Subtotal nephrectomy alters tubular function: Effect of phosphorus restriction

Kidney International, Vol. 52 (1997), pp. 1550—1560

Subtotal nephrectomy alters tubular function: Effect of phosphorus restriction DENISE LAOUARI, GERARD FRIEDLANDER, MARTINE BURTIN, CAROLINE SILVE, MICHELE DECHAUX, MICHELE GARABEDIAN, and CLAIRE KLEINKNECHT INSERM U 426, Faculté Xavier Bichat; Faculté Necker; and CNRS/URA583, Hôpital Necker, Paris, France

Subtotal nephrectomy alters tubular function: Effect of phosphorus restriction. Few studies have examined tubular function after subtotal nephrectomy (Nx) and conservative treatments. The effects of 70% and 80% Nx (associated with dietary phosphate restriction in the latter case) on the apical brush border membrane (BBM) enzymes 5'-nucleotidase, yglutamyl-transferase and alkalinc-phosphatase, and one BBM Na-phosphate cotransporter (NaPi-2) were studied in rats after a six week period. Changes in activity and mRNA abundance of the BBM enzymes and in NaPi-2 protein and mRNA abundance were compared with changes in the

distal markers of Na,K-ATPase activity and epidermal growth factor (EGF) production. The activity, but not the mRNA of BBM enzymes, was

moderately reduced by the 70% Nx. Both the mRNA and activity of yglutamyl-transferase and alkaline-phosphatase were decreased in the 80% Nx, and the NaPi-2 mRNA, protein and Na,K-ATPase activities were also reduced. These effects (except for 5'nucleotidase and Na,K-ATPase)

were partly reversed by phosphate restriction. Overproduction of EGF occurred after the 70% Nx, was blunted in the 80% Nx, and then partially restored by phosphate restriction. Aggravation of tubular alteration was associated with enhanced renal hyperplasia (increased DNA mass), reduced GFR and hyperphosphatemia, and high PTH levels, but reduced cAMP excretion. Improvement following phosphate restriction was associated with reduced hyperplasia and lowering of phosphatemia and PTH levels. These data demonstrate that Nx selectively affected BBM function through transcriptional changes that were partially reversed by phosphate

restriction. Regulatory factors involved in these changes may include intracellular phosphate content and growth factors, but not the PTH effects that are impaired in chronic renal failure.

according to tubular enlargement, expansion of the extracellular matrix and histological abnormalities such as dilation, atrophy and tubulointerstitial fibrosis. Tubular activity and function have been little examined in CRF. An increase in oxygen consumption [6] and ammoniagenesis [7] per residual nephron as well as adaptative changes in renal handling of minerals [8] have been reported after subtotal nephrectomy (Nx). Few data are available on the changes in the activity of tubular cell membranes where important transporters and energy regulators reside. An increase in renal Na,K-ATPase activity has been reported after Nx [9]. However, functional changes in the apical brush border membrane (BBM), reflecting changes in proximal function, are poorly documented in

CRF. It is not known whether CRF affects BBM enzymes involved in metabolic processes. Proximal phosphate (P) reabsorption is reported to be reduced in CRF [10], but the molecular process involved in this alteration is unknown. It is possible that the sodium-dependent P cotransporter (NaPi-2) may have a role

in altered P transport in CRF since NaPi-2, cloned from rat kidney, has been identified in renal BBM and shown to be regulated by dietary P and PTH status [11, 12]. Changes in renal BBM function induced by CRF after Nx were

therefore determined by measuring the activity and mRNA expression of three enzymes located in BBM, and the protein and

mRNA expressions of the NaPi-2 cotransporter. The enzymes The mechanism by which renal function declines in chronic under study were: (a) 5'-nucleotidase, an ectopeptidase involved renal failure (CRF) remains unclear and has been investigated in nucleotide catabolism and cell energy regeneration [13]; (b) following subtotal renal ablation. Most studies have focused on y-glutamyltransferase, which is probably involved in amino acid the importance of glomerular changes in the initiation and uptake and/or glutathione metabolism [14]; and (c) alkaline progression of renal damage [1]. However, it appears that tubular phosphatase, which participates in protein dephosphorylation damage is at least as important as glomcrulosclerosis in the course [15]. The activity of these enzymes in the liver was also deterof renal deterioration [2—4]. That tubular changes play a role in mined in order to evaluate the tissue specificity of the response. the deteriorating process can be suggested, since tubular mass is The changes in Na,K-ATPase activity, a basolateral enzyme the major constituent of kidney mass and renal compensatory heterogeneously expressed along the nephron [9], and changes in growth is a critical factor for renal deterioration [51. epidermal growth factor (EGF) production, which is expressed in Tubular modifications have until recently mainly been assessed distal tubules [16], were determined for comparative purposes. The effects of two levels of renal ablation, that is, 70% and 80% Key words: nephreetomy, tubular function, phosphorus restriction, chronic renal failure, hyperplasia, brush border membrane. Received for publication December 30, 1996 and in revised form July 9, 1997 Accepted for publication July 19, 1997

© 1997 by the International Society of Nephrology

ablation of renal mass, which was associated with dietary P restriction in latter case, were studied to evaluate Nx-induced changes in tubular function. The situation of P restriction was chosen since it is known to modify renal P transport and metabolism [17, 181, and to retard renal deterioration [19]. We show that subtotal nephrectomy reduced the expression of NaPi-2 and of two BBM enzymes in a tissue-specific way. Dietary

