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BASIC SCIENCE |
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*Department of Anatomy, Charité, University Medical School of Berlin, and Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany; and Departments of Physiology and Anatomy, University of Zürich, Switzerland.
Correspondence to: Prof. Dr. Sebastian Bachmann, AG Anatomie der Charité, Campus Mitte, Philippstr. 12, D-10098 Berlin, Germany. Phone: +49-30-450-528-001; Fax: +49-30-450-528-922; E-mail: sbachm{at}charite.de
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Introduction |
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At the molecular level, the regulation of NaPi-IIa is presently under active investigation. Open questions refer to the mechanisms of transmitting signals for retrieval and intracellular trafficking of the transporter (11). Insights came from patient data on Fanconi type syndromes and from knockout mouse models with gene defects interfering with endocytosis, calciotropic hormones, and the handling of various ligands to proximal tubule receptors (9,10). A knockout of the clcn5 gene encoding the vesicular chloride channel ClC-5 produced characteristics of Dent disease, which are based on a defective acidification of endosomes and an impaired vesicle trafficking (9,12). In this model, hyperphosphaturia was described along with a concentration of NaPi-IIa in the apical endocytic compartment. It was assumed that these impairments were related to elevated urinary and hence luminal PTH concentrations. In line with this hypothesis, the amount of megalin, an endocytic multiligand receptor that also degrades PTH from the proximal tubule filtrate (10,13), was downregulated in this model (9,14). It was argued, therefore, that higher PTH availability was related to the downregulation of NaPi-IIa in steady state (9). Experimentally induced retrieval of NaPi-IIa was observed in these mice nevertheless, although at reduced efficiency. The role of receptor-associated protein (RAP), which has been identified as a chaperone-like protein interfering with megalin and related receptors, has further been discussed in this context. The RAP knockout mouse showed a retardation in PTH-induced effect that may be related to an altered distribution of megalin (15,16). An interaction of megalin with a membrane protein of the proximal tubule has further been reported (17).
The rationale of the study presented here was to investigate the role of megalin in PTH-dependent inactivation of NaPi-IIa by the use of mice with a kidney-specific megalin defect induced by conditional gene targeting. These mice have been characterized in a previous study of our group (18). The renal inactivation of megalin permits the parallel study of NaPi-IIa in megalin-expressing and megalin-deficient proximal tubule cells in response to the hormonal stimulus. We have addressed the question whether the absence of the receptor interferes with the kidney’s ability to regulate its major NaPi cotransporter. Related changes in the endocytic capacity of the proximal tubule were analyzed in detail.
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Materials and Methods |
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To study the effect of PTH, mice were divided into two groups of Cre(+) or Cre(-) mice (n = 20 each), each receiving either an intraperitoneal injection of vehicle (0.9% NaCl) or PTH (1-34) fragment (Sigma; 1 µg per 10g body mass) 30 min before sacrifice, as described (6). Each group was further divided into two subgroups, each composed of n = 10, which after treatment either served for biochemical analysis, or for perfusion-fixation and histochemical analysis. Before sacrifice mice were anesthetized with sodium pentobarbital. To study fluid-phase endocytosis, horseradish peroxidase (HRP; Sigma; 120 µg/g body weight) was given to mice (each n = 3) under anesthesia via laparotomy and intravenous injection between 10 and 30 min before perfusion-fixation.
Kidney Slices
For slice preparation, kidneys from untreated Cre(+) and Cre(-) mice were prepared and slices incubated as described (16). One-millimeter-thick kidney slices were left untreated for control, or treated with PTH (1-34) fragment, 8-Br-cAMP, 8-Br-cGMP, or 1,2-dioctanoyl-sn-glycerol (DOG). During the course of the experiment, all solutions were gassed with 5% CO2/ 95% O2, and the pH was kept constant at pH 7.4 ± 0.2. Experiments were performed at least in double each with two kidneys used from two different mice. Untreated and PTH-treated slices were used in all experiments as internal controls.
