Biodistribution of quantum dots in the kidney after intravenous injection.

The biodistribution of nanoparticles is a major subject of current nanomedical research. To date, however, the exact investigation of nanoparticle fate in the microenvironment of a main excretory organ, the kidney has largely been neglected. In this study, the biodistribution of polyethylene glycol-coated quantum dots (Qdots) with special focus on their interaction with the kidney is investigated. Upon intravenous injection, nanoparticles showed effective blood circulation in mice and significant renal accumulation after two hours. Histological analysis of the kidney revealed that Qdots were strongly associated to the intraglomerular mesangial cells. This preferential deposition of nanoparticles in the kidney mesangium is highly promising, since it could be of utmost value for site-specific treatment of severe kidney diseases like diabetic nephropathy in the future.

Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.

The mechanisms leading to the recovery of the kidney after ischemic acute renal failure are poorly understood. To explore the role played by mitogenesis and dedifferentiation in this repair process and to identify whether the genetic response of the nephron segments reflects the level of susceptibility to injury, the temporal and nephron segment expressions of various proteins implicated in mitogenesis, differentiation, and injury were determined. Proliferating cell nuclear antigen (PCNA), a marker for the G1-S transition in the cell cycle and hence mitogenesis, was detected primarily in the S3 segment of the proximal tubule, with maximal expression at 2 d postischemia. Vimentin, normally present in mesenchymal cells but not epithelial cells, and hence a marker for the state of differentiation, was prominently expressed in the S3 segment 2-5 d postischemia. In the S3 segments in the outer stripe of the medulla cells that stained positively for PCNA also stained positively for vimentin. Clusterin, a marker for cell injury, was expressed primarily in the S3 segment and in the distal tubule with distinct staining patterns in each segment. None of the cells that stained with clusterin antibodies were positively stained with PCNA or vimentin antibodies. Likewise, none of the PCNA or vimentin-positive cells expressed clusterin at detectable levels. Thus, in the S3 segment, where there is significant ischemic injury, surviving cells express markers indicating that they undergo mitogenesis and dedifferentiate in the postischemic period. While there is some expression of c-Fos in the S3 segment, c-Fos was expressed predominantly, at 1 and 3 h postischemia, in the nuclei of the distal nephron, particularly in the thick ascending limb. The data support the view that the mature renal S3 segment epithelial cell can be a progenitor cell.

The swelling-activated chloride channel ClC-2, the chloride channel ClC-3, and ClC-5, a chloride channel mutated in kidney stone disease, are expressed in distinct subpopulations of renal epithelial cells.

The mammalian genome encodes at least nine different members of the ClC family of chloride channels. So far only two of them could be localized on a cellular level in the kidney. We now report on the precise intrarenal localization of the mRNAs coding for the chloride channels ClC-2, ClC-3 and ClC-5. Expression of ClC-2 mRNA, encoding a swelling-activated chloride channel, could be demonstrated in the S3 segment of the proximal tubule. The chloride channel ClC-3 mRNA and ClC-5 mRNA, coding for a chloride channel mutated in kidney stone disease, were both expressed in intercalated cells of the connecting tubule and collecting duct. Whereas ClC-3 mRNA expression was most prominent in the cortex of rat kidneys, ClC-5 mRNA was expressed from the cortex through the upper portion of the inner medulla. A detailed analysis revealed that ClC-3 was expressed by type B intercalated cells, whereas ClC-5 was expressed by type A intercalated cells. These findings have important implications for the pathogenesis of hereditary kidney stone disease caused by mutations in the CLCN5 gene.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

TRPP2 channel regulation.

Polycystin-2, or TRPP2 according to the TRP nomenclature, is encoded by PKD2, a gene mutated in patients with autosomal-dominant polycystic kidney disease. Its precise subcellular location and its intracellular trafficking are a matter of intense debate, although consensus has emerged that it is located in primary cilia, a long-neglected organelle possibly involved in sensory functions. Polycystin-2 has a calculated molecular mass of 110 kDa, and according to structural predictions it contains six membrane-spanning domains and a pore-forming region between the 5th and 6th membrane-spanning domain. This section irst introduces the reader to the field of cystic kidney diseases and to the PKD2 gene, before the ion channel properties of polycystin-2 are discussed in great detail.

Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo?