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Laouari et a!: Altered tubular function in remnant kidney

P restriction improved the activity and/or expression of these BBM components. METHODS

Animals and diets

Male Sprague-Dawley rats (Iffa Credo, Saint Aubin les Elboeuf, France) weighing 130 to 150 g were used in all studies and

were subjected to renal ablation [3, 9]. Approximately 70% or

1551

collected from the jugular vein. Plasma was analyzed for creatinine, urea, Na, K and calcium (Ca) content; blood pH and ionized calcium were measured in five animals in each group. The rats were killed towards midday on the day following the second biological study. They were fed a fixed amount of food (18 g) overnight in order to standardize their metabolic state. Euthanasia was performed by i.p. injection of nembutal (50 mg/kg body

wt). Blood was taken from the jugular vein and plasma 1,25(OH)2D3, PTH, P, triglycerides and cholesterol levels were

80% of total kidney mass was removed using a two stage determined. The abdomen was then opened and a lobe of liver

procedure. Firstly, the two poles of the left kidney were consec- and the whole kidney(s) were rapidly excised and immersed into utively excised and weighed. The amount of excised parenchyma liquid nitrogen (N). Frozen tissues were later weighed, pulverized,

was 40% or 60% of the left kidney mass, based on the mean kidney weight (0.47

0.02 g/100 g body wt) of five control rats

raised under the same conditions. Bleeding was prevented by finger pressure at sites of excision and coating cut surfaces with collagen powder (Pangen, Fournier, France). Secondly, the right kidney was removed four days later. Control rats underwent

and freeze-dried in liquid N using a Spex 6700 Freezer (Mill

Industries mc, Edison NJ, USA). Renal powder was kept in liquid N and different aliquots were used for: (1) determination of the

protein content and activity of the three BBM enzymes [5'-

nucleotidase (5'-Nu), y-glutamyltransferase (yGT) and alkaline phosphatase (Pase)I as well as Na,K-ATPase; (2) determination laparotomy and kidney decapsulation in parallel with uremic rats. of NaPi-2 cotransporter protein abundance in crude membranes; All animals received a standard chow diet until the second (3) simultaneous measurement of protein and DNA mass indicatoperation, a normal phosphate diet for five days (NP diet) and ing the degree of hypertrophy and hyperplasia; and (4) measurethen the NP diet or a low phosphate diet (LP diet) (INRA, Jouy ment of mRNA for the NaPi-2 cotransporter and BBM enzymes en Josas, France) thereafter according to the experimental group. after RNA extraction. The liver powders were only analyzed for The NP diet that was administered during the recovery period protein content and 5'-Nu, y-GT and Pase activities. served as a control diet and contained 0.46 g/100 g (%)P. The LP

diet, which contained 0.1% P, was obtained by removing NaH2PO4 (1.7%) from the diet and adding 0.3% NaCl so that the Na content remained constant (0.25%) in the two diets. Calcium

Techniques

Plasma and urine measurements. Plasma creatinine, urea, cholesterol and electrolyte levels were measured on a Hitachi autodiets, respectively. Both diets contained 0.5% potassium. Vitamin matic analyzer (Boehringer Mannheim, Meylan, France). Plasma D3 content was three times the recommended level, that is, 100 levels were determined on deproteinized plasma samples and on IU/100 g diet, to compensate for any vitamin deficiency provoked non-deproteinized urinary samples using the Fiske and Subarow by CRF. technique. Urinary Ca concentration was measured by a colonAll rats were housed in individual cages in an animal house at metric assay using Biotrol calcium monoreactif (Merck-Biotrol, 23 1°C and on a 12-hour light/dark cycle. Food intake was Nogent, France). Urinary protein concentration was determined measured daily throughout the six week experiment. All uremic by the colorimetric Biuret method. Urinary EGF concentration rats were fed ad lihitum whereas control rats were pair-fed with was determined by RIA using the mouse EGF reagent pack uremic rats with the most severe renal ablation. This meant that (Amersham, Les Ulis, France) containing '251-EGF and rabbit control rats were fed the mean quantity of food ingested by antiserum to mouse EGF. All urine samples were concentrated by corresponding uremic rats on the previous day. Nutritional ma- lyophilization in the latter case in order to ensure that the results nipulation (LP vs. NP diet) did not appear to influence the renal for U rats were reliable. Urinary cAMP was measured by the response, since neither food intake nor weight gain were affected 3H-cAMP assay system (Amersham, Les Ulis, France). by P restriction when measurements were taken at three and six Plasma immunoreactive parathyroid hormone (PTH) was meaweeks (data not given). sured by RIA using 1251-PTH and antibody anti-rat PTH (Mallinckrodt Medical, Evry, France). Plasma 1,25(OH)7D1 conExperimental protocol centration (N = 5/group) was measured by high-pressure liquid Renal enzymatic activity and associated variables were studied chromatography [20]. Tissue measurements. Crude homogenates were prepared by concomitantly under two different conditions. (1) The severity of renal ablation was examined by comparing control sham-operated homogeneizing frozen samples (100 to 150 mg) in isotonic merats (CNP; N 10) with uremic rats subjected to 70% Nx (uNP; dium (1: 20 wt/vol) containing 5 mrvi Tris, 1 mvi EDTA, 3 mM N = 5) or 80% Nx (UNP; N = 12). All rats received the NP diet. MgCl2 and 0.25 M sucrose (pH 7.4). Kidney homogenates were (2) The effect of P restriction was examined by comparing the centrifuged at 1,500 g for five minutes and supernatants were kept UNP and CNP groups with corresponding groups fed the LP diet, for protein content and enzymatic determination. Liver protein namely, the ULP (N 14) and CLP (N = 10) groups. content and enzymatic assays were performed using aliquots of Two biological studies were performed at three (first) and six fresh homogenate. Protein content was measured after precipitaweeks (second) after the onset of the protocol. Twenty-four-hour tion of proteins with 0.6 M trichloacetic acid according to the urine collections were made, and creatinine, protein and electro- Lowry method. lyte, cyclic AMP (cAMP) and EGF concentrations were deter'The assay for 5'-nucleotidase (EC3.1.3.5) was performed mined. Rats were anesthetized with ether so that blood pressures using a modification of the radioassay of Gentry and Olsson [21], could be taken using the tail cuff method and blood could be based on the release of 3H-adenosine from the enzyme substrate

