Suppressive effects of tenofovir disoproxil fumarate, an antiretroviral prodrug, on mineralization and type II and type III sodium-dependent phosphate transporters expression in primary human osteoblasts.

Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug commonly used for the management of Human Immunodeficiency Virus (HIV) in highly active antiretroviral therapy (HAART) and of chronic Hepatitis B Virus (HBV) infections. Long-term TDF-treated subjects present decrease of bone mineral density and rarely severe osteomalacia. Although these adverse effects have been attributed to the impaired proximal tubule function, a possible direct involvement of TDF on osteoblasts should be taken into account. The aim of this study was to evaluate whether sodium phosphate transporters NPT2A (sodium-dependent phosphate transport protein 2A), NPT2C (sodium-dependent phosphate transport protein 2C), PIT1 (sodium-dependent phosphate transporter 1), and PIT2 (sodium-dependent phosphate transporter 2) were expressed in primary human osteoblasts (HOBs), whether their expression was related to HOBs differentiation and whether TDF could affect mineralization and gene expression. PIT1 and PIT2 were expressed under proliferating conditions and increased after induction of mineralization, while NPT2A and NPT2C were almost undetectable. In HOBs TDF exposure induced a significant dose-dependent decrease in mineralization. Moreover, TDF caused a reduction of COL1A1 and of ATF4 expression in differentiated HOBs. In summary, HOBs do not express NPT2A and NPT2C and do express PIT1 and PIT2, suggesting a role of these two latter in human osteoblast mineralization. TDF impairs osteoblast mineralization, confirming a direct negative effect on bone. Therefore, in clinical practice, bone damage must be suspected and evaluated also in patients receiving TDF without kidney function alterations.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate.

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.

Role of RIHP and renal tubular sodium transporters in volume retention of pregnant rats.

BACKGROUND: Normal pregnancy is characterized by sodium and water conservation and an increase in plasma volume that is required for an uncomplicated pregnancy. Renal interstitial hydrostatic pressure (RIHP) is significantly decreased in pregnant rats. This decrease in RIHP may play an important role in the sodium and water retention that characterizes normal pregnancy. Paradoxically this enhanced renal sodium and water reabsorption appear to conflict with the consistent findings of a general decrease in abundance of renal tubular sodium transporters during normal pregnancy. The objective of this review is to examine the apparent discrepancy between the increases in renal tubular sodium and water reabsorption, facilitated by decreases in RIHP, and the seemingly discordant decreases in abundance of renal tubular transporters during normal pregnancy in rats. METHODS: Western blots and immunohistochemistry were used to evaluate abundance and localization of renal tubular transporters. RIHP was measured directly and continuously via a polyethylene (PE) matrix that was implanted in the left kidney of rats at the age of 11 to 16 weeks. RESULTS: Average basal RIHP and fractional excretion of sodium (FENa) were found to be significantly lower (P < .05) in midterm pregnant (MP; n = 18) and late-term pregnant (LP; n = 20) rats compared with nonpregnant (NP; n = 16) rats (3.5 +/- 0.3 mm Hg and 1.46 +/- 0.24% for MP; 3.3 +/- 0.1 mm Hg and 1.41 +/- 0.21% for LP; and 7.6 +/- 0.6 mm Hg and 3.67 +/- 0.24% for NP). Cortical Na+-K+-ATPase and Na-Pi2a cotransporter (Na-Pi) protein expression tend to decline with pregnancy. Also cortical Na+-H+ exchanger-1 (NHE-1) protein expression declines steadily during the course of pregnancy from MP to LP compared with that in NP rats, and cortical Na+-H+ exchanger-3 (NHE-3) protein expression is significantly lower in MP and LP compared with NP rats. CONCLUSIONS: We propose that during normal uncomplicated pregnancy, simultaneous decreases in RIHP and in net abundance of renal tubular sodium transporters occur. The effects of decreased RIHP exceed those of the reduction in net abundance, and presumably activity, of renal tubular transporters resulting in an enhanced net sodium and water retention during pregnancy.

