Ya Su

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

Human H+ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1

The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1–a4. This study investigated two naturally occurring point mutations in a4s COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H+ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1s catalytic activity. In yeast expression studies, protein levels, H+ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H+ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H+ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H+ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).

Mutation Conferring Apical-Targeting Motif on AE1 Exchanger Causes Autosomal Dominant Distal RTA

Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.

V1 and V0 Domains of the Human H+-ATPase Are Linked by an Interaction between the G and a Subunits

The specialized H+-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown wide-spread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H+-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H+-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V1 and V0 domains in man, which is known to be required for H+-ATPase assembly and regulation.

Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney

The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143–151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3 ± 0.2 μM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.

Characterisation of the gene for Drosophila amphiphysin.

A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes. Messenger RNA of this amphiphysin is expressed widely during embryogenesis and has elevated expression in a number of sites including the foregut, hindgut and epidermis, but not in the central nervous system. Taken together, these data are consistent with a role for Drosophila amphiphysin in endocytosis, but the details of this role may differ from that of vertebrate amphiphysins.

Urinary Exosomes Contain MicroRNAs Capable of Paracrine Modulation of Tubular Transporters in Kidney

Exosomes derived from all nephron segments are present in human urine, where their functionality is incompletely understood. Most studies have focused on biomarker discovery rather than exosome function. Through sequencing we identified the miRNA repertoire of urinary exosomes from healthy volunteers; 276 mature miRNAs and 345 pre-miRNAs were identified (43%/7% of reads). Among the most abundant were members of the miR-10, miR-30 and let-7 families. Targets for the identified miRNAs were predicted using five different databases; genes encoding membrane transporters and their regulators were enriched, highlighting the possibility that these miRNAs could modulate key renal tubular functions in a paracrine manner. As proof of concept, cultured renal epithelial cells were exposed to urinary exosomes and cellular exosomal uptake was confirmed; thereafter, reduced levels of the potassium channel ROMK and kinases SGK1 and WNK1 were observed in a human collecting duct cell line, while SPAK was unaltered. In proximal tubular cells, mRNA levels of the amino acid transporter gene SLC38A2 were diminished and reflected in a significant decrement of its encoded protein SNAT2. Protein levels of the kinase SGK1 did not change. Thus we demonstrated a novel potential function for miRNA in urinary exosomes.

Physical and Functional Links between Anion Exchanger-1 and Sodium Pump

Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in α-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the β1 subunit of Na(+),K(+)-ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and β1 colocalized in renal α-intercalated cells and coimmunoprecipitated (together with the catalytic α1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and β1 interact directly, with a Kd value of 0.81 μM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for β1 binding. siRNA-induced knockdown of β1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of β1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of β1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.