(Ca) concentrations were 0.53% and 0.44% in the NP and LP

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Laouari et a!.' Altered tubular function in remnant kidney

H-AMP. Specificity was assessed using cs/3-methylene-adenosinediphosphate (83 I.LM), which inhibits ecto-5'-nucleotidase.

analysis on crude renal membranes prepared as previously de-

• y-Glutamyltransferase (EC 2.3.2.2) activity was determined using the technique of Orlowski and Meister [22], which is based on the liberation of p-nitroanilide from L-y-glutamyl-p-nitroani-

of fresh cortex. Crude membrane samples containing 10 jig

lide with glycylglycin as the second substrate. Specificity was assessed using acivicin (AT125; 5 mrvi), which is a strong inhibitor of this enzyme.

scribed [28], but in this case frozen renal powder was used instead

protein were mixed with loading buffer (1 vol/i vol) devoid of /3 mercaptoethanol. Proteins were electrotransferred onto nitrocellulose membranes once the sodium dodecyl sulfate polyacryamide (10%) gel electrophoresis was completed. Membranes were incubated overnight with a 1/4,000 dilution of polyclonal antibody (provided by Dr. Biber, Zurick, Switzerland) raised against the

• Alkaline phosphatase (EC3.13.1) activity was determined from the amount of p-nitrophenyl liberated after enzymatic C-terminal region of NaPi-2. Labeled antigens were revealed hydrolysis of p-nitrophenyl-phosphate (PNPP), as described by Kempson et al [23]. Specificity was assessed using levamisole (1 mM), an inhibitor of this enzyme. • Na,K-ATPase (EC3.6.1.3) activity was determined by measurement of P liberated after hydrolysis of ATP according to the technique of Post and Sen [24]. Specificity was assessed using ouabain (4 mM), the specific inhibitor for this enzyme. Control assays showed that enzyme activities in frozen samples varied with protein content and that intra- and intervariability of the assays for frozen powder was less than 10%. This indicates that the between group differences were associated with the factor under examination (Nx, P restriction).

using anti-rabbit IgG horseradish peroxidase-linked whole anti-

body from sheep and enhanced chemiluminescence Western blotting solutions (Amersham, Les Ulis, France). Stained membranes were exposed to sensitive films and the intensities were quantified by densitometric scanning. Preliminary Western blot analysis confirmed the validity of frozen sample data as compared to fresh sample data since immunodetection was proportional to

protein deposition (range of 5 to 20 jig protein), and a major protein of approximately 84 kDa was detected under non-reducing conditions, as previously described [29].

Expression of results • The same kidney sample was used for measurement of Enzyme activities were measured by the difference between protein, DNA and RNA content, and calculation of the ratio of activities in the presence and absence of the inhibitor, and were protein/DNA and RNA!DNA. Nucleic acids were extracted and expressed as unit (U)/mg protein. One unit represented the measured as previously described [25]. appearance of 1 jimole of product per minute. All assays, apart • Total RNA from whole kidney was extracted by the phenol! from that for 5-nucleotidase, were performed with an excess of guanidium thiocyanate method with an "RNA plus" Kit (Bio- substrate so that activities were as near to Vmax as possible. mRNA data were expressed as the ratio of radioactivity for probe Systems, Montreuil, France). RNase protection assays were performed as previously described [261. All labeled antisense each protein-mRNA to radioactivity for GAPDH-mRNA, since probes were generated by in vitro transcription using T7 RNA the latter was unaffected by experimental conditions (uremia and polymerase (Promega France, Charbonnières) and the 32P-UTP P restriction). Possible losses of material during extraction and gel riboprobe was obtained from a cDNA template. The activity of deposit and inter-gel variability were corrected using this ratio. each riboprobe was approximately ito 5 X i0 cpm!pi. A rat yGT The data for NaPi-2 protein abundance were expressed as the riboprobe of 376 nucleotide (nt) was obtained from a plasmid ratio of intensity of 84 kDa protein from each rat sample provided by Dr. Laperche [27] and linearization by Psp5 II intensity of 84 kDa protein from one CNP rat sample serving as (protected fragment of 353 nt). The riboprobes for 5'-Nu and reference for each membrane examined. Fractional excretion of P (FEe) was the ratio of urinary P NaPi-2 were obtained by reverse transcription and polymerase chain reaction (PCR) using specific primer pairs. The PCR filtered P, where the filtered value was the product of creatinine products were cloned into pCR li by TA cloning® (InVitrogen clearance (Car) by the plasma P concentration. By, Leek, The Netherlands). The plasmid for 5'Nu was linearized Daily urinary excretions of EGF and cAMP were assumed to with ClaI (379 nt riboprobe, 312 nt protected fragment) and the represent renal production, provided plasma concentrations and, plasmid for NaPi-2 plasmid was linearized with SpE1 (362 nt hence, the filtered load of these compounds were negligible. The riboprobe, 238 nt protected fragment). The plasmid for rat latter variables were expressed per renal unit, that is, per nephron, alkaline phosphatase provided by Merck, Sharp and Dohme assuming that total nephrons per kidney was 28,000 in normal rats Research Laboratories (West Point, PA, USA) was treated with [6], and 8,400 (30%) and 5,600 (20%) of this value in the 70% and