Semiquantitative RT-PCR
Kidneys were rapidly dissected, homogenized, and lysed, and total RNA was prepared with the RNeasy-total-RNA kit (Quiagen). After digestion of genomic DNA by treatment with DNase, cDNA was synthesized by reverse transcription of 5 µg total RNA, with a cDNA synthesis kit (Invitrogen). cDNA fragments between bases 1276 to 1576 (GenBank accession no. L33878) for NaPi-IIa and from base 742 to 1047 (no. M17701) for GAPDH were amplified. The following sets of oligonucleotide primers were used: NaPi-IIa 5'-gTC CAg AgC AgC TCC gTg TT-3' and 5'-CAg CAA ACC AgC ggT ACT Tg-3' (300 bp); GAPDH: 5'-TAT CCg TTg Tgg ATC TgA C-3' and 5'-Tgg TCC Agg ggT TTC TTA C-3' (304 bp). Amplification was done with Taq polymerase (Life Technologies) over 23 to 35 cycles with an automated thermal cycler (Perkin-Elmer). Each cycle comprised denaturation at 94°C, annealing at 60°C, and extension at 72°C for 1 min each. The number of cycles was adapted for both products to obtain linear correlation between signal intensities of RT-PCR product and cDNA concentration. PCR products were analyzed by agarose gel electrophoresis and stained with ethidium bromide.
BBM Preparation
After removal, kidneys were shock-frozen and processed as described (6,19). In brief, kidneys were homogenized and BBM fractions prepared (19). Purity of the preparation was checked by comparing the activity of the BBM marker enzymes, 
-glutamyltransferase [-GT; (20)] and alkaline phosphatase [AP; (21)] in BBM fractions versus total homogenate. -GT and AP kits (-glutamyl transpeptidase [GGT] and ALP IFCC liquid; Roche) were used for determination. Total protein concentration was measured with a BCA kit (Pierce).
Western Blot Tests
Samples of the total homogenates and BBM fractions were electrophoresed under reducing conditions on 10% SDS polyacrylamide gels. Each lane was loaded with 40 µg of protein, and the proteins were electrotransferred onto nitrocellulose membranes. Equal loading was controlled by use of Ponceau red stain. Membranes were blocked and incubated with primary polyclonal rabbit anti-rat NaPi-IIa antibody [dilutions 1:500 to 1:4,000; (2)] and secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako; 1:2,000 dilution), followed by chemiluminescence reagent (ECL, Amersham). The resulting signals were densitometrically analyzed with a scanner and Bio-Profil Bio1D software. Results were summed intensity values; controls were set to 100% as a mean internal standard, and experimental levels were compared with standard.
Histochemistry of Perfusion-Fixed Mice
Kidneys were perfusion-fixed by retrograde cannulation of the abdominal aorta with 3% paraformaldehyde as described (22). Immunolabeling was performed on cryostat or paraffin sections of 5 µm thickness, blocked with 5% milk powder in PBS, incubated for 2 h with primary antibodies (sheep anti-megalin [1:2,500 dilution (15)] and rabbit anti–NaPi-IIa [1:400 dilution]), followed by secondary antibodies (Cy3-coupled donkey-anti sheep IgG and Cy2-coupled swine-anti rabbit IgG [Dianova]). Specificity of the double-staining procedures was controlled by parallel incubation of consecutive sections, each incubated only with one single probe. For HRP histochemistry, cryostat sections were incubated with diaminobenzidine added with H2O2. Controlling for general BBM antibody staining intensity we have tested antibodies against sodium sulfate cotransporter (NaSi-1) and sodium hydrogen exchanger-3 (NHE-3) as described (16).
Histochemistry of Slices
After incubation, slices were transferred to fixative (16), mounted onto cork plates, and frozen. Cryostat sections were then incubated with NaPi-IIa antibody (dilution 1:1500) followed by swine anti-rabbit IgG conjugated to FITC (Dako). For double staining of NaPi-IIa and 
-actin filaments, rhodamine-phalloidin was used (Molecular Probes; dilution 1:50). Reagents were added as previously described (16).