In patients with cutaneous T-cell lymphomas (CTCL), soluble interleukin-2 receptor serum levels (sIL-2R) were determined by ELISA technique, and natural killer cell (NK) activity, by a 4-h chromium-51 release assay. Decrease of NK activity correlated with the augmentation of serum sIL-2R. After a 4-d stimulation with interleukin 2 CTCL patients' peripheral mononuclear cells (PMC) showed an increase of cytotoxic activity similar to that in healthy donors' PMC. Normal donors' PMC demonstrated a diminished IL-2-induced cytotoxic activity in 25% CTCL serum (sIL-2R of 3000, 7330, and 10700 U/ml, respectively) compared to control serum (sIL-2R of 400, 340, and 420 U/ml, respectively). IL-2-dependent proliferation of 2-d phytohemagglutinin (PHA) blasts was lower in CTCL serum than in control serum. sIL-2R was enriched from one CTCL patient's serum by IL-2 affinity chromatography. Transfection of the Tac gene into NIH/3T3 fibroblasts resulted in the production of a recombinant sIL-2R. The presence of enriched native or recombinant sIL-2R inhibited interleukin-2-dependent generation of cytotoxic activity and PHA blast proliferation. We suggest that elevated sIL-2R levels account for diminished NK activity by neutralizing interleukin 2 in CTCL patients.

Expression of clusterin in Crohn's disease of the terminal ileum.

Crohn's disease (CD) is a chronic inflammatory intestinal disorder with disturbance and injury of the intestinal mucosal barrier, in which various proinflammatory molecules as well as molecules with antiinflammatory activity and cytoprotective function are found to be expressed. We investigated whether clusterin, a multifunctional cytoprotective protein, is upregulated in Crohn's disease, because augmented expression of clusterin is seen in many organs following various forms of tissue injury. Human actively and inactively inflamed ileal tissues from CD patients as well as normal intestinal specimens from control patients (normal ileum) were investigated by Western blot analysis, immunohistochemisty and in situ hybridization. As compared with controls, a strongly enhanced expression of clusterin was found in CD tissues, correlating with disease activity. Immunohistochemistry and in situ hybridization analysis revealed foci of crypts almost completely lined by clusterin expressing enterocytes in CD, a feature that was never seen in controls. Such crypts appeared especially within the morphologically intact mucosa apart from erosive or ulcerative lesions. Besides epithelia, clusterin was also expressed by inflammatory mononuclear cells. Enhanced expression of clusterin by crypt epithelia might reflect a cytoprotective function of the protein in order to prevent further injury of the intestinal mucosal barrier in CD.

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells.

OBJECTIVES: Polycystin-1 (PC1), a signalling receptor regulating Ca(2+)-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca(2+) homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca(2+) entry (NCCE) and Ca(2+) oscillations, with downstream effects on cell proliferation. RESULTS AND DISCUSSION: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca(2+) oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca(2+) oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca(2+) oscillations and a molecular mechanism to explain the association between abnormal Ca(2+) homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.

The proximal tubule phenotype and its disruption in acute renal failure and polycystic kidney disease.

In light of recent developments in the fields of genetics, molecular, cell and developmental biology, the kidney is receiving increasing attention as a model system for organ development and human diseases. Gene disruption experiments have provided evidence for the essential role of a number of proteins in the earliest phase of nephron development, but very little is known about the identity of such proteins in more advanced stages. This minireview will focus on the proximal tubule and its role in the pathology of ischemic acute renal failure and polycystic kidney disease. Like all other nephron segments, the proximal tubule develops from the metanephrogenic mesenchyme. So far the only genetic model which affects the function of the proximal tubule is a strain of knockout mice with an inactivation of the HNF1 gene. After ischemic renal damage the proximal tubule responds with a different genetic program than the distal tubule. Evidence from human polycystic kidney disease and several animal models of polycystic kidney disease suggests that proximal tubules are affected differently by polycystic kidney disease than distal tubules and collecting ducts.

Update in podocyte biology.

Knowledge of podocyte biology is growing rapidly. Podocytes are crucially involved in most hereditary diseases affecting the glomerulus, which all exhibit podocyte-specific defects, that is, foot process effacement and protein leakage. Efforts to understand molecular mechanisms causing these derangements are increasingly successful and will allow a better targeting of interventions to halt the progression of chronic renal disease.

Regulation of clusterin expression following spinal cord injury.

