Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

Expression and function of sodium transporters in two opossum kidney cell clonal sublines

The present study describes characteristic features of two clonal subpopulations of opossum kidney (OK) cells (OKLC and OKHC) that are functionally different but morphologically identical. The most impressive differences between OKHC and OKLCcells are the overexpression of Na+-K+-ATPase and type 3 Na+/H+ exchanger by the former, accompanied by an increased Na+-K+-ATPase activity (57.6 ± 5.6 vs. 30.0 ± 0.1 nmol Pi · mg protein−1 · min−1); the increased ability to translocate Na+ from the apical to the basolateral surface; and the increased Na+-dependent pHi recovery (0.254 ± 0.016 vs. 0.094 ± 0.011 pH units/s). V max values (in pH units/s) for Na+-dependent pHi recovery in OKHCcells (0.00521 ± 0.0004) were twice ( P < 0.05) those in OKLC (0.00202 ± 0.0001), with similar K m values (in mM) for Na+(OKLC, 21.0 ± 5.5; OKHC, 14.0 ± 5.6). In addition, we measured the activities of transporters (organic ions, α-methyl-d-glucoside, l-type amino acids, and Na+) and enzymes (adenylyl cyclase, aromaticl-amino acid decarboxylase, and catechol- O-methyltransferase). The cells were also characterized morphologically by optical and scanning electron microscopy and karyotyped. It is suggested that OKLC and OKHC cells constitute an interesting cell model for the study of renal epithelial physiology and pathophysiology, namely, hypertension.

JB3, an IGF-I Receptor Antagonist, Inhibits Early Renal Growth in Diabetic and Uninephrectomized Rats

Biochemical evidence suggests that insulin-like growth factor I (IGF-I) may play an important role as a mediator of kidney growth. In the present study, an IGF-I receptor antagonist (JB3) was synthesized, and its effect on the renal growth that follows the induction of diabetes or unilateral nephrectomy (UNx) was examined. JB3 was generated by solid phase peptide synthesis. Its activity as an IGF-I antagonist was confirmed in an opossum kidney cell line from its inhibitory effect on the increase in thymidine incorporation into DNA induced by recombinant human IGF-I. Male Wistar rats were anesthetized with halothane and subjected to either the induction of diabetes by streptozotocin (intravenous 60 mg/kg) for 4 d (control animals received citrate buffer) or UNx for 11 d (control animals were sham operated). JB3 was delivered by subcutaneous infusion using an osmotic minipump implanted 3 d before the induction of diabetes or UNx. Kidney wet weight, DNA, and protein all were significantly higher 4 d after the induction of diabetes (24%) or 11 d after UNx (55%). Dose-response studies (1 to 30 μg/kg per day) showed JB3 administration to inhibit the increase in kidney growth in both diabetic and UNx rats. The increase in kidney wet weight, DNA, and protein was significantly lower in UNx rats that were treated with JB3 10 μg/kg per day (P < 0.05) than in saline vehicle controls but was abolished in diabetic rats that were treated with JB3 3 μg/kg per day (P < 0.01). Increasing the dose of JB3 to 30 μg/kg per day was associated with a decrease in its inhibitory effect, resulting in bell-shaped dose-response curves. JB3 administration had no effect on the blood glucose concentration or food consumption by either diabetic or nondiabetic animals. The results support the concept of IGF-I as an important mediator of the early renal growth that follows the induction of diabetes or UNx in the rat.

Type II Na-Pi cotransport is regulated transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells

The purpose of the present study was to determine whether isohydric changes in HCO3 concentration and Pco 2directly affect apical Na-dependent Pi(Na-Pi) cotransport in OK cells (opossum kidney cell line). Cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (high-HCO3/CO2condition), or 22 mM NaHCO3/5% CO2, pH 7.4 (low-HCO3/CO2condition) (for 14–24 h). Incubation in lower HCO3/CO2concentrations increased Na-Picotransport 1.5-fold. The increased Na-Pi cotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and a two- to threefold increase in NaPi-4 mRNA abundance. The increase in NaPi-4 mRNA could be completely prevented by incubation in the presence of a transcriptional inhibitor, suggesting that the increase in NaPi-4 mRNA results from an increased NaPi-4 mRNA transcription. In agreement, the NaPi-4 promoter activity was stimulated by 50% at lower HCO3/CO2concentrations. In conclusion, our data demonstrate that isohydric changes in HCO3 concentration and Pco 2exert a significant, direct cellular effect on Na-Pi cotransport and NaPi-4 protein expression in OK cells by affecting NaPi-4 mRNA transcription.