Ultrastructural Analysis
Conventional electron microscopy (EM) was performed on perfusion-fixed tissue embedded in Epon 812 (Serva). For immuno-EM, small tissue samples were cryo-substituted in a freeze-substitution unit (Leica) and infiltrated with London LR-White hydrophilic resin (Science Services). Ultrathin sections were incubated with anti–NaPi-IIa (1:300 dilution) or anti-megalin antibody (1:5,000 dilution) followed by 5 nm gold-labeled goat anti-rabbit IgG (Amersham) or 12 nm gold-labeled donkey anti-sheep IgG (Dianova), respectively, and subsequently silver impregnated (AuroProbe, Amersham). For morphometry, EM pictures of the luminal cell pole and BBM were taken at random from PTH or vehicle treated Cre(+) mice (n = 3 each) to evaluate immunogold staining intensity in the respective compartment. A total of 96 micrographs, each containing a single cell from proximal S1/S2 segments with versus without remnant megalin expression as identified by the presence or absence of DAT, was evaluated.
In Situ Hybridization
Digoxigenin (DIG)-11-UTP–labeled riboprobes were synthesized by in vitro transcription (DIG RNA labeling kit [Sp6/T7]; Roche) with a full-length mouse NaPi-IIa cDNA in the pSport 1 vector (Invitrogen). After linearization with NotI or SalI, sense or antisense riboprobes were generated with T7 and SP6 RNA polymerases, respectively, as described (22). Seven-micron-thick paraffin sections from perfusion-fixed tissue were treated according to established methods (23). Signal was generated with 4-nitroblue tetrazolium chloride. For control, sense probes were applied in parallel with antisense probes. For double staining with anti-megalin antibody and fluorescence-labeled secondary antibody, incubations were started thereafter and performed as detailed above.
Statistical Analyses
Biochemical and morphometric values are means ± SEM. Statistical significance of differences was evaluated by the Wilcoxon signed rank test, Mann-Whitney Wilcoxon test, and unpaired t test, as appropriate.
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Results |
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). Signal was high in the inner cortical labyrinth, weaker in midcortical and superficial convolutions, and absent from the straight segments. Similar results were obtained by in situ hybridization for NaPi-IIa mRNA. In contrast, concomitant immunostaining for megalin invariably labeled all parts of the proximal tubule with no evidence for regional heterogeneity (Figure 1, a and a'). The kidney-specific inactivation of megalin in Cre(+) mice was incomplete, probably owing to a variable efficiency of the Cre recombinase (18), which resulted in a remnant expression of megalin in approximately 10% to 25% of the nephrons (Figure 1, b and b'). Individual nephrons were showing half megalin-positive, and half negative profiles of the proximal tubule (Figure 4, b and b', and Figure 5a). NaPi-IIa staining in Cre(+) was concentrated in the same areas of the cortex as in Cre(-); however, staining was also detectable in mid- and outer cortical proximal convolutions, and BBM signal was particularly strong throughout, whereas intracellular labeling was weaker than in controls. This was also reflected by the quantitative evaluation of Western blots showing a 69.5% higher abundance of NaPi-IIa in BBM of Cre(+) compared with Cre(-) in steady state, and extracts from total kidney homogenate showed increases as well (Figure 6). Significantly decreased urinary phosphate excretion (-53.5%) in Cre(+) further supported the enhanced availability of the cotransporter at its site of action (Figure 7b). By contrast, strength and distribution of NaPi-IIa mRNA expression were apparently independent of the presence of megalin, because these were similar in both strains as studied by in situ hybridization and semi-quantitative RT-PCR evaluation, and sites with remnant megalin expression showed the same NaPi-IIa mRNA signal as megalin-deficient tubules in comparable position (Figure 2, a and a', and Figure 7a). The immunoreactive signal intensity of other transporters of BBM such as NaSi-1 and NHE-3 was unaltered as well (Figure 8).