Pvuii and Bg/II to generate a 373 nt riboprobe and a 366 nt

80% Nx groups, respectively. The data for EGF were also

expressed per g renal protein since the latter expression takes C. Dani (Nice, France), an 851pb fragment was subeloned into renal hypertrophy into account for this distal function. Renal growth was assessed from the increase in renal DNA per pBSK (Ozyme, Montigny Le Bretonneux, France) and linearization of the plasmid with PvuII and Sy1 generated a 194 nt kidney (index of hyperplasia) and the increase in the renal protein riboprobe and a 164 nt protected fragment. to DNA ratio (index of cell hypertrophy). Each RNA sample (10 ig for yGT and NaPi-2, and 20 g for Pase and 5'-Nu) was hybridized overnight at 50°C with one Statistics labeled riboprobe of BBM protein and labeled GAPDH riboData are presented as means SEM. Two way ANOVA was probe. The radioactive mRNA hybrids were separated by electro- used to study the effects of uremia and P restriction and a one way phoresis, following RNase digestion, and radioactivity was quan- ANOVA was used to compare differences between CNP, uNP tified (cpm!g total RNA) with an Instant Imager (Packard, and UNP groups. Comparisons of the results between pairs of groups were made using the Fischer test and the nonparametric Rungis, France). • The NaPi-2 protein abundance was measured by Western blot Mann-Whitney U-test in the case of PTH due to wide intragroup protected fragment. The plasmid for rat GAPDH was provided by

Laouari et a!: Altered tubular ftnction in remnant kidney

1553

variations. The minimum level of significance for analyses was set rats (UNP vs. uNP, P < 0.01), and were partially restored in ULP at P < 0.05. Correlation coefficients were calculated by the rats.

method of least squares. The linear regression for PTH was Renal growth Kidney weight (data not shown) was higher in uNP than in CNP RESULTS rats (0.43 0.02 vs. 0.34 0.01, g/100 g body wt) and was higher Brush border membrane function still in UNP rats (0.79 0.05 g/100 g body wt). Kidney weight was unaffected by P restriction in control rats, but was reduced in ULP Enzyme activity. 5'-Nu activity (Fig. Ia) activity was similar in the two Nx groups (uNP and UNP groups) and was significantly as compared to UNP rats (0.49 0.05 vs. 0.79 0.05 g/100 g body lower than in normal CNP rats (Fig. 1). P restriction did not affect wt, P < 0.01). Renal protein mass was also higher in uNP than in 5'Nu activity in either control or uremic rats. y-GT activity (Fig. CNP rats (346 25 vs. 254 6 mg/kidney, P < 0.01) and was IB) fell as the severity of Nx increased (UNP vs. uNP, P < 0.01). comparable in UNP and CNP rats. P restriction did not affect P restriction did not affect yGT activity in the control rats but renal protein mass in control rats. Protein mass was lower in ULP significantly improved the yGT activity in uremic rats (ULP rats than in UNP rats (186 12 vs. 240 18 mg/kidney). DNA mass per kidney (Fig. 6A) increased with the severity of vs. UNP, P < 0.01). Values in ULP rats were lower than in CLP

performed using transformed data.

