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*
Department of Anatomy and Medical Research Center, Klinikum Mannheim,
University of Heidelberg, Germany
Department of Physiology, University of Zurich, Switzerland
Department of Anatomy, Charité, Humboldt
University, Berlin, Germany.
Correspondence to Prof. Dr. S. Bachmann, Campus Virchow Klinikum, A.G. Anatomie der Charite, Elektronenmikroskopie, Augustenburger Platz 1, 13353 Berlin, Germany. Phone: +49-30-450-56501; Fax: +49-30-450-62922; E-mail: sbachm{at}charite.de
Abstract
Abstract. Renal phosphate (Pi) absorption is mediated via the type II sodium/Pi cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules. Simultaneous detection of NaPi-2 mRNA by in situ hybridization and of NaPi-2 immunoreactivity by immunohistochemistry was performed to investigate the distribution of the cotransporter in healthy control rats and during progression of autosomal dominant polycystic kidney disease (ADPKD). The purpose of the study was to disclose a relation between proximal tubular cell differentiation and NaPi-2 expression. In controls, NaPi-2 expression was present in the entire proximal tubule. In the Han:SPRD (cy/+) model for ADPKD, the proximal nephron is primarily affected by the cystic changes. Epithelial proliferation and impaired epithelial-matrix interaction result in a loss of cell differentiation that eventually leads to cystic enlargement of the nephron. Normal expression of NaPi-2 in this model was found only in tubules with intact BBM. Loss of BBM and cellular interdigitation were paralleled by the loss of NaPi-2 in situ hybridization and immunoreactive signals. These changes were moderate and focal in 2-mo-old rats and generalized all over the cortex after 8 mo. Advanced renal damage in the older PKD group was associated with mild phosphaturia, which suggests functional insufficiency of tubular NaPi-2 reabsorption. These data show how proliferative changes and loss of tubular epithelial differentiation in ADPKD may prevent functional expression of the NaPi-2 system in the proximal tubule in a rapidly progressive manner. NaPi-2 in proximal tubule BBM is suggested to play an important role in impaired tubular absorption of Pi in renal disease.
The Han:SPRD (cy/+) rat model for autosomal dominant polycystic kidney disease (ADPKD) produces cystic changes of the nephron that are similar to those that occur in human PKD. Pathologic changes initially affect the proximal tubule and involve the typical features of cystic epithelial transformation, such as increased cell proliferation, loss of differentiation, and a final arrest in a dedifferentiated, simplified cellular morphology (1,2). The model is particularly suitable for the study of transitory stages between intact and progressively cystic tubules. In advanced stages, the Han:SPRD rat shows signs of renal osteopathy, which suggests an impairment of sodium/phosphate homeostasis and secondary hyperparathyroidism (3). The major renal Na/Pi cotransport system (type II Na/Pi cotransporter [NaPi-2]) is located in the brush border membrane (BBM), the Golgi apparatus, and lysosomes of the proximal tubule (4,5,6). Renal Pi reabsorption is tightly regulated by Pi intake (7), acid/base metabolism (8), parathyroid (PTH) and glucocorticoid hormones (9), and genetic abnormalities (10), all of which have been shown to influence Pi transport; the resulting changes were related to NaPi-2 protein and/or mRNA abundances. Animal models for genetically altered Pi handling, such as the Hyp mouse model for X-linked hypophosphatemic rickets, show reduced NaPi-2 expression in the BBM (11). Impaired Pi reabsorption and reduced NaPi-2 protein abundance can also be due to an age-related problem (12). In the present study, we investigated the mRNA expression and immunoreactivity (IR) of NaPi-2 in relation to the gradual changes in proximal tubular differentiation as they occur in rat ADPKD. Clinical parameters were evaluated to monitor renal functional impairment and Pi balance.