We have investigated the localization and regulation of a putative extracellular chaperone, clusterin, in the rat spinal cord after lesion. In control animals, clusterin is expressed in motoneurons, in meningeal and ependymal cells, and in astrocytes mainly located beneath the pial surface. Beginning at day 2 after hemisection at segmental level C6, clusterin levels increase in GFAP-positive astrocytes within the lesioned segment. Three weeks after trauma, clusterin mRNA and protein are elevated in neurons close to the lesion site and in glial elements within scar tissue and within degenerating fiber tracts rostral and caudal to the lesion. This study provides evidence for a role of clusterin in the subacute and late phase of spinal cord injury.

A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail.

Expression of newly cloned cDNAs in mammalian cell lines is an essential tool for the functional analysis of the proteins encoded by these cDNAs. In many instances, however, evaluation of the protein is difficult because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins on Western blots or for immunocytochemistry or immunoprecipitation. A number of gene fusion systems have been employed in which a known peptide is fused to the expression product of interest and the fusion protein is purified using affinity chromatography and identified in extracts or by immunocytochemistry using antibodies directed against the affinity handle peptide. The DNA-binding domain of the yeast transcription factor GAL4 is widely used to construct fusion proteins with putative transcription factors to evaluate potential trans-acting domains. Because of the lack of commercially available anti-GAL4 antibodies, the further biochemical characterization of these fusion proteins has remained difficult. We describe the construction of two mammalian expression vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for pM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast transcription factor GAL4 with a Flag peptide (consisting of the 11-amino-acid leader peptide of the gene 10 product from bacteriophage T7) at the NH2-terminus and a tail of six histidines at the COOH-terminus. Unique restriction sites allow both the construction of fusion proteins with the GAL4 DNA-binding domain and the replacement of the GAL4 fragment with another insert.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common monogenetic diseases in humans. The discovery that mutations in the PKD1 and PKD2 genes are responsible for ADPKD has sparked extensive research efforts into the physiological and pathogenetic role of polycystin-1 and polycystin-2, the proteins encoded by these two genes. While polycystin-1 may mediate the contact among cells or between cells and the extracellular matrix, a lot of evidence suggests that polycystin-2 represents an endoplasmic reticulum-bound cation channel. Cyst development has been compared to the growth of benign tumors and this view is highlighted by the model that a somatic mutation in addition to the germline mutation is responsible for cystogenesis (two-hit model of cyst formation). Since in vitro polycystin-1 and polycystin-2 interact through their COOH termini, the two proteins possibly act in a common pathway, which controls the width of renal tubules. The loss of one protein may lead to a disruption of this pathway and to the uncontrolled expansion of tubules. Our increasing knowledge of the molecular events in ADPKD has also started to be useful in designing novel diagnostic and therapeutic strategies.

An ever-expanding story of cyst formation.

Autosomal-dominant polycystic kidney disease represents one of the most common monogenetic human disorders. The cloning of the PKD1 and PKD2 genes, which are mutated in far more than 90% of the patients affected by this disease, has generated high hopes for a quick understanding of the pathogenesis of cyst formation. However, these expectations have not yet been fulfilled, since the function of both polycystin-1 and polycystin-2, the two proteins encoded by PKD1 and PKD2, still remains a puzzle. In this review, we will highlight some of the characteristics of polycystic kidney disease, briefly touch on polycystin-1, and then go on to describe recent results of experiments with polycystin-2, since the latter is the major focus of our work. We will discuss new evidence which suggests that autosomal-dominant polycystic kidney disease actually behaves recessively on a cellular level. Finally, a model will be presented that tries to explain the available data.

Genomic structure and chromosomal location of the rat gene encoding the zinc finger transcription factor Kid-1.

We have previously cloned and sequenced a novel zinc finger cDNA, Kid-1, from the rat. Because of its developmentally regulated expression pattern and its suppression after renal injury, as well as its kidney-predominant expression, we propose that Kid-1 is likely to play an important role in renal gene regulation. Kid-1 encodes a predicted protein with 13 zinc fingers at the carboxy end and Krüppel-associated box (KRAB) A and B regions at the amino terminus. Expression of a Kid-1-GAL4 chimeric protein results in strong transcriptional repression of cotransfected constructs containing GAL4 binding sites and a chloramphenicol acetyl transferase gene driven by either a minimal promoter or a SV40 enhancer. We now report the cloning, structural organization, and chromosomal localization of the Kid-1 gene. The Kid-1 gene is composed of four exons and three introns, closely reflecting the organization of the Kid-1 protein. The KRAB A and B regions are encoded by the second and third exons, respectively. The entire zinc finger region is encoded by the fourth exon. Using a combination of linkage analysis and somatic cell hybrid analysis, Kid-1 was mapped to rat chromosome (RNO) 10. Kid-1, Il3, and Sparc form a tight linkage group on RNO10. Regional sublocalization to RNO10q21.3-q22 was established by fluorescence in situ hybridization.