Nx, indicating that hyperplasia was enhanced after severe Nx. Phosphate restriction did not affect DNA mass in control rats but blunted the elevation of DNA mass in UNP rats. This indicated induced a marked improvement in Pase activity in ULP rats (ULP that hyperplasia was lower in ULP than in UNP rats. The renal protein:DNA ratio (Fig. 6B) was slightly higher in rats vs. UNP, P < 0.01). However, values in the ULP rats were still uNP than in CNP rats and was significantly reduced in UNP rats. 50% less than normal. mRNA enzyme expression. The ratio for 5'Nu to GAPDH P restriction had no effect on the protein/DNA ratio in either the mRNA expression (Fig. 2A) was unaffected by the severity of Nx. control or uremic rats. P restriction had no effect on the mRNA ratio in either control or uremic rats. The ratio of yGT:GAPDH mRNA expression (Fig. Renal function, plasma parathyroid hormone and 2B) was only slightly reduced in the 70% Nx rats whereas it was calcium/phosphate balance markedly decreased in the 80% Nx rats (UNP vs. uNP, P < 0.01). Plasma creatinine levels increased with the severity of Nx but This ratio was not affected by P restriction in either the control or were not significantly affected by P restriction (Table 1). Creatiuremic groups. The ratio for Pase to GAPDH mRNA expression nine clearance (data not shown) was slightly reduced in uNP rats 0.1 mI/mm), fell markedly in UNP rats (Fig. 2C) tended to be lower in uNP than in CNP rats and fell (1.3 0.1 vs. 1.80 dramatically in UNP rats. P restriction did not affect the Pase/ (0.42 0.05 mI/mn) and did not improve in ULP rats (0.41 0.04 GAPDH mRNA ratio in CLP rats but induced a significant mi/mm). Other indices of renal insufficiency such as proteinuria, improvement in this ratio in U rats (ULP vs. UNP, P < 0.01). blood pressure and cholesterolemia demonstrated a similar patNaPi-2 cotransporter. NaPi-2 protein abundance (Fig. 3A) was tern (data not shown). not significantly altered in uNP rats but fell fourfold in UNP rats. Plasma PTH levels also increased with the severity of renal P restriction induced a 25% increase in protein abundance in the insufficiency. PTH levels were not affected by P restriction in CLP rats and almost normalized the protein abundance in ULP control rats but fell dramatically in uremic rats. cAMP excretion rats (ULP vs. CNP, NS). The NaPi-2/GAPDH mRNA ratio (Fig. per nephron, which is an index of renal PTH response, was 3B) was unaltered in uNP rats and was severely reduced in UNP increased in uNP as compared to CNP rats; it was at comparable rats. P restriction induced an improvement in this ratio in the levels in UNP and CNP rats. Phosphate restriction induced uremic rats, and values for this ratio in th ULP rats were almost moderate decreases in cAMP excretion in both control and restored to normal levels in ULP rats. Phosphate restriction did uremic rats. Plasma P and total and ionized Ca (Ca2) levels were at not affect the mRNA ratio in control rats. approximately normal levels in uNP rats. In contrast, plasma P Na,K-ATPase activity concentrations were elevated and total Ca and Ca2 levels were Na,K.-ATPase activity was not significantly altered in uNP rats reduced in UNP rats. The P restriction did not affect plasma P or but was significantly decreased in the UNP rats. Phosphate total Ca levels in the control rats, but induced a decrease in restriction did not modi!i activities in either the control or uremic plasma P associated with normalization of total Ca and improvement of Ca2 + levels in uremic rats. rats (Fig. 4). rats. Pase activity (Fig. 1C) was reduced in the 70% Nx group (uNP) and fell dramatically in the 80% Nx group (UNP). P restriction did not affect Pase activity in the control rats but

Epidermal growth factor production Epidermal growth factor (EGF) excretion per nephron (data

not shown) was increased in Nx as compared to control rats 0.04 pg/nephron/day) and was greater in uNP rats (4.6 0.6 pg/nephron) than in UNP rats (2.9 0.2 pg/nephron, UNP vs. (0.95

Correlations beween brush border membrane variables and plasma parathyroid hormone or phosphate concentrations The activity of yGT and Pase varied inversely and linearly with the logarithmic expression of PTH and plasma P when the values

obtained for all rats were analyzed in a single set (Table 2). uNP, P < 0.01. Fig. 5). Phosphate restriction had no effect on Weaker correlations were found between these plasma variables EGF excretion per nephron. Epidermal growth factor excretion, and the abundance of enzyme mRNA. Correlations between each expressed per g renal protein (Fig. 5), was markedly increased in of these plasma variables and protein or NaPi-2 mRNA abunuNP rats. These effects were blunted but still observable in UNP dance were always stronger. There was no significant correlation

1554

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Laouari et al: Altered tubular function in remnant kidney

Fig. 2. mRNA ratio for (A) 5'-nucleotidase, (B) '-glutamyltransferase or (C) alkaline phosphatase, to GAPDH, in normal rats (CNP), 70% Nx rats (uNP), 80% Nx rats (UNP) on normal phosphate (NP) diet and in normal

rats and 80% Nx rats on a low phosphate (LP) diet (CLP and ULP). *Uremic groups comprising 70% Nx (u) or 80% Nx (U) versus corresponding control (C) groups, P < 0.01 (**); ThNP versus UNP, P < 0.01 (f+); 5rats on LP diet versus corresponding rats on NP diet, P <0.01 ().

1555

Laouari et at: Altered tubular function in remnant kidney

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NP diet and n normal rats and 80% Nx rats on LP diet (CLP and ULP). This expression is an index of renal EGF production relative to renal protein increment. *Urcmic groups (u or U) versus corresponding control (C) groups, P < 0.05 (i), P < 0.01 (*); uNP versus UNP, P < 0.01 5rats on LP diet versus corresponding rats on NP diet, P < 0.01 (*5).

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between 5'Nu activity or mRNA and either of these two variables (data not shown).

Correlation beween NaPi-2 protein abundance and fractional excretion of phosphate

Vitamin D1 Wide individual variations in plasma 1,25(OH)2D3 levels were recorded for the small number of rats examined. Values in UNP 17 ng/liter) and ULP rats (62 12 ng!liter) were not (75

Fractional excretion of phosphate (FEe) is presented in Figure significantly different but tended to be lower than in CNP rats 7A. Both degrees of Nx (uNP and UNP) induced a significant and (112 17 nglliter).

comparable rise in FEE. Fractional excretion of phosphate fell

dramatically following P restriction in both the control and uremic rats. The fractional excretion of P showed a weak but significant inverse relationship with NaPi protein abundance (Fig. 7B) when the values obtained for all rats were analyzed in a single set.