Materials and Methods
Rats from the Han:SPRD strain exhibiting PKD were bred in the local animal facility as described (Klinikum Mannheim, FRG (1). Animals were maintained and treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the German Law on the Protection of Animals. Heterozygously affected males with ADPKD (cy/+) were checked for renal cysts by abdominal palpation. Heterozygously unaffected, age-matched littermates (-/-) were used as controls. Rats had free access to tap water and standard rat chow containing 19% protein (Altromin, Lage, Germany). For morphologic and histochemical analysis, 2- and 8-mo-old heterozygously affected rats and control animals of corresponding age were chosen. For complementary information on the progression of the disease, clinical parameters were determined in 2-, 6-, and 8 mo-old rats of both categories (total n = 63). For determination of serum (S) creatinine (Cr), urea, phosphate, and parathyroid hormone (PTH) levels and of urinary Cr, sodium, potassium, and protein levels, eight rats from each group were used. Blood samples were drawn from the retroorbital venous plexus under light ether anesthesia. To obtain urinary (U) parameters, rats were placed in metabolic cages, and after an accustoming period, samples were collected over 24 h. Parameters were determined in the Department of Clinical Chemistry of the University of Heidelberg according to standard methodology (12). Fractional excretion (FE) of Pi and Na+ was calculated as (UPi,Na/SPi,Na)/(UCr/SCr).
To obtain tissue for morphology, six animals from each group of animals were perfusion-fixed as described (1). Kidneys were removed and snap-frozen in liquid nitrogen-cooled isopentane. Messenger RNA expression for NaPi-2 was investigated by in situ hybridization (ISH) using nonisotopic riboprobes made from rat NaPi-2 cDNA (13). To generate hybridization probes, a full-length fragment of rat NaPi-2 cDNA was used; NotI and T7 polymerases were used to obtain sense probe, and SalI and SP6 polymerases were used to obtain antisense probe, respectively. Incorporation of digoxigenin-labeled UTP (Roche Diagnostics, Mannheim, Germany) by in vitro transcription and hybridization steps was carried out as described (1,5). After the washing procedure, sections were incubated with an alkaline phosphatase-detection system (Roche). Each run of hybridization comprised several animals from each group, and color development was interrupted at submaximal intensity levels in a standardized manner. For concomitant immunohistochemical labeling, slides were subsequently incubated with a rabbit anti-rat NaPi-2 antibody (dilution 1:100 in phosphate-buffered saline [PBS]/1% BSA) for 2 h at room temperature followed by overnight incubation at 4°C. After rinsing in PBS, bound antibody was labeled by biotinylated anti-rabbit IgG second antibody (Dako, Glostrup, Denmark; 1:50 in PBS/BSA) and streptavidin-Texas red fluorophore (Amersham Buchler, Braunschweig, Germany; 1:100 in PBS). After additional rinsing, slides were coverslipped and studied by combined epifluorescence and bright field microscopy using a Leica DMRB fluorescence microscope supplemented with interference contrast optics.
Statistical Analyses
Statistical differences between normal and PKD rats were evaluated using
one-way analysis of variance, and, when allowed by the F value,
results were compared by the two-tailed unpaired t test. For
comparison of the values, we used the means along with the range of the tested
parameters. Significance was accepted at P < 0.05.
Results
Clinical parameters indicated moderate renal damage in the young and advanced damage in the older PKD groups. Plasma creatinine levels were not significantly different in the young rats but increased in the older PKD rats compared with the respective controls (+58%; P < 0.005); likewise, plasma urea levels were increased by 59% (P < 0.005). Proteinuria was increased 4.7-fold only in the older PKD group (P < 0.005). The older PKD rats further showed significant diuresis (enhanced 4-fold; P < 0.005), natriuresis (enhanced 4.4-fold; P < 0.005) and kaliuresis (enhanced 2.5-fold; P < 0.005) compared with the respective controls. Plasma PTH values were statistically indifferent, although individual animals of the older PKD group showed markedly elevated levels. Compared with controls, plasma Pi levels were moderately decreased by 14% (P < 0.05) in the older PKD rats, and phosphaturia was increased 2.3-fold (P < 0.005). The FE of Pi was 1.1 ± 0.9% in the young controls and 2.1 ± 1.4% in the young PKD rats (NS) and was 2.45 ± 0.9% in the aged controls and 12.8 ± 4.7% in the aged PKD rats (P < 0.005). For comparison, the FE of sodium was 1.5 ± 0.2% in the young controls and 2.2 ± 0.2% in the young PKD rats (P < 0.05) and was 1.7 ± 0.2% in the older controls and 3.3 ± 0.2% in the older PKD rats (P < 0.005).