The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression.

We have previously reported the cloning, sequencing, and partial characterization of Kid-1, a zinc finger-encoding cDNA from the rat kidney. The Kid-1 protein and approximately one-third of all other zinc finger proteins contain a highly conserved region of approximately 75 amino acids at their NH2 terminus named Krüppel-associated box (KRAB), which is subdivided into A and B domains. The evolutionary conservation, wide distribution, and genomic organization of the KRAB domains suggest an important role of this region in the transcriptional regulatory function of zinc finger proteins. The functional significance of the KRAB domain was evaluated by studying transcriptional activities of yeast GAL4-rat Kid-1 fusion proteins containing various regions of the non-zinc-finger domain of Kid-1. Transcriptional repressor activity of GAL4-Kid-1 fusion proteins maps to the KRAB-A domain. The KRAB-A domain of another zinc finger protein, ZNF2, also has repressor activity. Site-directed mutagenesis of conserved amino acids in this motif results in decreased repressor activity. Thus, we have established a functional significance for the KRAB-A domain, a consensus sequence common in zinc finger proteins.

The Krüppel-associated box (KRAB)-zinc finger protein Kid-1 and the Wilms' tumor protein WT1, two transcriptional repressor proteins, bind to heteroduplex DNA.

Zinc finger proteins of the Cys2His2 class represent a large group of DNA-binding proteins. A major subfamily of those proteins, the Krüppel-associated box (KRAB) domain-containing Cys2His2-zinc finger proteins, have been described as potent transcriptional repressors. So far, however, no DNA-binding sites for KRAB domain-containing zinc finger proteins have been isolated. Using a polymerase chain reaction-based selection strategy with double- and single-stranded DNA, we failed to reveal a binding site for Kid-1, one member of KRAB-zinc finger proteins. Binding of Kid-1 both to single- and homoduplex double-stranded DNA was negligible. We now present evidence that Kid-1 binds to heteroduplex DNA. Similar to Kid-1, the non-KRAB-zinc finger protein WT1 also bound avidly to heteroduplex DNA (both the -KTS and +KTS splice variant of WT1), whereas the POU domain protein Oct-6, the ets domain protein Ets-1 and the RING finger of BRCA-1 did not bind to heteroduplex DNA. Binding of WT1 to heteroduplex DNA was markedly reduced in naturally occurring mutants. The recognition of certain DNA structures by transcriptional repressor proteins may therefore represent a more common phenomenon than previously thought.

The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton.

Despite the recent positional cloning of the PKD1 and PKD2 genes, which are mutated in the great majority of patients with autosomal-dominant polycystic kidney disease (PKD), the pathogenic mechanism for cyst formation is still unclear. The finding, that the PKD1 and PKD2 proteins interact with each other through their COOH termini, suggests that both proteins are part of the same protein complex or signal transduction pathway. Using a yeast two-hybrid screen with the PKD2 protein, we isolated the PKD2-interacting protein Hax-1. The specificity of the interaction was demonstrated by the fact that PKD2L, a protein closely related to PKD2, failed to interact with Hax-1. Immunofluorescence experiments showed that in most cells PKD2 and Hax-1 colocalized in the cell body, but in some cells PKD2 and Hax-1 also were sorted into cellular processes and lamellipodia. Furthermore we demonstrated an association between Hax-1 and the F-actin-binding protein cortactin, which suggests a link between PKD2 and the actin cytoskeleton. We speculate that PKD2 is involved in the formation of cell-matrix contacts, which are dysfunctional without a wild-type PKD2 protein, thus leading to cystic enlargement of tubular structures in the kidney, liver, and pancreas.

Expression of the transcriptional repressor protein Kid-1 leads to the disintegration of the nucleolus.

The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.