A mutation creating an out-of-frame alternative translation initiation site in the GRHPR 5'UTR causing primary hyperoxaluria type II.

Primary hyperoxaluria type II is a recessive genetic disorder caused by mutations in the GRHPR gene. Although several dozen mutations have been described, all affect coding or transcript splicing. A man suspected of having primary hyperoxaluria type II was heterozygous for a novel single-nucleotide deletion (c.694delC) in GRHPR affecting Gln(232) , which introduced a pre-mature termination (p.Gln232Argfs*3). Two 5'untranslated region (UTR) variants of unknown significance were also noted. We show that these two variants occur in cis, on the opposite allele, and introduce - immediately upstream of the canonical translation initiation site - a novel out-of-frame translational start site. In vitro studies using the GRHPR 5'UTR fused to a luciferase reporter show that the variant start site pre-empted initiation at the canonical translational start site, and this was corroborated within the broader context of 1.3 kb of the GRHPR proximal promoter. This latter mechanism may be underappreciated in general; reports of clinically significant functional variation of this type are extremely rare.

Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer.

Insulin-like growth factor-I (IGF-I)-mediated signaling is thought to be involved in the regulation of multiple cellular functions in different tumors including renal cell carcinoma (RCC). Blocking IGF-I signaling by any of the several strategies abolishes or delays the progression of a variety of tumors in animal models. Herein, we demonstrate that in RCC cell lines, IGF-I-mediated signaling is found to be inhibited in the presence of wild type von Hippel-Lindau (VHL) tumor suppresser gene. Moreover, molecular modeling and biochemical approaches have revealed that beta-domain of the VHL gene product by interacting directly with protein kinase Cdelta inhibits its association with IGF-IR for downstream signaling. We also demonstrated that RCC has IGF-I-mediated invasive activity where protein kinase Cdelta is an important downstream molecule, and this invasiveness can be blocked by wild type VHL. These experiments thus elucidate a novel tumor suppresser function of VHL with its unique kinase inhibitory domain.

An important von Hippel-Lindau tumor suppressor domain mediates Sp1-binding and self-association.

VHL is the causative gene for both von Hippel-Lindau (VHL) disease and sporadic clear-cell renal cancer. We showed earlier that VHL downregulates vascular endothelial growth factor transcription by directly binding and inhibiting the transcriptional activator Sp1. We have now mapped the VHL Sp1-binding domain to amino acids 96-122. The 96-122 domain is disproportionately affected by substitution mutations, which interfere with the VHL-Sp1 interaction. Deletion of the 96-122 domain prevents VHL effects on Sp1 DNA binding and on VHL target gene expression, indicating the domain contributes importantly to VHL tumor suppressor activity. Nevertheless, prevention of the VHL-Sp1 interaction only partially abrogates VHL's transcriptional repressor activity, supporting the existence of VHL transcriptional effectors in addition to Sp1. VHL also directly interacts with the Sp1 zinc fingers and self-associates via the 96-122 domain, which furthermore suggest the domain may bind other metalloproteins and contribute to VHL dominant-negative effects.

Advances in the molecular basis of renal neoplasia.

The past 2 years have provided exciting progress in elucidating the molecular basis of renal cancer. Work on the von Hippel-Lindau tumor suppressor, pVHL, in clear-cell renal cancer is already suggesting new potential therapies, and should have important implications in the pathogenesis of renal cystic disease and tumor angiogenesis. In addition, study of the Wilms' tumor suppressor, WT1, is revealing much about the pathogenesis of Wilms' tumor, urogenital development, and glomerular podocyte biology. c-met, the gene encoding the hepatocyte growth factor receptor, has recently been identified as a causative gene for hereditary papillary renal cancer. This review will highlight these and other new molecular advances in the renal cancer field.

Cellular activation triggered by the autosomal dominant polycystic kidney disease gene product PKD2.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC alpha and PKC epsilon, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts.

Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C zeta.

