Localization of phosphatidylinositol phosphate kinase IIgamma in kidney to a membrane trafficking compartment within specialized cells of the nephron

Abstract

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kgamma. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kalpha and PIP4Kbeta, PIP4Kgamma is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kgamma, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kgamma had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kalpha, bacterially expressed recombinant PIP4Kgamma was completely inactive but did have the ability to associate with active PIP4Kalpha in vitro. Overall our data suggest that PIP4Kgamma may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

Fig. 1.

PIP5K2C is differentially expressed in adult mouse tissues. A: expression of PIP5K2A, PIP5K2B, and PIP5K2C determined by quantitative PCR using the comparative threshold cycle (C_T) method. Normalized expression is presented as relative fold increase above PIP5K2A in testis (n = 9). B: in situ hybridization using PIP5K2C-specific probes on thin sections of adult mouse brain (a), liver (b), and kidney (c; not to scale). Control incubations using an excess of cold probe indicate nonspecific binding (d–f). C: autoradiographic emulsion staining of mouse kidney cortex tubules, silver grains (arrows) indicating PIP5K2C mRNA expression (tu, tubule; gl, glomerulus). Scale bar = 20 μm.

Fig. 2.

Specificity of anti-PIP4Kγ antibody. Detection of PIP4Kγ, but not the PIP4Kα or PIP4Kβ isoforms, was established in various procedures. A: equivalent amounts of purified recombinant protein of each isoform were visualized on SDS-PAGE (i) and detected by Western blotting (ii). B: HeLa cells overexpressing green fluorescent protein (GFP)-tagged PIP4Kγ or PIP4Kα were fixed and stained with anti-PIP4Kγ antibody. Scale bar = 10 μm. C: Surface plasmon resonance analysis using PIP4Kα, PIP4Kβ, and PIP4Kγ adsorbed to an nitrilotriacetic acid biosensor. Anti-PIP4Kγ antibody was passed over the biosensor at 0 s and exchanged for wash buffer at 45 s. Data were normalized to 6xHis tag control protein binding.

Fig. 3.

PIP4Kγ is expressed in adult mouse tissues. Lysates were probed with PIP4Kγ-specific antibody (i) and with neutralized antibody after saturation with immunizing peptide (ii). Whole tissue lysates (A) and protein preparations from different kidney regions (B) were probed with the same antibody. Equivalent protein loading was based on total lysate concentration. Purified recombinant PIP4Kγ was used to identify endogenous PIP4Kγ at the correct molecular mass (arrow).

Fig. 4.

PIP4Kγ expression in the adult mouse kidney. Tissue immunohistochemistry using antibody specific to PIP4Kγ identified the expression of endogenous protein in kidney sections. A: positive PIP4Kγ signal detected by reporter enzyme (i: scale bar = 10 μm) or epifluorescence (ii; scale bar = 40 μm). Negative control reactions are also shown (primary antibody preincubated with antigenic peptide). B: high resolution imaging indicated that PIP4Kγ (green) was differentially distributed in the representative kidney regions indicated after removal of autofluorescence. Nuclei (red) are counterstained as contrast. Scale bar = 40 μm.

Fig. 5.

Endogenous PIP4Kγ is differentially expressed in the adult mouse nephron. Actin (red) staining of cortical regions (A) identified luminal brush borders (arrow) in proximal tubules (d; distal tubule, p; proximal tubule), and localization of PIP4Kγ (green) in distal tubules (B). PIP4Kγ was not coincident with AQP1 (red) in the cortex (C) but was present in Tamm-Horsfall protein (THP; red) positive tubules in the outer medulla (D–F). PIP4Kγ was also seen in THP-negative tubules in this region (F; arrows). Double staining for PIP4Kγ (green) and AQP2 (red) in different kidney regions (G–L) revealed tubules positive for PIP4Kγ but negative for AQP2 in cortex and outer medulla (G, H; arrows). Isolated cells in AQP2-positive tubules also expressed PIP4Kγ (I–L; arrows). A, B, D–F, and K: images from horizontal sections. C, G–J, and L: images from sagittal sections. Scale bars = 20 μm.

Fig. 6.

Distribution of PIP4Kγ in kidney thick ascending limb (TAL) cells in situ. Endogenous PIP4Kγ was detected in thin sections of the outer medulla region of adult mouse kidney. Saggital kidney section shows longitudinal TAL tubule sections (A, C), and horizontal kidney section shows TAL tubule cross-sections (B and D). Scale bars = 10 μm. Statistical analysis of PIP4Kγ distribution across TAL tubule cross-sections (such as in B), by fluorescence intensity, indicated peaks in the luminal region (E; n = 25).

Fig. 7.

Overexpressed PIP4Kγ localizes to a specific cellular compartment. Transiently transfected cells, expressing GFP-tagged PIP4Kγ (green), were costained with antibodies to specific markers for different cellular compartments (red). No specific colocalization was observed with the endoplasmic reticulum marker calreticulin (A). Partial colocalization was observed with the cis-Golgi marker GM130 (B),and this juxtaposed labeling was maintained during Golgi dissociation by treatment with Brefeldin A (C). Scale bars = 10 μm.

Fig. 8.

Activity and binding characteristics of recombinant PIP4Ks. PIP4Kα and PIP4Kγ recombinant protein was purified from a bacterial source. A: PIP4K assay using equivalent amounts of recombinant protein from each PIP4K, showing generation of PtdIns(4,5)P₂. B: immunoprecipitation (IP) of recombinant PIP4K protein using PIP4Kα-specific antibody. IPs are from equivalent amounts of PIP4Kα, PIP4Kγ, or a mixture of the 2 isoforms, in buffer. Control IP contains no recombinant protein. Western blots (WB) were probed with either anti-PIP4Kα (i) or anti-PIP4Kγ (ii). Recombinant protein markers (rec) indicate PIP4K-positive bands (65 kDa).