In healthy controls of both age groups, NaPi-2 mRNA expression as revealed by ISH, and NaPi-2 IR were detected in the entire proximal tubule. No other tubular segments were labeled. In the cortex, microanatomical distribution of both NaPi-2 ISH and IR signals was inhomogeneous with the strongest signal intensity located in juxtamedullary tubules of the cortical labyrinth, whereas tubules of the outer cortical region were less intensively labeled (Figure 1). As revealed by double-labeling histochemistry, differences were also observed with regard to the signal intensity in immunoreactive versus ISH-labeled tubular profiles. Proximal straight tubules of the medullary rays showed strong ISH label but weak IR (Figure 1). In the outer stripe of the outer medulla, NaPi-2 IR was enhanced in proximal straight tubules that derived from juxtamedullary nephrons but not in those that came from more superficially located nephrons; by contrast, NaPi-2 mRNA signal was faint throughout the outer stripe (Figures 1 and 2, B and B'). The onset of both NaPi-2 ISH and IR signals coincided sharply with the onset of the proximal tubule epithelium at the urinary pole (Figure 2, A and A'). The end of both signals was at the transition of the outer medullary straight part of the proximal tubule to the thin limb segment (Figure 2, B and B'). Signal for NaPi-2 mRNA was localized in the entire cytoplasm of the proximal tubule cell, whereas IR generally was restricted to the BBM and to few intracellular structures likely to correspond to the subapical vesicular compartment and lysosomes. No principal differences were encountered in the distribution or intensity of both signals when comparing young and aged control rats.
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In 2-mo-old PKD rats, NaPi-2 mRNA and IR were normal in numerous nephrons with intact or only moderately dilated proximal tubular segments (Figure 3, A and A'). Cystically altered profiles, however, showed marked changes of cotransporter expression. According to a previously established classification of cyst types (1,2), plaque-like focal sites of less-differentiated proximal tubular epithelium with concomitant loss of BBM and greatly increased cell proliferation rate represent the initial stages of cystic alteration, and these sites were lacking or showing substantially reduced NaPi-2 IR and ISH signal wherever the cells had lost their BBM (Figures 2, D and D', and 3, A and A'). Transitions from still intact portions of tubular epithelia to the widely dilated, NaPi-2-negative cystic epithelia were usually sharp (Figure 2, C and C'). Larger portions of cystically dilated proximal tubular epithelia (also typed "abnormal" epithelia (2)) revealed more extensive loss of BBM and concomitant diminution in NaPi-2 signal. Wherever the BBM was lost, IR was almost abolished; however, remnants of apical fluorescence occurred in epithelia lacking their brush border but otherwise showing undiminished mRNA signal (Figure 2, E and E'). This remnant NaPi-2 IR was apparently located in the (still preserved) apical vesicular or endocytotic compartment. Larger cysts lined by atrophic epithelium were completely deficient of NaPi-2 signals (Figure 3). A less frequently occurring type of cystically altered proximal segments with a hypertrophic/hyperplastic phenotype characterized by densely packed cells, thickened epithelium and lack of BBM (1,2) expressed NaPi-2 mRNA to the maximum extent. Unexpectedly, NaPi-2 IR was present as a significant apical and lateral fluorescence margin, even though cell differentiation in these epithelia was markedly reduced as compared with the original phenotype (Figure 2, G and G').
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Microanatomically, the observed nephron changes were moderate and focal in 2-mo-old rat kidneys but generalized all over the cortex in the 8-mo-old (Figure 3). Midcortical and juxtamedullary nephrons were affected before superficial nephrons were. Kidneys from the older rats were characterized by the markedly advanced loss of intact nephrons and the formation of larger cysts, and these changes were accompanied by manifest reductions in NaPi-2 ISH and IR signal density.
Coincident with cystic epithelial changes, interstitial fibrosis developed progressively with age. The extent of peritubular fibrotic deposits was not necessarily correlated with altered NaPi-2 expression, because in highly fibrotic areas of the 8-mo-old PKD kidneys, single scattered profiles of proximal tubule revealed normal NaPi signals (Figure 2, F and F').
