Well-defined aminooxy terminated N-(2-hydroxypropyl) methacrylamide macromers for site specific bioconjugation of glycoproteins.

Syntheses and characterization of aminooxy terminated polymers of N-(2-hydroxyproyl) methacrylamide (HPMA) of controlled molecular weight and narrow molecular weight distribution are presented here. Design of a chain transfer agent (CTA) containing N-tert-butoxycarbonyl (t-Boc) protected aminooxy group enabled us to use reversible addition-fragmentation (RAFT) polymerization technique to polymerize the HPMA monomer. An amide bond was utilized to link the aminooxy group and the CTA through a triethylene glycol spacer. As a result, the aminooxy group is linked to the poly(HPMA) backbone through a hydrolytically stable amide bond. By varying the monomer to initiator ratios, polymers with targeted molecular weights were obtained. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) and mass spectrometry (ESI and MALDI-TOF). The t-Boc protecting group was quantitatively removed to generate aminooxy terminated poly(HPMA) macromers. These macromers were converted to rhodamine B terminated poly(HPMA) by reacting N-hydroxysuccinimide (NHS) ester of the dye with the terminal aminooxy group to form a stable alkoxyamide bond. Utility of these dye-labeled polymers as molecular probes was evaluated by fluorescence microscopy by studying their intracellular uptake by renal epithelial cells. These aminooxy terminated poly(HPMA) were also tested as biocompatible carriers to prepare chemoselective bioconjugates of proteins using transferrin (Tf) as the protein. Oxidation of the sialic acid side chains of Tf generated aldehyde functionalized protein that was reacted with aminooxy terminated poly(HPMA), which resulted in protein-polymer bioconjugates carrying oxime linkages. These bioconjugates were characterized by gel electrophoresis and MALDI-TOF mass spectrometry.

Fibrocystin/polyductin, found in the same protein complex with polycystin-2, regulates calcium responses in kidney epithelia.

Recent evidence suggests that fibrocystin/polyductin (FPC), polycystin-1 (PC1), and polycystin-2 (PC2) are all localized at the plasma membrane and the primary cilium, where PC1 and PC2 contribute to fluid flow sensation and may function in the same mechanotransduction pathways. To further define the exact subcellular localization of FPC, the protein product encoded by the PKHD1 gene responsible for autosomal recessive polycystic kidney disease (PKD) in humans, and whether FPC has direct and/or indirect cross talk with PC2, which, in turn, is pivotal for the pathogenesis of autosomal dominant PKD, we performed double immunostaining and coimmunoprecipitation as well as a microfluorimetry study of kidney tubular epithelial cells. FPC and PC2 are found to completely or partially colocalize at the plasma membrane and the primary cilium and can be reciprocally coimmunoprecipitated. Although incomplete removal of FPC by small interfering RNA and antibody 803 to intracellular epitopes of FPC did not abolish flow-induced intracellular calcium responses, antibody 804 to extracellular epitopes of FPC blocked cellular calcium responses to flow stimulation. These findings suggest that FPC and polycystins share, at least in part, a common mechanotransduction pathway.

Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by progressive cyst formation and ultimate loss of renal function. Increased cell proliferation is a key feature of the disease. Here, we show that the ADPKD protein polycystin-2 (PC2) regulates the cell cycle through direct interaction with Id2, a member of the helix-loop-helix (HLH) protein family that is known to regulate cell proliferation and differentiation. Id2 expression suppresses the induction of a cyclin-dependent kinase inhibitor, p21, by either polycystin-1 (PC1) or PC2. The PC2-Id2 interaction is regulated by PC1-dependent phosphorylation of PC2. Enhanced Id2 nuclear localization is seen in human and mouse cystic kidneys. Inhibition of Id2 expression by RNA interference corrects the hyperproliferative phenotype of PC1 mutant cells. We propose that Id2 has a crucial role in cell-cycle regulation that is mediated by PC1 and PC2.

Dimeric architecture of the human bumetanide-sensitive Na-K-Cl Co-transporter.