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To test the effect of an impaired endocytosis on the trafficking and inactivation of NaPi-IIa in proximal tubule, PTH was administered intraperitoneally for 30 min and kidneys were evaluated by immunohistochemistry and Western blot test. In controls the expected internalization of NaPi-IIa immunoreactive signal into the subapical compartment of the proximal tubule with concomitant reduction in BBM signal intensity was observed in agreement with previous results (24) (Figure 3, a and b'). This effect was undetectable in tubules lacking megalin expression (Figure 3, c and d'). Profiles with partial remnant expression confirmed this result revealing that megalin signal and NaPi-IIa retrieval were interrupted coordinately (Figure 3, d and d'). Ultrastructural analysis that used immunogold staining extended this finding showing that an increase in intracellular NaPi-IIa signal, indicative of a retrieval of the cotransporter into the subapical compartment, was only found in cells with intact DAT (Figure 5, b and c). Morphometry confirmed these observations (Table 1). Our results thus suggest a cell-autonomous defect of PTH-induced NaPi-IIa internalization that depends on the presence of megalin, rather than on systemically mediated changes.
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To examine if changes in the coupling of PTH receptors to previously identified intracellular signaling cascades could be responsible for the observed differences in PTH-induced NaPi-IIa internalization (11), we stimulated these cascades directly. Incubation of kidney slices from Cre(-) mice with the protein kinase C pathway activator DOG (1 µM), 8-Br-cAMP (1 mM), or 8-Br-cGMP (1 mM) led to internalization of NaPi-IIa from the brush border after 45 min as demonstrated earlier (16). However, no internalization occurred after 45 min in kidney slices from Cre(+) mice suggesting that the defect lies not in coupling of PTH receptors to signaling pathways but in a common mechanism for all endocytic signals (Figure 9, a through e').
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Discussion |
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A typical membrane protein like NaPi-IIa may not per se be viewed as a conventional ligand for megalin, and in fact, immunoprecipitation studies failed to show interaction between the two molecules (17). On the other hand, NaPi-IIa requires a broad spectrum of protein interaction for adequate trafficking (3), and its inactivation has been shown to depend on endocytosis, which suggests a role for megalin (24).
Analyzing the defects in NaPi-IIa regulation caused by the genetic manipulation in Cre(+) mice, cell-autonomous effects are to be distinguished from global systemic effects induced by the genetic manipulation. At steady state, a number of renal changes have resulted from the inactivation of the megalin gene. There was moderate, but significant remnant expression of megalin, which permitted the parallel study of megalin-deficient versus intact nephrons within one sample, and may functionally stand for some compensatory capacity protecting against physiologic imbalance unless additional effects were superimposed, as shown previously for the vitamin D metabolism (18). Accordingly, serum calcium, Pi and PTH levels were not significantly altered at steady state. Still, the absence of megalin resulted in a severe global reabsorption deficiency and low molecular weight proteinuria as in the full knockout mouse, and reduced phosphaturia (25,26). Together, the systemic parameters in Cre(+) nonetheless do not suggest direct influences on the observed defects in NaPi-IIa regulation.
Among the principal factors indicating cell-autonomous impairment of NaPi-IIa regulation in Cre(+), the obvious structural alteration of the proximal tubule cell and the resulting effect on the reabsorption of proteins must be considered. In fact, the number of luminal invaginations and endocytic vesicles were substantially reduced in Cre(+) mice, and as a particular structure, the DAT representing a variable membrane pool between endosomes and recycling vesicles (8) were completely absent from proximal tubule cells lacking megalin. HRP, the marker for fluid phase endocytosis that codistributes with NaPi-IIa during PTH-induced inactivation of the transporter (24), was strikingly absent from megalin-deficient cells. This implies that not only the ligand-specific uptake of proteins (10), but also the general, nonspecific reabsorption from the filtrate was disrupted. The reduction of the vacuolar apparatus may be causally related with reduced protein reabsorption, as previously suggested (7), and a consequent impairment of NaPi-IIa trafficking appears plausible. The comparable phenotype of the chloride channel (ClC-5)-deficient mouse resembles the Cre(+) model with respect to proteinuria, downregulation of megalin, impairment of receptor-mediated and fluid-phase endocytosis, and shows reduced internalization of NaPi-IIa (9); however, these changes are not associated with obvious structural alterations, and they are more gradual than in Cre(+), even though they seem to indirectly depend on megalin availability as well (14).