The transcription factor Sp1 is ubiquitously expressed and plays a significant role in the constitutive and induced expression of a variety of mammalian genes and may even contribute to tumorigenesis. Here, we describe a novel pathway whereby Sp1 promotes the transcription of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent angiogenic factor, by interacting directly and specifically with protein kinase C zeta (PKC zeta) isoform in renal cell carcinoma. PKC zeta binds and phosphorylates the zinc finger region of Sp1. Moreover, in the presence of the wild type von Hippel-Lindau gene product, the interaction of Sp1 with PKC zeta is inhibited, and in this manner steady state levels of Sp1 phosphorylation are decreased significantly. Co-transfection of renal cell carcinoma cells and human fibrosarcoma cells with a plasmid overexpressing PKC zeta and VPF/VEGF promoter luciferase constructs results in activation of Sp1-mediated transcription, whereas expression of a dominant-negative mutant of PKC zeta repressed this activation. Taken together, our results suggest a new pathway of cell signaling through PKC zeta and provide an insight into PKC zeta and Sp1-dependent transcriptional regulation of VPF/VEGF expression and thus tumor angiogenesis.

Transforming growth factor alpha is a target for the von Hippel-Lindau tumor suppressor.

The von Hippel-Lindau (VHL) tumor suppressor gene has a critical role in the pathogenesis of clear cell renal cell carcinoma (RCC), because VHL mutations have been found in both VHL disease-associated and sporadic RCC. Overexpression of transforming growth factor (TGF)-alpha has been observed in numerous RCC tumors and cell lines, and TGF-alpha has been demonstrated to support RCC cell growth through an autocrine loop. We demonstrate here that VHL substantially decreases TGF-alpha message and protein by shortening TGF-alpha mRNA half-life. By Northern analysis TGF-alpha mRNA steady-state levels were suppressed 5-fold in permanent 786-0 RCC cell lines expressing wild-type VHL compared with 786-0 cells expressing an empty vector or a mutant VHL protein lacking COOH-terminal residues 116-213 (deltaVHL). By Western analysis, VHL also substantially down-regulated the unprocessed, cell-associated Mr 20,000 TGF-alpha protein. Moreover, secreted TGF-alpha was undetectable in VHL-expressing cells. In contrast, VHL did not down-regulate the TGF-alpha receptor, epidermal growth factor receptor, either at the mRNA or protein level. Nuclear run-on in vitro transcription experiments in 786-0 cells showed that VHL did not affect transcriptional control of the endogenous TGF-alpha gene. However, actinomycin D experiments revealed a long TGF-alpha mRNA half-life in 786-0 cells that was significantly decreased by wild-type VHL but not by deltaVHL. We have, therefore, identified TGF-alpha, an important growth factor for RCC, as a new target gene for VHL and demonstrated that VHL acts by decreasing TGF-alpha mRNA stability.

The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity.

The von Hippel-Lindau tumor suppressor gene (VHL) has a critical role in the pathogenesis of clear-cell renal cell carcinoma (RCC), as VHL mutations have been found in both von Hippel-Lindau disease-associated and sporadic RCCs. Recent studies suggest that vascular endothelial growth factor (VEGF) mRNA is upregulated in RCC- and von Hippel-Lindau disease-associated tumors. We have therefore assessed the effect of the VHL gene product on VEGF expression. VEGF promoter-luciferase constructs were transiently cotransfected with a wild-type VHL (wt-VHL) vector in several cell lines, including 293 embryonic kidney and RCC cell lines. wt-VHL protein inhibited VEGF promoter activity in a dose-dependent manner up to 5- to 10-fold. Deletion analysis defined a 144-bp region of the VEGF promoter necessary for VHL repression. This VHL-responsive element is GC rich and specifically binds the transcription factor Sp1 in crude nuclear extracts. In Drosophila cells, cotransfected VHL represses Sp1-mediated activation but not basal activity of the VEGF promoter. We next demonstrated in coimmunoprecipitates that VHL and Sp1 were part of the same complex and, by using a glutathione-S-transferase-VHL fusion protein and purified Sp1, that VHL and Sp1 directly interact. Furthermore, endogenous VEGF mRNA levels were suppressed in permanent RCC cell lines expressing wt-VHL, and nuclear run-on studies indicated that VHL regulation of VEGF occurs at least partly at the transcriptional level. These observations support a new mechanism for VHL-mediated transcriptional repression via a direct inhibitory action on Sp1 and suggest that loss of Sp1 inhibition may be important in the pathogenesis of von Hippel-Lindau disease and RCC.

Sp1 is a critical regulator of the Wilms' tumor-1 gene.

