Peter Gerke

NEPH1 defines a novel family of podocin interacting proteins

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid‐resistant proteinuria. In addition, mice lacking NEPH1 develop a nephrotic syndrome that resembles NPHS mutations, suggesting that all three proteins are essential for the integrity of glomerular podocytes. Podocin interacts with the C‐terminal domain of nephrin and facilitates nephrin‐dependent signaling. NEPH1, a member of the immunoglobulin superfamily, is structurally related to nephrin. We report now that NEPH1 belongs to a family of three closely related proteins that interact with the C‐terminal domain of podocin. All three NEPH proteins share a conserved podocin‐binding motif; mutation of a centrally located tyrosine residue dramatically lowers the affinity of NEPH1 for podocin. NEPH1 triggers AP‐1 activation similarly to nephrin but requires the presence of Tec family kinases for efficient transactivation. We conclude that NEPH1 defines a new family of podocinbinding molecules that are potential candidates for hereditary nephrotic syndromes not linked to either NPHS1 or NPHS2.

Homodimerization and Heterodimerization of the Glomerular Podocyte Proteins Nephrin and NEPH1

Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig superfamily. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pattern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phylogenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin revealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational glycosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and heterodimeric interactions that may facilitate cis- and trans- homodimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm.

The kidney as a second site of human C-reactive protein formation in vivo.

C‐reactive protein (CRP) is the main acute phase reactant in humans. Its production is presumably restricted to the liver but extrahepatic expression by inflamed tissue has not been studied indetail. By real‐time PCR and immunohistochemistry we here show that renal cortical tubular epithelial cells (TEC) express CRP mRNA and protein within 6 h after stimulation with conditioned medium (CM) or IL‐6, but not IL‐1α or TNF‐α. Western blot analysis with monoclonal anti‐CRP antibody that recognizes native CRP revealed protein secretion into supernatants of CM‐stimulated TEC cultures. While hepatoma‐derived Hep3B cells could be induced similarly, peripheral blood mononuclear cells could not. CRP mRNA transcripts were observed in nephrectomized renal allografts with severe acute rejection but not with chronic allograft nephropathy (CAN). Of 19 needle biopsies of acutely rejecting kidney transplants, 15 demonstrated CRP mRNA production with the relative expression levels increasing with the severity of rejection. On the other hand, none of 7 graft biopsies with acute tubular necrosis (ATN) or CAN showed CRP mRNA expression. By using monoclonal anti‐CRP antibody, cortical tubules as well as glomerular cells were shown to locally express CRP in rejecting, but not in ATN kidneys. We conclude that inflamed kidneys represent a so far unknown site of CRP formation in vivo. These data shed new light on the acute phase reaction not merely representing a systemic inflammatory pathway but probably being part of the local immune response.

Nephrocystin interacts with Pyk2, p130Cas, and tensin and triggers phosphorylation of Pyk2

Juvenile nephronophthisis type 1 is caused by mutations of NPHP1, the gene encoding for nephrocystin. The function of nephrocystin is presently unknown, but the presence of a Src homology 3 domain and its recently described interaction with p130Cas suggest that nephrocystin is part of the focal adhesion signaling complex. We generated a nephrocystin-specific antiserum and analyzed the interaction of native nephrocystin with endogenous proteins. Immunoprecipitation of nephrocystin revealed that nephrocystin forms protein complexes with p130Cas, proline-rich tyrosine kinase 2 (Pyk2), and tensin, indicating that these proteins participate in a common signaling pathway. Expression of nephrocystin resulted in phosphorylation of Pyk2 on tyrosine 402 as well as activation of downstream mitogen-activated protein kinases, such as ERK1 and ERK2. Our findings suggest that nephrocystin helps to recruit Pyk2 to cell matrix adhesions, thereby initiating phosphorylation of Pyk2 and Pyk2-dependent signaling. A lack of functional nephrocystin may compromise Pyk2 signaling in a subset of renal epithelial cells.

β-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity

β-Arrestins mediate internalization of plasma membrane receptors. Nephrin, a structural component of the glomerular slit diaphragm, is a single transmembrane spanning receptor and belongs to the family of adhesion molecules. Its mutation causes a hereditary nephrotic syndrome. We report the previously undescribed interaction of β-arrestin2 with the nephrin C terminus. The phosphorylation status of nephrin Y1193 regulates inversely the binding of β-arrestin2 and podocin. The Src-family member Yes, known to enhance podocin–nephrin interaction by nephrin phosphorylation, diminishes β-arrestin2–nephrin interaction. β-Arrestin2 induces nephrin endocytosis and attenuates nephrin signaling. This finding suggests that nephrin Y1193 serves as a molecular switch that determines the integrity of the slit diaphragm by functional competition between β-arrestin2 and podocin. This concept offers a molecular pathomechanism of slit diaphragm distortion and opens therapeutic avenues for glomerular diseases.

