Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct.

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.

Expression of the chemokine receptor CCR6 in human renal inflammation.

BACKGROUND: Nodular inflammatory cell infiltrates with defined microarchitecture, i.e. tertiary lymphoid organs, develop in the tubulointerstitium during chronic renal inflammation. CCR6 and the corresponding ligand CCL20 are involved in the formation of gut-associated lymphatic tissue. We hypothesized that CCR6 might be involved in the formation of nodular infiltrates in the kidney. METHODS: CCR6- and CD20-positive B cells were localized in renal biopsies with IgA nephropathy (n = 13), membranous nephropathy (n = 12), crescentic glomerulonephritis (cGN, n = 11) and chronic interstitial nephritis (n = 13), and in pre-implantation biopsies as controls (n = 8). The mRNA expression of CCR6 and the ligand CCL20 was quantified by real-time RT-PCR in 51 renal biopsies of the same disease entities. RESULTS: In the pre-transplant biopsies, CCR6 was expressed by endothelial cells of peritubular and glomerular capillaries. In patients with glomerulonephritis, infiltrating cells were positive particularly in areas of nodular inflammatory cell accumulations. A major part of the CCR6-positive cells were CD20-positive B cells, but a part of the CD3-positive T cells were also found to be positive. The constitutive expression of CCR6 on the endothelium of glomerular capillaries was lost in biopsies with progressive injury. Tubular epithelial cells expressed CCR6 in inflamed kidneys, most commonly on the basolateral side. CONCLUSIONS: CCR6 and the corresponding ligand CCL20 might therefore be involved in the recruitment of T and B cells to organized nodular infiltrates in chronic renal inflammation. The functional role of endothelial CCR6 needs to be evaluated in further studies.

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney.

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice.

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na(+)-phosphate cotransporter (NaP(i)-IIa) in the brush border membrane (BBM). The activity and localization of NaP(i)-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP(i)-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na(+)/H(+)-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP(i)-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP(i)-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1-34) led to internalization of NaP(i)-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3-34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP(i)-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP(i)-IIa and, specifically, couples the apical PTHR to PLC.

Unchanged expression of the sodium-dependent phosphate cotransporter NaPi-IIa despite diurnal changes in renal phosphate excretion.

Renal phosphate excretion is subjected to circadian rhythmicity. The bulk of filtered inorganic phosphate (P(i)) is reabsorbed by the sodium-dependent phosphate cotransporter NaPi-IIa. The regulation of proximal tubular phosphate reabsorptive capacity is largely attributed to the altered abundance of NaPi-IIa residing in the brush border membrane (BBM) of proximal tubular cells. Therefore, we examined if the diurnal rise in renal phosphate excretion is accompanied by a corresponding change in NaPi-IIa expression. Renal phosphate excretion, creatinine clearance, and serum phosphate were determined at consecutive time points in rats, starting from 8 a.m. until 5 p.m. During this period, renal phosphate excretion (fractional P(i) excretion) increased more than eightfold until 5 p.m. compared to the morning values at 8 a.m. In addition, serum phosphate and creatinine clearance as well as the calculated tubular phosphate threshold increased. Neither immunoblot analysis of BBMs nor immunohistochemical staining for NaPi-IIa yielded evidence for a lower abundance of NaPi-IIa in kidneys collected in the afternoon compared to those in the morning. However, kidneys sampled in the afternoon showed a small decrease (14%) in (32)P uptakes into BBM vesicles (BBMVs). Thus, the diurnal rise in renal phosphate excretion was associated with a mild reduction in the sodium-dependent phosphate transport rate in proximal tubular BBMs. There was no apparent downregulation of NaPi-IIa abundance and only a small reduction in Na(+)-dependent Pi-transport activity. Thus, the diurnal changes in urinary phosphate excretion appear to be mainly related to changes in serum phosphate and tubular threshold but not to NaPi-IIa expression.

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC).

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Luminal kidney and intestine SLC6 amino acid transporters of B0AT-cluster and their tissue distribution in Mus musculus.

