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Caplanusi A, Fuller AJ, Gonzalez-Villalobos RA, Hammond TG, Navar LG. Metabolic inhibition-induced transient [Ca.sup.2+] increase depends on mitochondria in a human proximal renal cell line. Am J Physiol Renal Physio1293: F533-F540, 2007. First published May 23, 2007; doi:10.1152/ajprenal.00030.2007.--During ischemia or hypoxia an increase in intracellular cytosolic [Ca.sup.2+] induces deleterious events but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the [Ca.sup.2+] overload via mitochondrial [Na.sup.+]-[Ca.sup.2+] exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial cells (proximal tubular origin) were subjected to MI and changes in cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]), [Na.sup.+], and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5-fold increase in [[[Ca.sup.2+]].sub.i], reaching 136.5 [+ or -] 15.8 nM in the first 3.45 min. Subsequently [[[Ca.sup.2+]].sub.i] dropped and stabilized to 62.7 [+ or -] 7.3 nM by 30 min. The first phase of the transient increase was [La.sup.3+] sensitive, not influenced by diltiazem, and abolished when mitochondria were deenergized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The subsequent recovery phase was impaired in a [Na.sup.+]-free medium and weakened when the mitochondrial NCX was blocked with 7-chloro-5-(2-chlorophenyl)- 1,5-dihydro-4,1-benzothiazepin2(3H)-one (CGP-37157). Thus [Ca.sup.2+] entry is likely mediated by store-operated [Ca.sup.2+] channels and depends on energized mitochondria, whereas [[[Ca.sup.2+]].sub.i] recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial-mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca2+ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells. [Ca.sup.2+] influx; energized mitochondria; mitochondrial [Na.sup.+]-[Ca.sup.2+] exchanger; thapsigargin; CGP-37157

Megalin is a multiligand receptor heavily involved in protein endocytosis. We recently demonstrated that megalin binds and mediates internalization of ANG II. Although there is a strong structural resemblance between ANG II and ANG-(1-7), their physiological actions and their affinity for the angiotensin type 1 receptor (A[T.sub.1]R) are dissimilar. Therefore, the hypothesis of the present work was to test whether megalin binds and internalizes ANG-(1-7). The uptake of ANG-(1-7) was determined by exposure of confluent monolayers of BN/MSV cells (a model representative of the yolk sac epithelium) to fluorescently labeled ANG-(1-7) (100 nM) and measurement of the amount of cell-associated fluorescence after 4 h by flow cytometry. Anti-megalin antisera and an A[T.sub.1]R blocker (olmesartan) were used to interfere with uptake via megalin and the A[T.sub.1]R, respectively. ANG(1-7) uptake was prevented by anti-megalin antisera (63%) to a higher degree than olmesartan (13%) (P < 0.001). In analysis by flow cytometry of binding experiments performed in brush-border membrane vesicles isolated from kidneys of CD-I mice, anti-megalin antisera interfered with ANG-(1-7) binding more strongly than olmesartan (P < 0.05 against positive control). Interactions of megalin with ANG-(1-7) at a molecular level were studied by surface plasmon resonance, demonstrating that ANG-(1-7) binds megalin dose and time dependently and with an affinity similar to ANG II. These results show that the scavenger receptor megalin binds and internalizes ANG-(1-7). endocytosis; epithelial cells; angiotensin type 1 receptor; olmesartan