Inter-organ specificity In the liver, the activities of 5'-Nu and of alkaline phosphatase

(Pase) did not differ between the uremic and corresponding control rats, whereas y-glutamyltransferase (yGT) activity was

1556

Laouari et al: Altered tubular function in remnant kidney

A

Nx. The activity of Na,K-ATPase was also reduced by severe Nx, and epithelial growth factor (EGF) production was increased by

8

moderate Nx. Dietary phosphate (P) restriction partly reversed the Nx-induced alterations in tubular function, although the response depended on the variable examined. The heterogeneous changes in activity and mRNA expression

*

of the three BBM enzymes studied after Nx underline the

-,-

selectivity of the response. The 5'-nucleotidase (5'Nu) activity, unlike 5'Nu mRNA abundance, was slightly reduced in both the

70% and 80% Nx groups, whereas both activity and mRNA

P

(ID

E

z

0

DNA increment

I4

CNP

uNP

UNP

CLP

ULP

2.1

4.2

7.1

2.0

5.3

expression of alkaline phosphatase (Pase) and y-glytamyltransferase (yGT) declined with increasing Nx severity (Figs. I and 2). The improvement induced by P restriction on BBM enzymes was

also selective, since no changes were observed in 5'Nu activity while the yGT activity and both Pase activity and mRNA rose. These results clearly indicate that the BBM alterations after Nx

were not due to the degradation of BBM. This statement is supported by the strong relationship observed between changes in enzyme activity and mRNA expression. This suggests that changes in enzyme activity were mainly mediated by transcriptional regulation of genes encoding for these proteins. The fact that changes in enzymatic activity were of a greater magnitude than those for

mRNA could be explained by the capacity for translation to 60

amplify transcription.

**

B

Changes in the expression of the BBM cotransporter NaPi-2 were roughly parallel to those for the enzymes. Two differences were noted: (a) NaPi-2 protein and mRNA were only reduced by severe Nx (Fig. 3); (b) NaPi-2 protein expression, unlike enzyme expression, was increased in normal rats under P restriction. This

50

E 40

** ++

0

p

30 < z

a

20

specificity of the changes, since neither S'nucleotidase (5'Na) nor yGT expressions were affected by P restriction. However, we did

not find any change in NaPi-2 mRNA expression, which is at

0

0 10

A

0

CNP

uNP

UNP

CLP

ULP

Fig. 6. (A) Renal DNA mass and (B) protein:DNA ratio in normal rats (CNP), 70% Nx (uNP) and 80% Nx rats (UNP) on NP diet and in normal rats and 80% Nx rats on LP diet (CLP and ULP). *UremiC groups (u or U) versus corresponding control (C) groups, P < 0.05 (*), P < 0.01 (**);

uNP versus UNP, P < 0.01 (); *rats on LP diet versus corresponding rats on NP diet, P < 0.01 ().

significantly

latter effect has been previously described by Levi et al, who showed that rats chronically fed a low P (0.1%) versus a high P (1.2%) diet showed a twofold increase in NaPi-2 mRNA and a fivefold increase in NaPi-2 protein associated with an increased BBM cotransport rate [11]. Their study also underlined the

higher in uremic than in control rats (Fig. 8).

variance with these authors. This may have been due to the lower level of P restriction (maximum level 0.5% vs. 1.2%) and/or the fasting protocol used in the present study. An increase in NaPi-2 protein abundance without an alteration in mRNA abundance has also been found after acute decreases in dietary P intake, and was attributed to increased insertion of the cotransporter in the apical membrane [11]. A mechanism of this type may well provide an explanation for the findings reported in the present study.

The effects of severe Nx on NaPi-2 expression were more

impressive than those of P restriction, since there was a fourfold reduction of protein abundance and a twofold reduction of NaPi-2 mRNA. NaPi-2 expression was nearly corrected by the P restriction in these animals (Fig. 3). Changes in NaPi-2 protein abundance, as for BBM enzymes, thus may be mediated by transcriptional regulation of genes encoding for this cotransporter.

Nx induced alterations in P reabsorption [determined from increased fractional excretion of P (FEe) in the two Nx groups; Fig. 7AJ as previously demonstrated [10]. Changes in NaPi-2 DISCUSSION expression were sometimes/often related to changes in FE (Fig. This study shows that proximal enzyme and transport function 7B), and when this occurred it was found that the high FE1, was and distal function are altered after subtotal nephrectomy (Nx). associated with a low NaPi-2 protein abundance. However, these The mRNA expression and/or activity of brush border membrane two parameters were poorly correlated on the whole. It could he (BBM) enzymes as well as mRNA and protein expressions of argued that FE was underestimated in Nx rats because values for BBM sodium-phosphate cotransporter (NaPi-2) were reduced by filtered P are calculated from creatinine clearance Cur, which is Phosphate restriction did not affect liver enzymes in either uremic

or control rats.

1557

Laouari et al: Altered tubular function in remnant kidney

Table 1. Role of nephrectomy (Nx) and phosphate (P) restriction on renal function, plasma PTH, urinary cAMP production and on plasma phosphorus and calcium concentrations

Group CNP uNP UNP CLP ULP

48

Urinary cAMP/d pmol/nephron

Plasma PTH ng/liter

p.mol/liter 1

68 4U

131 7"

47 2

120 5"

36.4 98.2 506.6

6.0 22.9"

8.1 16.6

0.3

86.2"'

7.6

42.3 102.7

5.2 30.7""'

5.5 4.5

Plasma total

Plasma P

0

Plasma creatinine

. Plasma ionised Ca

Ca

mmol/liter 0.07 0.06

0.9"

2.03 1.94 3.52

0.4" 0.7"

2.28 2.98

0.06 0.12""'