The B degrees transport system mediates the Na(+)-driven uptake of a broad range of neutral amino acids into epithelial cells of small intestine and kidney proximal tubule. A corresponding transporter was identified in 2004 (A. Broer, K. Klingel, S. Kowalczuk, J. E. Rasko, J. Cavanaugh, and S. Broer. J Biol Chem 279: 24467-24476, 2004) within the SLC6 family and named B degrees AT1 (SLC6A19). A phylogenetically related transporter known as XT3 in human (SLC6A20) and XT3s1 in mouse was shown to function as an imino acid transporter, to localize also to kidney and small intestine and renamed SIT1 or Imino(B). Besides these two transporters with known functions, there are two other gene products belonging to the same phylogenetic B degrees AT-cluster, XT2 (SLC6A18) and rodent XT3 that are still "orphans." Quantitative real-time RT-PCR showed that the mRNAs of the four B degrees AT-cluster members are abundant in kidney, whereas only those of B degrees AT1 and XT3s1/SIT1 are elevated in small intestine. In brain, the XT3s1/SIT1 mRNA is more abundant than the other B degrees AT-cluster mRNAs. We show here by immunofluorescence that all four mouse B degrees AT-cluster transporters localize, with differential axial gradients, to the brush-border membrane of proximal kidney tubule and, with the possible exception of XT3, also of intestine. Deglycosylation and Western blotting of brush-border proteins demonstrated the glycosylation and confirmed the luminal localization of B degrees AT1, XT2, and XT3. In summary, this study shows the luminal brush-border localization of the Na(+)-dependent amino and imino acid transporters B degrees AT1 and XT3s1/SIT1 in kidney and intestine. It also shows that the structurally highly similar orphan transporters XT2 and XT3 have the same luminal but a slightly differing axial localization along the kidney proximal tubule.

Secreted frizzled-related protein-4 reduces sodium-phosphate co-transporter abundance and activity in proximal tubule cells.

The phosphatonin, secreted frizzled-related protein-4 (sFRP-4), induces phosphaturia and inhibits 25-hydroxyvitamin D 1alpha-hydroxylase activity normally induced in response to hypophosphatemia. To determine the mechanism by which sFRP-4 alters renal phosphate (P(i)) transport, we examined the effect of sFRP-4 on renal brush border membrane (BBMV) Na(+)-dependent P(i) uptake, and the abundance and localization of the major Na(+)-P(i)-IIa co-transporter in proximal tubules and opossum kidney (OK) cells. Infusion of sFRP-4 increased renal fractional excretion of P(i) and decreased renal beta-catenin concentrations. The increase in renal P(i) excretion with sFRP-4 infusion was associated with a 21.9 +/- 3.4% decrease in BBMV Na(+)-dependent P(i) uptake (P < 0.001) compared with a 39.5 +/- 2.1% inhibition of Na(+)-dependent P(i) transport in renal BBMV induced by PTH (P < 0.001). sFRP-4 infusion was associated with a 30.7 +/- 4.8% decrease in Na(+)-P(i)-IIa co-transporter protein abundance (P < 0.01) assessed by immunoblotting methods compared to a 45.4 +/- 8.8% decrease induced by PTH (P < 0.001). In OK cells, sFRP-4 reduced surface expression of a heterologous Na(+)-P(i)-IIa co-transporter. We conclude that sFRP-4 increases renal P(i) excretion by reducing Na(+)-P(i)-IIa transporter abundance in the brush border of the proximal tubule through enhanced internalization of the protein.

Parathyroid hormone treatment induces dissociation of type IIa Na+-P(i) cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.

The type IIa Na+-P(i) cotransporter (NaP(i)-IIa) and the Na+/H+ exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between NaP(i)-IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. NaP(i)-IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the NaP(i)-IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, NaP(i)-IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of NaP(i)-IIa without changes in the amount of NHERF1. The effect of PTH on the NaP(i)-IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of NaP(i)-IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of NaP(i)-IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 phosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, NaP(i)-IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and NaP(i)-IIa on the basis of their brush-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1.

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice.

Intake of a low-phosphate diet stimulates transepithelial transport of Pi in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaPi cotransporters NaPi type IIa (NaPi-IIa) and NaPi type IIb (NaPi-IIb), respectively. Low-Pi diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D3-1alpha-hydroxylase (1alphaOHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D]. Regulation of the intestinal absorption of Pi and the abundance of NaPi-IIb by 1,25(OH)2D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-Pi diet of NaPi-IIb in small intestine as well as NaPi-IIa in kidneys of either VDR- or 1alphaOHase-deficient mice. In both mouse models, upregulation by a low-Pi diet of the NaPi cotransporters NaPi-IIa and NaPi-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1alphaOHase-deficient mice, the same changes in NaPi-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaPi cotransport in small intestine (via NaPi-IIb) and kidney (via NaPi-IIa) by low dietary intake of Pi cannot be explained by the 1,25(OH)2D-VDR axis.

Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1.

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock-out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.

Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells.

