Gerti Stange

Essential role for collectrin in renal amino acid transport

Angiotensin -converting enzyme 2 (ACE2) is a regulator of the renin angiotensin system involved in acute lung failure, cardiovascular functions and severe acute respiratory syndrome (SARS) infections in mammals. A gene encoding a homologue to ACE2, termed collectrin (Tmem27), has been identified in immediate proximity to the ace2 locus. The in vivo function of collectrin was unclear. Here we report that targeted disruption of collectrin in mice results in a severe defect in renal amino acid uptake owing to downregulation of apical amino acid transporters in the kidney. Collectrin associates with multiple apical transporters and defines a novel group of renal amino acid transporters. Expression of collectrin in Xenopus oocytes and Madin–Darby canine kidney (MDCK) cells enhances amino acid transport by the transporter B0AT1. These data identify collectrin as a key regulator of renal amino acid uptake.

The Na+-Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi

The principal mediators of renal phosphate (P(i)) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary P(i). Although their physiological importance has been confirmed in knockout animal studies, significant P(i) reabsorptive capacity remains, which suggests the involvement of other secondary-active P(i) transporters along the nephron. Here we show that a member of the SLC20 gene family (PiT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary P(i). In rats treated chronically on a high-P(i) (1.2%) diet, there was a marked decrease in the apparent abundance of PiT-2 protein in kidney slices compared with those from rats kept on a chronic low-P(i) (0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-P(i) diet, NaPi-IIa showed rapid downregulation after 2 h; PiT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PiT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PiT-2, until now considered as a ubiquitously expressed P(i) housekeeping transporter, is a novel mediator of P(i) reabsorption in the PT under conditions of acute P(i) deprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc.

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na+/phosphate co-transport and Na+/H+ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na+/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na+/H+ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca2+ concentration do not induce inhibition of Na+/H+ exchange; PTH-dependent inhibition of Na+/H+ exchange is not prevented by ionomycin or by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Ca2+ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40–50% inhibition of Na+/H+ exchange. An analysis of intracellular pH sensitivity of Na+/H+ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced Vmax of Na+/H+ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na+/H+ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).

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

Inorganic phosphate (Pi) 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 Pi 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 Pi intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1−/− under a Pi rich diet. This was paralleled by a higher urinary fractional Pi 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-Pi 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 Pi-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.

Protein kinase C activators induce membrane retrieval of type II Na+‐phosphate cotransporters expressed in Xenopus oocytes

1 The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium‐coupled inorganic phosphate (Na+‐Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+‐Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2 In this study, PKC‐induced inhibition of type II Na+‐Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi‐activated current. 3 Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+‐Pi cotransporters lost > 50% of Pi‐activated transport function when exposed to the PKC activators DOG (1,2‐dioctanoyl‐sn‐glycerol) or PMA (phorbol 12‐myristate 13‐acetate). DOG‐induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4α‐PDD (4α‐phorbol 12,13‐didecanoate) showed no significant loss of cotransporter function. 4 Oocytes expressing the rat renal Na+‐SO42‐ cotransporter alone, or coexpressing this with the type IIa rat renal Na+‐Pi cotransporter, showed no downregulation of SO42‐‐activated cotransport activity by DOG. 5 Steady‐state and presteady‐state voltage‐dependent kinetics of type II Na+‐Pi cotransporter function were unaffected by DOG. 6 DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC‐mediated endocytosis. 7 Immunocytochemical studies showed a redistribution of type II Na+‐Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG‐induced internalization of the transport protein. 8 These findings document a specific retrieval of exogenous type II Na+‐Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte.

OSR1-sensitive renal tubular phosphate reabsorption

Background: The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter. Methods: Immunohistochemistry and confocal microscopy were employed to determine renal localization of OSR1 and NaPiIIa. To elucidate the effect of OSR on NaPiIIa activity, cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1, and phosphate transport was estimated from phosphateinduced currents determined with dual electrode voltage clamp. To elucidate the in vivo significance of OSR1 serum phosphate and hormone concentrations as well as urinary phosphate output of mice carrying one allele of WNK-resistant OSR1 (osr1tg/+) were compared to the respective values of wild type mice (osr1+/+). Results: NaPiIIa and OSR1 were both expressed in proximal renal tubule cells. Coexpression of OSR1 significantly up-regulated phosphate-induced currents in NaPiIIa-expressing Xenopus oocytes. Despite decreased serum phosphate concentration urinary phosphate excretion was significantly increased and NaPiIIa protein abundance in the brush border membrane significantly reduced in osr1tg/+ mice as compared to osr1+/+ mice. Serum PTH and calcitriol levels were similar in osr1tg/+ mice and in osr1+/+ mice, serum FGF23 concentration was, however, significantly higher in osr1tg/+ mice than in osr1+/+ mice. Conclusions: OSR1 is expressed in proximal renal tubules and participates in the regulation of FGF23 release and renal tubular phosphate transport.

Renal expression of FGF23 and peripheral resistance to elevated FGF23 in rodent models of polycystic kidney disease

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.

Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis

Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had reached 10-15 mg/24 h. Surface expression of proximal Na(+)-coupled transporters and water channels was in part [Na(+)-P(i) cotransporter IIa (NaPi-IIa) and aquaporin-1 (AQP1)] increased by megalin deficiency alone, but unchanged (Na(+)/H(+) exchanger 3) or reduced (NaPi-IIa and AQP1) in GN irrespective of the endocytosis defect. In distal epithelia, significant increases in proteolytic cleavage products of alpha-epithelial Na(+) channel (ENaC) and gamma-ENaC were observed, suggesting enhanced tubular sodium reabsorption. The effects of glomerular proteinuria dominated over those of blunted proximal endocytosis in contributing to ENaC cleavage. Our data indicate that ENaC-mediated sodium entry may be the rate-limiting step in proteinuric sodium retention. Enhanced proteolytic cleavage of ENaC points to a novel mechanism of channel activation which may involve the action of filtered plasma proteases.

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/Pi cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent Pi and Na+ affinities for Pi-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.