The primary mediator of NaCl reabsorption in the renal distal tubule is the human bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter (hNKCC2), located at the apical membrane of the thick ascending limb of Henle's loop. The physiologic importance of this transporter is emphasized by the tubular disorder Bartter syndrome type I, which arises from the functional impairment of hNKCC2 as a result of mutations in the SLC12A1 gene. The aim of the present study was to investigate the oligomeric state of hNKCC2 to understand further its operational mechanism. To this end, hNKCC2 was heterologously expressed in Xenopus laevis oocytes. Chemical cross-linking with dimethyl-3,3-dithio-bis-propionamidate indicated that hNKCC2 subunits can reversibly form high molecular weight complexes. Co-immunoprecipitation of tagged hNKCC2 subunits further substantiated a physical interaction between individual hNKCC2 subunits. The size of the hNKCC2 multimers was determined by sucrose gradient centrifugation, and a preference for dimeric complexes (approximately 320 kD) was demonstrated. Finally, concatemeric constructs consisting of two wild-type subunits or a wild-type and a functionally impaired hNKCC2 subunit (G319R) were expressed in oocytes. Subsequently, the concatemers were functionally characterized, resulting in a significant bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.2 nmol/oocyte per 30 min for the wild-type-wild-type concatemer, which was reduced to 1.3 +/- 0.1 nmol/oocyte per 30 min for the wild-type-G319R concatemer. In conclusion, this study suggests that hNKCC2 forms at least functional dimers when expressed in Xenopus laevis oocytes of which the individual subunits transport Na(+) independently.

Mutations in the human Na-K-2Cl cotransporter (NKCC2) identified in Bartter syndrome type I consistently result in nonfunctional transporters.

Bartter syndrome (BS) is a heterogeneous renal tubular disorder affecting Na-K-Cl reabsorption in the thick ascending limb of Henle's loop. BS type I patients typically present with profound hypokalemia and metabolic alkalosis. The main goal of the present study was to elucidate the functional implications of six homozygous mutations (G193R, A267S, G319R, A508T, del526N, and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter (hNKCC2) identified in patients diagnosed with BS type I. To this end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the corresponding mutants was injected in Xenopus laevis oocytes and transporter activity was measured after 72 h by means of a bumetanide-sensitive (22)Na(+) uptake assay at 30 degrees C. Injection of 25 ng of hNKCC2 cRNA resulted in bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNA yielded no significant bumetanide-sensitive (22)Na(+) uptake. Expression of wild-type and mutant transporters was confirmed by immunoblotting, showing significantly less mutant protein compared with wild-type at the same cRNA injection levels. However, when the wild-type cRNA injection level was reduced to obtain a protein expression level equal to that of the mutants, the wild-type still exhibited a significant bumetanide-sensitive (22)Na(+) uptake. Immunocytochemical analysis showed immunopositive staining of hNKCC2 at the plasma membrane for wild-type and all studied mutants. In conclusion, mutations in hNKCC2 identified in type I BS patients, when expressed in Xenopus oocytes, result in a low expression of normally routed but functionally impaired transporters. These results are in line with the hypothesis that the mutations in hNKCC2 are the underlying cause of the clinical abnormalities seen in patients with type I BS.

Functional implications of mutations in the human renal outer medullary potassium channel (ROMK2) identified in Bartter syndrome.

Bartter syndrome is an autosomal recessive heterogeneous renal tubular disorder affecting NaCl reabsorption in the thick ascending limb of Henle's loop (TAL). The aim of this study was to elucidate the functional implications of mutations in the predominant human ROMK isoform in TAL, hROMK2, involved in Bartter syndrome type II. cRNA of flag-tagged hROMK2 and eight mutants identified in seven non-related patients was expressed in Xenopus laevis oocytes. hROMK2 activity was measured by two-electrode voltage-clamp analysis and defined as the Ba2+ -sensitive current at a holding potential of -75 mV. The subcellular localization of hROMK2 in oocytes was studied by immunocytochemistry. Injection of 25 pg hROMK2 cRNA resulted in an inwardly rectifying Ba2+ -sensitive current of 522+/-43 nA ( n=22). The mutants could be divided into three distinct groups. First, at 25 pg injection mutants W80C, V103E and T313/350X exhibited no significant currents and could only be detected intracellularly. Upon 8 ng injection, plasma membrane presence was observed as well as currents up to 60% of wild-type current. Second, mutants V53E and V296G exhibited no Ba2+ -sensitive current, but were present in the plasma membrane at 0.1 ng and 8 ng injection levels. Third, mutants P91L and A179T were detectable on the plasma membrane (0.1 ng) and yielded currents of 98% and 80% of wild-type, respectively, at 25 pg injection. S294C yielded currents that were 45% of wild-type and were detected both on and just below the plasma membrane at 0.1 ng injection. This study has unraveled three distinct mechanisms by which mutations in hROMK2 could impair channel function in Bartter syndrome. Future experiments on kidney epithelial cell lines will have to confirm this classification, after which specific pharmacological treatments could be considered for each group of mutations.