Kidney Int 1988; 33: Holley RW, BohJen P, Fava R, Baldwin JH, Kleeman G, Armour R. Purification of kidney epithelial cell growth inhibitors. Proc Natl Acad Sa USA 1980; 77: Fine LG, Holley RW, Nasri H, Badie-Dezfooly B. BSC-I growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: Relationship to Na + /H + antiport activity. Proc Natl Acad Sci USA 1985, 82: Kanda S, Saha PK, Nomata K et al. Transient increase in renal epidermal growth factor content after unilateral nephrectomy in the mouse. Ada Endocrinol 1991; 124: Flyvbjerg A, Thorlacius-Ussing O, Naeraa R, Ingerslev J, Orskov H. Kidney tissue somatomedin C and initial renal growth in diabetic and uninephrectomised rats. Diabetologia 1988; 31: Banfic H, Kukolja S Plasma from uninephrectomized rats stimulates production of inositol trisphosphates and inositol tetrakisphosphate in renal cortical slices. Biochem J 1988; 255: Banfic H. Extracellular Ca 2+ influences 1,2-diacylglycerol and inositol monophosphate formation in renal cortical slices stimulated with plasma obtained from uninephrectomized rats. Biochem Biophys Ada 1989; 1002: Kanda S, Kanetake H, Saito Y. A study of growth regulators in the rabbit liver for the renal cortical tubular cells (Abstract). Kidney Int 1990; 37: Yamamoto N, Kanetake H, Yamada J. In vitro evidence from tissue cultures to prove existence of rabbit and human renotropic growth factor. Kidney Int 1983; 23: Received for publication Accepted in revised form