2.1"

2.61

2.74

0.02 0.02

2.20 0.09" 2.58 2.61

0.95

0.03 0.05 0.03"

1.05 1.05

0.03 0.04

1.15 1.09

0.02 004C'

Normal (CNP, N = 10), 70% Nx (uNP, N = 5) and 80% Nx rats (UNP, N =10) were fed the normal phosphate (NP) diet. Other control (C) rats and 80% Nx rats were fed the low protein (LP) diet: CLP (N = 10) and ULP (N = 10) groups respectively. Tonised Ca concentrations were determined for five rats per group. Significant difference between uNP or UNP and CNP rats

"P < 0.05 "P < 0.01 C

Significant difference between UNP and uNP rats, P < 0.01 "Significant difference between rats on NP diet and corresponding rats on LP diet, P < 0.01

Table 2. Correlations between renal BBM function (enzymes, NaPi-2) and plasma PTH or P concentrations

r vs. Ln PTH vs. plasma P

0.63

0.62

Enzymatic activity Pase yGT P r

<10


0.71

0.63

Enzyme-mRNA

P


P

r 0.41 0.55

Protein Na-Pi-2

Pase

yGT

iO i0-

8X

mRNA Na-Pi-2

r

P

r

P

r

P

0.60 0.57

3 x iO

0.82 0.74


0.68 0.73


i0

<10

Linear regressions were obtained by plotting the data for renal variables (all rats: CNP, uNP, UNP, CLP and ULP) against corresponding values for plasma P or the logarithmic transformation of plasma PTI-1. All correlations were negative, r is the correlation coefficient and P is the significance of the correlation. Data for 5' Nu activity and mRNA have not been given since no correlations were found.

known to overestimate GFR in renal failure. Although this effect

Interestingly, overproduction was blunted by severe Nx and was

might mask differences in FE between the 70% and 80% Nx partially restored under P restriction. groups, differences would not be as great as those for NaPi-2 The factors involved in the alterations of tubular function in protein abundance in these two groups. This suggests that NaPi-2 is only one of a number of transporters involved in P reabsorption in the whole kidney. Only severe Nx affected Na,K-ATPase activity in the present study (Fig. 4). These data concur with those of a previous study where it was demonstrated that renal Na,K-ATPase activity per tubular area was unchanged in moderate CRF [9]. A reduction in

CRF still have to be identified. A role for PTH in BBM function is suggested by the present work, since there was a good inverse correlation between changes in PTH concentrations and BBM variables (Table 2). Furthermore, parathyroidectomy (PTX) increases protein expression of NaPi-2 whereas these effects are reversed by PTH treatment [12]. However, several of our findings

tissue cells in CRF [301, and hence is not tissue-specific. Furthermore, reduced Na,K-ATPase activity in the tissues of uremic rats was not associated with changes in enzyme mRNA expression and protein abundance [311, and suggests there are post-translational events or toxic inhibitory effects due to uremia. We suggest that uremic factors played an inhibitory role in the present study, since a reduction in Na,K-ATPase activity was only observed after 80% Mx that resulted in severe CRF.

reduced by severe Nx associated with hyper-PTH, whereas only protein abundance was affected by PTH [121. This suggests that

argue against a role for PTH in BBM function in the present Na,K-ATPase activity has been reported in various organs or experiment. Firstly, both protein and NaPi-2 mRNA levels were

The renal production of EGF, a marker of distal tubular function was increased by Nx and moderate Mx in particular (Fig. 5). This contrasts with a reduction in BBM function (Figs. I to 3).

factors other than PTH were involved in NaP1-2 regulation. Secondly, the increase in NaPi-2 expression induced by P restriction in normal rats was independent of changes in PTH. (Fig. 3A).

Kilav et al have also reported an adaptative response to P restriction after PTX [33]. Thirdly, the elevation of PTH in CRF rats is associated with a down-regulation of PTH-PTHrP receptors in kidney [34, 35] as in other tissues [35]. This results in an

impaired renal response to PTH, which is characterized by unchanged or reduced basal adenyl-cyclase activity despite high

Other renal proteins may increase when BBM function is re- PTH levels [34, 36j. Our data on cAMP excretion (Table 1) would duced; Liang and Barnes have reported increased expression of therefore indicate that the PTH-mediated response of cAMP was osteopontin (a renal chemokine) associated with reduced renal not altered in rats with moderate Nx (uNP rats), impaired after Pase expression in aging rats with renal failure [32]. The increase severe Nx (UNP rats) and still impaired following P restriction in renal EGF production after moderate Nx is in agreement with since cAMP excretion was still reduced despite lowered PTH previous data showing EGF accumulation after unilateral Mx [16]. concentrations. The maintenance of impaired renal effects of

Laouan et al: Altered tubular function in remnant kidney

-o

r

C

-V

r C-)

-D

C C

C)

2 -o

2 -o

C

z-D

*

0

0 th

1

PTH after P restriction is in agreement with the findings of Urefla et al, who reported that adenyl-cyclase activity remained dimin-

ished following PTX in Nx rats [36]. It is probable that PTH-

t

-u

r

C

r

2 -o

=

-o

2

C)

C)

(0 N.)