The Na(+)/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of P(i) in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary P(i) intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na(+)-transporting systems such as NHE3 and Na(+)-K(+)-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D(1)-like receptor agonists fenoldopam (10 microM) and SKF-38393 (10 microM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D(2)-like selective agonist quinpirole (1 microM) had no effect. The D(1) and D(2) agonists did not affect the renal Na(+)/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D(1)-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 microM chelerythrine) or ERK1/2 (20 microM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 microM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D(1)-like receptors, an effect that is mediated by PKA.

Potential role of ABCA7 in cellular lipid efflux to apoA-I.

ABCA7 is homologous to ABCA1 and has recently been shown in cell culture to bind apolipoprotein A-I (apoA-I) and to promote the efflux of phospholipids. However, it is not known if ABCA7 promotes lipid efflux in vivo. When expressed in HEK293 cells, both human and mouse ABCA7 promoted phospholipid efflux to apoA-I but no detectable cholesterol efflux. However, genetic knockdown of ABCA7 in mouse peritoneal macrophages did not affect phospholipid or cholesterol efflux to apoA-I. Moreover, in ABCA1-knockout macrophages, there was no detectable apoA-I-stimulated phospholipid efflux, inconsistent with a residual role of ABCA7. In contrast to plasma membrane localization of ABCA7 in transfected embryonic kidney cells, immunofluorescence microscopy of endogenous ABCA7 in macrophages showed a predominantly intracellular localization of the protein. Strikingly, immunofluorescence studies of adult mouse kidney revealed an apical brush border membrane localization of ABCA7 in the proximal tubule, suggesting that ABCA7 may come in contact with apoA-I in the glomerular filtrate. Although ABCA7 does not contribute to apolipoprotein-mediated lipid efflux in resting macrophages, its cell surface location in the kidney suggests that it could serve such a role in tissue microenvironments.

Novel aspects in regulated expression of the renal type IIa Na/Pi-cotransporter.

Proximal tubular phosphate (P(i)) reabsorption is a key element in overall phosphate homeostasis; physiologic/pathophysiologic alterations are related to the control of brush border membrane expression (regulated endocytosis) of the type IIa sodium (Na)/phosphate(P(i))-cotransporter (NaPi-IIa). The carboxy terminus of NaPi-IIa contains sequences important for its apical delivery/expression; the last three amino acids are involved in PSD95/DglA/ZO-1 (PDZ) interactions involving NaPi-IIa, Na/H exchanger-regulatory factor 1 (NHERF1/2), and PDZK1/2 (apical scaffold). Regulated endocytosis of NaPi-IIa [e.g., parathyroid hormone (PTH)-induced] is reduced in megalin-deficient mice; internalization occurs via clathrin-coated structures, early endosomes, and finally leads to lysosomal degradation. NaPi-IIa contains, in the third intracellular loop, a sequence motif required for internalization. Different hormonal [e.g., PTH, atrial natriuretic peptide (ANP), also nitric oxide (NO)] and nonhormonal factors activate a variety of intracellular signaling cascades [protein kinase A (PK-A), protein kinase C (PK-C), protein kinase G (PK-G), extracellular receptor kinase (ERK)-1/2] leading (by unknown mechanisms) to NaPi-IIa internalization. Different phosphatonins [e.g., fibroblast growth factor (FGF)-23, frizzled related protein (FRP)-4, matrix extracellularphosphoglycoprotein (MEPE)], associated with different pathophysiologic states of renal P(i)-handling, seem also to control apical expression of NaPi-IIa. Internalization of NaPi-IIa first requires its removal from the apical scaffold. This scaffold can also be considered as a regulatory scaffold containing also protein kinase A (PK-A)-anchoring proteins (AKAPs, ezrin) and the apical PTH receptor. The role of the different components of the regulatory scaffold in regulated endocytosis of NaPi-IIa is at present unknown.

Kidney-specific inactivation of the megalin gene impairs trafficking of renal inorganic sodium phosphate cotransporter (NaPi-IIa).