NEPH2 Is Located at the Glomerular Slit Diaphragm, Interacts with Nephrin and Is Cleaved from Podocytes by Metalloproteinases

The NEPH family comprises three transmembrane proteins of the Ig superfamily interacting with the glomerular slit diaphragm proteins podocin and ZO-1. NEPH1 binds to nephrin, another component of the slit diaphragm, and loss of either partner causes heavy proteinuria. NEPH2, which is strongly conserved among a large number of species, is also expressed in the kidney; however, its function is unknown. The authors raised NEPH2 antisera to demonstrate NEPH2 expression in a variety of mouse tissues, including the kidney and a podocyte cell line. The authors localized the expression of NEPH2 to the glomerular slit diaphragm by electron microscopy and show NEPH2 homodimerization and specific interactions with the extracellular domain of nephrin in vitro and in vivo. NEPH1, however, failed to interact with NEPH2. The authors detected immunoreactive NEPH2 in urine of healthy subjects, suggesting that the extracellular domain is cleaved under physiologic conditions. These findings were confirmed in vitro in podocyte cell culture. Shedding is increased by tyrosine phosphatase inhibitors and diminished by GM6001, an inhibitor of metalloproteinases. Overexpression experiments indicate an involvement of the MT1-matrix metalloproteinase. The results suggest a role for NEPH2 in the organization and/or maintenance of the glomerular slit diaphragm that may differ from the functions of NEPH1 and nephrin.

The polycystin-1 C-terminal fragment triggers branching morphogenesis and migration of tubular kidney epithelial cells

Mutations of either PKD1 or PKD2 cause autosomal dominant polycystic kidney disease, a syndrome characterized by extensive formation of renal cysts and progressive renal failure. Homozygous deletion of Pkd1 or Pkd2, the genes encoding polycystin-1 and polycystin-2, disrupt normal renal tubular differentiation in mice but do not affect the early steps of renal development. Here, we show that expression of the C-terminal 112 amino acids of human polycystin-1 triggers branching morphogenesis and migration of inner medullary collecting duct (IMCD) cells, and support in vitro tubule formation. The integrity of the polycystin-2-binding region is necessary but not sufficient to induce branching of IMCD cells. The C-terminal domain of polycystin-1 stimulated protein kinase C-alpha (PKC-alpha), but not the extracellular signal-regulated kinases ERK1 or ERK2. Accordingly, inhibition of PKC, but not ERK, prevented polycystin-1-mediated IMCD cell morphogenesis. In contrast, HGF-mediated morphogenesis required ERK activation but was not dependent on PKC. Our findings demonstrate that the C-terminal domain of polycystin-1, acting in a ligand-independent fashion, triggers unique signaling pathways for morphogenesis, and likely plays a central role in polycystin-1 function.

Neuronal Expression and Interaction with the Synaptic Protein CASK Suggest a Role for Neph1 and Neph2 in Synaptogenesis

Formation, differentiation, and plasticity of synapses require interactions between pre‐ and postsynaptic partners. Recently, it was shown that the transmembrane immunoglobulin superfamily protein SYG‐1 is required for providing synaptic specificity in C. elegans. However, it is unclear whether the mammalian orthologs of SYG‐1 are also involved in local cell interactions to determine specificity during synapse formation. We used in situ hybridization, immunohistochemistry, and immunogold electron microscopy to study the temporal and spatial expression of Neph1 and Neph2 in the developing and adult mouse brain. Both proteins show similar patterns with neuronal expression starting around embryonic days 12 and 11, respectively. Expression is strongest in areas of high migratory activity. In the adult brain, Neph1 and Neph2 are predominantly seen in the olfactory nerve layer and the glomerular layer of the olfactory bulb, in the hippocampus, and in Purkinje cells of the cerebellum. At the ultrastructural level, Neph1 and Neph2 are detectable within the dendritic shafts of pyramidal neurons. To a lesser extent, there is also synaptic localization of Neph1 within the stratum pyramidale of the hippocampal CA1 and CA3 region on both pre‐ and postsynaptic sites. Here it colocalizes with the synaptic scaffolder calmodulin‐associated serin/threonin kinase (CASK), and both Neph1 and Neph2 interact with the PDZ domain of CASK via their cytoplasmic tail. Our results show that Neph proteins are expressed in the developing nervous system of mammals and suggest that these proteins may have a conserved function in synapse formation or neurogenesis. J. Comp. Neurol. 498:466–475, 2006.

Increased Expression of Secreted Frizzled-Related Protein 4 in Polycystic Kidneys

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.

Nephrogenic systemic fibrosis following exposure to gadolinium-containing contrast agent

Nephrogenic systemic fibrosis (NSF) is a disease recently described in patients with kidney failure. It is characterized by scleroderma-like thickening of the skin, subcutaneous edema and ensuing joint contractures leading to profound disability. Furthermore, involvement of internal organs has been described. Whereas the pathogenesis is not known to date, recent reports have linked NSF to high doses of gadolinium-containing contrast agents given at magnetic resonance angiography (MRA). We describe a patient with severe NSF. The patient had received erythropoietin and had undergone vascular interventions which are suspected risk factors for this disease. Notably, the disease developed shortly after the application of gadolinium at an MRA, giving support to the recently published hypothesis that gadolinium-containing contrast agents are among the causative factors. We provide a short overview and hope to raise overall awareness towards this entity and the use of MRA contrast agents in renal patients.