Impact of reduced nephron mass on cyclosporine- and/or sirolimus-induced nephrotoxicity.

BACKGROUND: We evaluated the impact of reduced nephron mass on nephrotoxicity by cyclosporine A (CsA) and/or sirolimus (SRL). METHODS: Renal function was tested in salt-depleted rats bearing two kidneys (2K), one kidney, or half a kidney (1/2K) and treated for 7 or 28 days with CsA (5 mg/kg) and/or SRL (0.8 mg/kg). We also measured the expression of aquaporin-2, sodium/phosphate cotransporter (NaPi)-2, paracellin-1, and kidney injury molecule (KIM)-1 by real-time polymerase chain reaction. RESULTS: At 7 days in 2K, serum creatinine clearance (CrCl) was decreased only in CsA/SRL-treated group (P<0.05) compared with controls; in 1/2K, CrCl was decreased in all groups, but most dramatically in CsA/SRL group (P<0.05). Extended 28-day therapy worsened CrCl in all 1/2K groups (P<0.01). Although the expression of aquaporin-2, NaPi-2, and paracellin-1 mRNAs tended to increase in kidneys with a reduced nephron mass, NaPi-2 mRNA levels decreased in 1/2K rats exposed to CsA/SRL for 28 days (P<0.05). In contrast, low KIM-1 mRNA expression in control 2K rats increased fourfold in untreated 1/2K (P<0.05), and 50- to 200-fold in CsA/SRL-treated 1/2K (P=0.01). CONCLUSIONS: Nephrotoxicity is significantly worsened by reduced nephron mass, which correlates with increased expression of KIM-1 and inhibited expression of NaPi-2.

El eje hueso-riñón en el control del fósforo sérico y la mineralización ósea / Bone-Kidney axis in the control of serum phosphorus and bone mineralization

El eje hueso-riñón ha sido pensado como un mecanismo por el cual el esqueleto se comunica con el riñón para coordinar la mineralización de la matriz extracelular ósea con el manejo renal del fosfato. Osteoblastos /osteocitos están bien preparados para coordinar las homeostasis sistémica de fósforo y la mineralización ósea, ya que ellos expresan todos los componentes implicados en un posible eje hueso-riñón, incluyendo al PHEX, FGF-23, MEPE, y DMP1. Los efectos autocrinos de proteínas de la familia SIBLING como MEPE y DMP1 sobre los osteoblastos podrían regular la producción de proteínas de matriz extracelular que intervienen en la mineralización. El riñón provee uno de los efectores de este eje que regula el balance de fosfato a través de la expresión apical de los cotransportadores sodio/fosfato NaPi-IIa y NaPi-IIc en el túbulo proximal. Central en este eje es el FGF-23, producido por los osteoblastos que tiene acciones fosfatúricas sobre el riñón. Cuando se descubrió que el FGF23, la primera fosfatonina era de origen osteoblástico/osteocitico, quedó establecido el eje hueso-riñón. Probar definitivamente la existencia de este eje hueso-riñón y definir exactamente su rol fisiológico requerirá de investigaciones adicionales

The bone-kidney axis has been thought as a mechanism for the skeleton to communicate with the kidney to coordinate the mineralization of extracelular matrix with the renal handling of phosphate. Osteoblasts / osteocytes are well suited for coordinating systemic phosphate homeostasis and mineralization, since they express all of the implicated components of a possible bone-kidney axis, including PHEX, FGF-23, MEPE, and DMP1. In addition, autocrine effects of SIBLING proteins as MEPE and DMP1 on osteoblasts could regulate the production of ECM proteins that regulate mineralization. The kidney provides one of the effectors of the axis that regulates phosphate balance through the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Central in this axis is FGF-23, produced by osteoblasts that has phosphaturic actions on the kidney. When FGF23, the first phosphatonin, was discovered to be of osteoblastic/osteocyte origin, the bone kidney axis was established. Proving the existence of this bone-kidney axis and defining its physiological role will require additional investigations