Renal reabsorption of inorganic phosphate is mediated by the type IIa sodium phosphate cotransporter (NaPi-IIa) of the proximal tubule. Changes in renal phosphate handling are mainly attributable to altered NaPi-IIa brush border membrane (BBM) expression. Parathyroid hormone (PTH) induces inactivation of NaPi-IIa by endocytic membrane retrieval and degradation. The key elements triggering this process are not clear to date. Megalin serves as a receptor for the endocytosis of multiple ligands and is coexpressed with NaPi-IIa in the proximal tubule. Investigated was the role of megalin in the regulation of NaPi-IIa in steady state and during inactivation. Kidneys and tubular BBM fractions from mice with a renal-specific megalin gene defect and from controls were analyzed by light and electron microscopic histochemical techniques and Western blot test. Steady-state levels of NaPi-IIa in BBM were significantly enhanced, mRNA levels preserved, and phosphaturia reduced in the absence of megalin. Fluid-phase endocytosis was prevented and the apical endocytic apparatus markedly reduced. Systemic administration of PTH resulted in a defective retrieval and impaired degradation of NaPi-IIa. In vitro, the application of various stimuli of the PTH-induced signaling cascade had no effect either. Adequate steady-state expression of NaPi-IIa and the capacity of the proximal tubule cell to react on PTH-driven inactivation of NaPi-IIa by endocytosis and intracellular translocation require the presence of megalin.

Expression of visinin-like protein-3 in mouse kidney.

In renal proximal brush borders the Na/Pi cotransporter NaPi-IIa is part of a heteromultimeric complex including the PDZ proteins PDZK1 and NHERF1, which interact with the C terminus of NaPi-IIa. In this study, a yeast two-hybrid screen against the N terminus of the Na/Pi cotransporter NaPi-IIa was performed. Thereby we identified visinin-like protein-3 (VILIP-3), a member of neuronal calcium sensors. In this study, expression and protein localization of VILIP-3 in the mouse kidney was performed by immunofluorescence and RT-PCR using laser-assisted microdissected nephron segments. VILIP-3 was found to be abundant in distal and collecting ducts where it partly colocalized with calbindin D28K. In addition VILIP-3 was observed in the brush borders of proximal tubular S1 and S3 segments of both superficial and deep nephrons.

Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal proximal tubule.

BACKGROUND: Dopamine is a principal natriuretic hormone in mammalian Na+ homeostasis. Dopamine acutely alters glomerular filtration rate (GFR) and decreases Na+ absorption in both the proximal and distal nephron. Proximal tubule natriuresis is effected through inhibition of the apical membrane Na/H exchanger NHE3. METHODS: We examined whether dopamine directly and acutely decreases apical membrane NHE3 protein using renal tissue in two in vitro systems: renal cortical slices and in vitro perfused single tubules. After incubation with dopamine, NHE3 activity was measured by 22Na flux and NHE3 antigen was measured by immunoblot in apical membrane and total cellular membranes. RESULTS: Direct application of dopamine to either cortical slices or microperfused tubules acutely decreases NHE3 activity and antigen at the apical membrane of the proximal tubule. No change in total cellular NHE3 was detected. CONCLUSION: One mechanism by which dopamine causes natriuresis is via direct and acute reduction of NHE3 protein at the apical membrane via changes in NHE3 protein trafficking.

PDZK1: I. a major scaffolder in brush borders of proximal tubular cells.

BACKGROUND: In proximal tubular cells, PDZK1 (NaPi-Cap1) has been implicated in apical expression of the Na+-dependent phosphate cotransporter (NaPi-IIa) via interaction with its C-terminus. PDZK1 represents a multidomain protein consisting of four PDZ domains and thus is believed to have a broader specificity besides NaPi-IIa. METHODS: We subjected single PDZ domains derived from PDZK1 either to yeast two-hybrid screens or yeast trap assays. Different pull-down assays and blot overlays were applied to corroborate the PDZK1-mediated interactions in vitro. Co-localization of interacting proteins with PDZK1 in proximal tubular cells was assessed by immunohistochemistry. RESULTS: In the yeast screens, the most abundant candidate protein to interact with PDZK1 was the membrane-associated protein of 17 kD (MAP17). Besides MAP17, C-terminal parts of following transporters were also identified: NaPi-IIa, solute carrier SLC17A1 (NaPi-I), Na+/H+ exchanger (NHE-3), organic cation transporter (OCTN1), chloride-formate exchanger (CFEX), and urate-anion exchanger (URAT1). In addition, other regulatory factors were found among the clones, such as a protein kinase A (PKA)-anchoring protein (D-AKAP2) and N+/H+ exchanger regulator factor (NHERF-1). All interactions of itemized proteins with PDZK1 were affirmed by in vitro techniques. Apart from PDZK1, strong in vitro interactions of NHERF-1 were also observed with the solute transporters (excluding MAP17) and D-AKAP2. All identified proteins were immunolocalized in proximal tubular cells, wherein all membrane proteins co-localized with PDZK1 in brush borders. CONCLUSION: We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.

PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells.

BACKGROUND: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4. METHODS: After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells. RESULTS: Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells. CONCLUSION: We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.

Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells.

An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.