Fig. 7. (A) Fractional excretion of P (FEe) and (B) relationship between changes in FE and changes in NaPi-2 protein in normal rats (CNP), 70% Nx (uNP) and 80% Nx rats (UNP) on NP diet and in normal rats and 80% Nx rats on LP diet (CLP and ULP). (A) *Uremic groups (ii or U) versus corresponding control (C) groups, P < 0.01 (); trats on LP diet versus corresponding rats on NP diet, P < 0.01 (a). (B) The correlation was — .08x) although values were statistically significant (r = 0.55; y = —50.01 much dispersed about the linear curve.

Liver Paso activity, mU/mg protein

a

I

0 0. 0 C)

2) aC

0 a

ID

S

0 Ft.)

-n

D)

z

a, In

r

Co

C)

0 0

100

0

p

B

.

.S .

0

U

.

.. 0 0 0

% Changes In FeP

0 0

U

Liver y-GT activity, mU/mg protein

S

F'.)

0 0

-s

b

w

C-)

2 -v

0

0

(0) '0,

0

Liver 5'-Nu activity, mU/mg protein

**

Pa P

P IS.)

0

FoP, ratio of excreted P/filtered P

P ci

**

1558

mediated effects on intracellular calcium were also impaired in Nx

rats. Although a rise in basal Ca2 levels has been reported in several tissues in CRF [371, evidence that this occurs in remnant kidneys is still lacking. Our data on cAMP excretion after P Fig. 8. (A) Activity of liver 5'-nucleotidase, (B) -glutamyltransferase and (C) alkaline phosphatase in normal rats (CNP), 70% Nx (uNP) and restriction suggest that elevations in intracellular Ca levels are 80% Nx rats (UNP) on a normal phosphate (NP) diet and in normal rats

unlikely to be reversed when PTH levels are decreased. and 80% Nx rats on a low phosphate (LP) diet (CLP and ULP). *Uremic It is postulated that changes in PTH levels merely accompany groups u or U versus corresponding C groups, P < 0.01 (**).

Laouari et al: Altered tubular function in remnant kidney

1559

and are not directly responsible for tubular alterations. Vitamin APPENDIX D3 [1,25(OH)7D3] may also play a rote in tubular alterations since 1,25(OH)2D3 has been shown to stimulate synthesis of renal Pase

Nx, subtotal nephrectomy; BBM, brush border membrane; NaPi-2,

in tubular cells [38]. However, the lack of parallel changes in sodium-potassium cotransporter; EGF, epidermal growth factor; GFR, i,25(OH)2D3 and renal Pase in our study argues against this glomerular filtration rate; PTH, parathyroid hormone; CRF, chronic renal hypothesis. The changes in plasma P levels were closely associated

failure; F, phosphate; Ca, calcium; NP, normal phosphate; LP, low

with those of BBM parameters (Table 2) and may reflect more severe intracellular changes in P concentrations. Shapiro et al have shown that renal intracellular P concentrations were increased in remnant kidneys despite normal phosphatemia and were reduced by P restriction [181. Since cellular P is involved in many metabolic processes, it may play a regulatory role in BBM

phosphate; CNP, control sham-operated rats fed a normal phosphate diet;

function. Renal compensatory growth in association with growth factors may also be implicated in the regulation of BBM enzymes since

REFERENCES

changes in hyperplasia (Fig. 6A) were closely associated with those for BBM enzymes. In addition, detrimental alterations in BBM enzymes were induced by Nx and improved following P restriction in the kidney but not in the liver (Fig. 8). Furthermore, the effects of P restriction on BBM enzymes were seen in remnant

but not in normal kidneys (Figs. I and 2), despite a reduction in intracellular P content in both cases as previously described [18]. Finally, the activity of Pase in renal tubular cells was found to be regulated by growth factors, that is, stimulated by TGFI3 and inhibited by EGF [39]. It is probable that growth factors do not play a crucial role in the regulation of NaPi-2 expression, unlike BBM enzyme expression, since changes in NaPi-2 after P restriction occurred in the normal kidney (Fig. 3A and [111). The small elevation in plasma creatinine observed following moderate Nx (Table I) suggested that alterations in BBM enzymes occurred when glomerular function was little impaired. In addition, BBM function improved under P restriction whereas no changes in glomerular function were observed. This suggests that renal BBM enzyme dysfunction is an early event of renal deterioration. In conclusion, subtotal nephrectomy resulted in profound alterations in BBM tubular function. These abnormalities cannot be accounted for by a single mechanism. The partial reversal of alterations by P restriction suggests that intracellular P may be implicated in these modifications. In contrast, it is unlikely that PTH is involved due to the development of renal PTH resistance in CRF. The specificity of the renal BBM enzyme response in hypertrophic kidney suggests that growth factors may also play complementary roles in the regulation of these enzymes. It is possible that BBM dysfunction may occur before morphological

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pour Ia Recherche Médicale and Laboratoires de Recherche Physiologiques. We thank Merck & Co., Inc. for providing the cDNA for rat alkaline phosphatase analyses, and are grateful to Dr. Biber for providing the NaPi-2 antibodies. We would also like to thank B. Escoubet and D. Prié for helping prepare the probes. A part of this work was presented at the 28th meeting of The American Society of Nephrology, San Diego, 1995.

uNP, 70% nephrectomized uremic rats; UNP, 80% nephrectomized uremic rats; ULP, 80% nephrectomized uremic rats fed a low phosphate diet; CLP, control animals fed a low phosphate diet; N, nitrogen; 5'-Nu, 5'-nucleotidase; y-GT, y-glutamyltransferase; Pase, alkaline phosphatase; Ccr, creatinine clearance; FEE, fractional excretion of phosphate.

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