Megalin is an abundant membrane protein heavily involved in receptor-mediated endocytosis. The major functions of megalin in vivo remain incompletely defined as megalin typically faces specialized milieus such as glomerular filtrate, airways, epididymal fluid, thyroid colloid, and yolk sac fluid, which lack many of its known ligands. In the course of studies on ANG II internalization, we were surprised when only part of the uptake of labeled ANG II into immortalized yolk sac cells (BN-16 cells) was blocked by specific peptide inhibitors and direct competitors of the angiotensin type 1 receptor. This led us to test if megalin was a receptor for ANG II. Four lines of direct evidence demonstrate that megalin and, to a lesser extent, its chaperone protein cubilin are receptors for ANG II. First, in BN-16 cells anti-megalin and anti-cubilin antisera interfere with ANG II uptake. Second, also in BN-16 cells, pure ANG II competes for uptake of a known megalin ligand. Third, in proximal tubule cell brush-border membrane vesicles extracted from mice, anti-megalin antisera interfere with ANG II binding. Fourth, purified megalin binds ANG II directly in surface plasmon resonance experiments. The finding that megalin is a receptor for ANG II suggests a major new function for the megalin pathway in vivo. These results also indicate that ANG II internalization in some tissues is megalin dependent and that megalin may play a role in regulating proximal tubule ANG II levels. endocytosis; epithelial cells; angiotensin type 1 receptor; intracellular angiotensin II

Although several heavy metal toxins are delivered to the kidney on the carrier protein metallothionein (MT), uncertainty as to how MT enters proximal tubular cells limits treatment strategies. Prompted by reports that MT-I interferes with renal uptake of the megalin ligand [[beta].sub.2]-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubiiin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a [K.sub.d] ~[10.sup.4] and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace >90% bound MT from rat renal brush-border membranes. Megalin ligands including [[beta].sub.2]-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fiuorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum. cadmium; cisplatin; cubilin; proximal tubules

Experiments were performed on rat kidney and plasma samples to examine whether renal endosomes contain angiotensin (ANG) peptides and whether these peptides are regulated by acute enalapril treatment and chronic changes in dietary salt intake. The possibility that an acute blockade of the renin-ANG system with an ANG converting enzyme (ACE) inhibitor can affect ANG II levels in the intracellular organelles was also explored. Results showed that ANG I and ANG II levels and ACE activity are present in renal endosomes and are affected by acute changes of the renin-ANG system, but not by chronic changes in salt intake.

Vasopressin (AVP)-elicited water transport via aquaporin2-containing vesicles is discussed. This transport, linking renal physiology/medicine to other areas of cell/molecular biology, especially neurobiology, and to moving of membrane transporter proteins by epithelial cells, has been studied. Focus is on AVP actions in mammals in the collecting duct, especially the inner medullary collecting duct (IMCD). IMCD cells have a complex integrated-signaling and vesicle-trafficking mechanism integrating the transport with other IMCD cell aspects and the kidney.

The subcellular localization and nephron segment of the rat renal chloride gene 5 (CLC5) mRNA and protein were investigated through immunohistochemistry and in situ hybridization. The loss of CLC5 has been associated with renal disorders such as Dent's diseases and Fanconi's syndrome. It was revealed that CLC5 was expressed in the intracellular portions of both S3 segment of proximal tubules and the medullary thick ascending limb and that loss in the function of CLC5 resulted to endosymal dysfunction and prevented ion transport and reabsorption.

The interaction of immunoglobulin light chains with cubilins has been studied. Immunoglobulin light chains cause nephrotoxicity in multiple myeloma by binding to renal proximal tubular receptors. Cubilins are giant glycoprotein receptors found exclusively in epithelial cells lining the renal proximal tubule and the visceral yolk sac. It may have a role in the endocytosis and cell trafficking of light chains when it is in coelution with kappa-light chains.

The role of gentamicin in inhibiting endosomal fusion and reversibility of gentamicin activity by administration of polyaspartic acid were analyzed in isolated kidneys from male Sprague-Dawley rats. Administration of gentamicin induced vacuolation in the isolated kidneys of male Sprague-Dawley rats and inhibited endosomal fusion. However, the inhibitory effects of gentamicin on endosomal fusion were not altered by the administration of polyaspartic acid.

Functional and structural status of the water channel-comprising membrane within the toad urinary bladder epithelial cells were studied. Comparisons between permeability characteristics and protein composition of several endosomal fractions was conducted in addition to the location of apically labeled proteins through a specialized endocytic pathway. It was concluded that a segment of the apically exposed membranes proteins move along with fluid-phase markers into endosomal compartments.