The enhanced concentration of immunoreactive NaPi-IIa in BBM and more widespread distribution of the signal in Cre(+) during steady state condition, typically associated with preserved mRNA levels (3), may thus result from an impairment of endocytic removal that would otherwise be active to regulate the constitutive turnover of the transporter. Surprisingly, the localization of NaPi-IIa in the ClC-5 knockout showed a conspicuous redistribution from the BBM toward the endosomal compartment in steady state. It has been argued that a defect in megalin-dependent inactivation of luminally bound PTH leading to inadequate stimulation of PTH receptors could have caused this effect; this interpretation was underlined by the moderate increase in urinary PTH and phosphaturia (9,13). Because excreted PTH levels in Cre(+) were much higher than in ClC-5 knockouts, whereas BBM NaPi-IIa expression and phosphaturia were changed in opposite direction, the profound alterations of the endocytic apparatus in Cre(+) would explain this discrepancy. In addition, the substantial intracellular alterations of the proximal tubule in Cre(+) mice may also have an effect on mechanisms controlling NaPi-IIa stability, because lysosomes were less frequent than in controls.
In agreement with these considerations, the failure to internalize NaPi-IIa in Cre(+) mice in response to PTH shown in the present in vitro and in vivo material is clearly dependent on megalin deficiency, as visualized at the cell level and underlined by ultrastructural and biochemical techniques. Comparison with the ClC-5 knockout mouse further specifies the need for intact megalin-dependent endocytosis to inactivate NaPi-IIa upon the application of PTH; in fact retrieval of NaPi-IIa from a stimulated baseline condition was similarly impaired as in the present model (9). The application of pharmacologic agents mimicking the effects of a signaling cascade triggered by the PTH receptor to inactivate NaPi-IIa failed to induce changes as well, so that obviously these pathways, which involve adenylate cyclase- and phospholipase C–generated cAMP, IP3, DAG, and a rise in cellular Ca2+ (11) were inefficient in the absence of megalin. Similar observations were made in the RAP knockout mouse with reduced megalin availability (16).
Considering a specific, direct relation between NaPi-IIa and megalin beyond general endocytosis mechanisms that could be relevant for internalization of the transporter, one may assume at least some protein-protein interaction should exist between the two gene products. However, no interaction has been demonstrated by several biochemical techniques up to date. Yet the manyfold and complexity of cytoplasmic adapter proteins, some of which have been defined for each, megalin and NaPi-IIa [for review, (10,27)], provide so far unrecognized means of an interaction that may support a more direct relation between megalin and NaPi-IIa during inactivation of the cotransporter. A relation between megalin deficiency and the expressional pattern of other local transporters such as NaSi-1 and NHE-3 was not evident from steady state analysis, as presented here. The question as to the acute regulation of NHE-3 by PTH and a possible involvement of megalin was not tested, because in the past we had not been able to observe an acute internalization of NHE-3 from the BBM in various comparable mouse models (unpublished observations).
We therefore suggest that proximal tubular NaPi-IIa handling is severely affected in a mouse model with kidney-specific inactivation of the megalin gene. Megalin deficiency induces an ultrastructural anomaly of the endocytic apparatus and interferes with fluid-phase endocytosis, resulting in an enhanced BBM insertion of NaPi-IIa, reduced phosphaturia, and an impaired PTH-induced retrieval of the cotransporter. The profound disruption of endocytosis appears to be the predominant cause for NaPi-IIa dysfunction, although further, as yet unknown interactions of the cotransporter and megalin may matter as well.
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Acknowledgments |
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