Discussion
The present results demonstrate the micromorphologic distribution of the inorganic phosphate cotransporter NaPi-2 in healthy and polycystic rat kidney using combined immunohistochemistry and ISH. The regional heterogeneity and inter- and intranephron variability of NaPi-2 IR and mRNA signals may be related to enhanced Pi reabsorption in the juxtamedullary convolutions (14), locally distinct hormonal or metabolic control of NaPi-2 synthesis (4, 15, 16), and differences in processing of the gene product (17). By contrast, studies of rats receiving a low- versus high-phosphate diet generally have shown parallel signal intensity of NaPi-2 IR and mRNA expression (4), which could reflect an effect of dietary extremes to synchronize transcription and translation efficiency. In contrast to a previous study (12), there was no evidence of agerelated differences in NaPi-2 expression between the control groups.
In the PKD groups, morphologic changes in the proximal tubule characterized previously by the manifestation of a loss of BBM, reduced basolateral interdigitation, increased cell proliferation (2), and an increased expression of the basement membrane compound collagen IV (1) were accompanied by a simultaneous decrease of NaPi-2 mRNA and IR. Alterations in circulating levels of PTH have previously been shown to suppress NaPi-2 IR (18). However, in the present study, PTH was only moderately elevated in the PKD groups. The arrest of the epithelial cells in a less differentiated state is an essential feature in PKD (19). Thus, impaired cellular differentiation and degraded luminal membrane specialization may have interfered with transcription and/or processing of NaPi-2 mRNA. It is possible that an impairment in exocytotic fusion events for NaPi-2 caused by the changes in the luminal membrane exerts a negative feedback on transcriptional events. Because the first signs of NaPi-2 synthesis during ontogeny depend on the formation of BBM (5), it is likely that consequent to the loss of cellular differentiation in PKD a state of regression that resembles the embryonic phenotype of proximal tubule epithelium with lacking NaPi-2 synthesis is reached. Regression preferentially began in the midcortical and juxtamedullary nephrons, which previously were shown to be the principal site for regulatory adaptations of NaPi-2 (6).
The hypertrophic/hyperplastic type of cyst epithelium showed an exceptional phenotype with strongly enhanced NaPi-2 mRNA levels and, even though a BBM was absent from these epithelia (1), intensive NaPi-2 IR at the apical cell pole probably accumulating in apical lysosomes (6). These epithelia also showed the highest levels of proliferation activity (2), but instead of forming large cysts, their diameter was rarely widened. According to their microanatomical position, these portions were derived from the proximal straight tubules. Because of the relatively rare occurrence, the potential functional impact of this pathologically altered epithelium should, however, be considered secondary in nature.
Progressive reduction in NaPi-2 expression along with proteinuria and altered plasma urea and creatinine levels indicates progressive renal damage; however, the phosphate metabolism was not fundamentally impaired in the PKD groups, even though severe renal osteopathy associated with secondary hyperparathyroidism was previously reported from Han:SPRD (cy/+) rats of higher age (3). By contrast, the older PKD rats of our study presented increased phosphaturia with no significant elevation of PTH levels; it therefore seems likely that at this age, filtration was still sufficient to prevent phosphate accumulation and that enhanced phosphaturia was due to insufficient reabsorption of the damaged proximal tubular segments.
In summary, the present results simultaneously show the distribution of NaPi-2 ISH and IR in rat kidney proximal tubule under normal conditions and during pathophysiologic changes that occur in Han:SPRD (cy/+) rats with ADPKD. Proliferative changes and structural dedifferentiation of the cystically affected nephrons provoke a loss of NaPi-2 synthesis that is markedly progressive with age. Clinical parameters indicate that during progression of the disease, impaired NaPi-2 expression can result in phosphate malabsorption and phosphaturia before attaining end-stage renal disease, which is likely to end up in an accumulation rather than a loss of phosphate.
Acknowledgments
This work was supported by funds from the Deutsche Forschungs-gemeinschaft (Ba 700/10 to 1). We thank Nicholas Obermüller for establishing the ISH protocol, and Benno Nafz for help in the statistical analysis.
References
This article has been cited by other articles:
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  R. Schmitt, E. Klussmann, T. Kahl, D. H. Ellison, and S. Bachmann Renal expression of sodium transporters and aquaporin-2 in hypothyroid rats Am J Physiol Renal Physiol, May 1, 2003; 284(5): F1097 - F1104. [Abstract] [Full Text] [PDF]
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