We performed deletion analysis of WT1-reporter constructs containing up to 24 kilobases of 5'-flanking and first intron WT1 sequence in stably transfected cultured cells as an unbiased approach to identify cis elements critical for WT1 transcription. Although not a tissue-specific element, a proximate 9-base pair CTC repeat accounted for approximately 80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNA-protein complex in gel shifts. Antibody supershift, oligonucleotide competition, and Southwestern studies indicated that the CTC-binding factor is the transcriptional activator Sp1. Sp1 binds the CTC repeat with an affinity, KD = 0.37 nM, at least as high as the consensus GC box. Similar CTC repeats are found in promoters of other growth-related genes. Because Sp1 is important for WT1 expression, we examined Sp1 immunohistochemistry in fetal and adult kidney. In a pattern that precedes that of WT1 message, Sp1 immunostaining was highest in uninduced mesenchyme, early tubules, developing podocytes, and mature glomeruli, but was minimal in mature proximal tubules. This work suggests abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 may have a wider role in nephrogenesis.

A far upstream cis-element is required for Wilms' tumor-1 (WT1) gene expression in renal cell culture.

To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells. In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture.

Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron.

We recently reported a novel intracellular mechanism of Na-K-adenosinetriphosphatase (Na-K-ATPase) regulation in the cortical collecting duct (CCD) by agents that increase cell adenosine 3',5'-cyclic monophosphate (cAMP), which involves stimulation of protein kinase A (PKA) and phospholipase A2 (PLA2). We now determined whether this mechanism also operates in other nephron segments. In the medullary thick ascending limb (MTAL) dopamine, the DA1 agonist fenoldopam, forskolin, or dibutyryl-cAMP inhibited Na-K-ATPase activity, similar to results in CCD. In both segments this effect was blocked by 20-residue inhibitory peptide (IP20), a peptide inhibitor of PKA, but not by staurosporine, a protein kinase C (PKC) inhibitor. PKC activators phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-dioctanoylglycerol had no effect on Na-K pump activity in either CCD or MTAL. In contrast, all three PKC activators inhibited pump activity in the proximal convoluted tubule (PCT), an effect reproduced only by dopamine or by parathyroid hormone [PTH-(1-34)]. In PCT the pump inhibition by dopamine or PTH-(1-34) was abolished by staurosporine but not by IP20. The PLA2 inhibitor mepacrine prevented the effect of all agents, and arachidonic acid produced a dose-dependent pump inhibition in each of the three segments studied. We conclude that intracellular mechanisms of Na-K-ATPase regulation differ along the nephron, as they involve activation of PKA in CCD and MTAL and of PKC in PCT. These two pathways probably share a common mechanism in stimulating PLA2, arachidonic acid release, and production of eicosanoids in both the proximal and distal nephron.

Intracellular signaling in the regulation of renal Na-K-ATPase. II. Role of eicosanoids.

We recently reported a novel intracellular mechanism of renal Na-K-ATPase regulation by agents that increase cell cAMP, which involves protein kinase A-phospholipase A2 and is mediated by one or more arachidonic acid metabolites (Satoh, T., H. T. Cohen, and A. I. Katz. 1992. J. Clin. Invest. 89:1496). The present studies were, therefore, designed to assess the role of eicosanoids in the modulation of Na-K-ATPase activity in the rat cortical collecting duct. The effect of various cAMP agonists (dopamine, fenoldopam, vasopressin, forskolin, and dibutyryl cAMP), which inhibited the pump to a similar extent (approximately 50%), was independent of altered Na entry as it was elicited in the presence of amiloride or nystatin, or when NaCl was replaced with choline Cl. This effect was completely blocked by SKF 525A or ethoxyresorufin, two inhibitors of the cytochrome P450-dependent monooxygenase pathway, or by pretreating the animals with CoCl2, which depletes cytochrome P450. Equimolar concentrations (10(-7) M) of the cyclooxygenase inhibitors indomethacin or meclofenamate caused only a partial inhibition of the cAMP agonists' effect on the pump, whereas nordihydroguaiaretic acid or A 63162, two inhibitors of the lipoxygenase pathway, were without effect. Furthermore, two products of this pathway, leukotriene B4 and leukotriene D4, had no effect on Na-K-ATPase activity, and ICI 198615, a leukotriene receptor antagonist, did not alter pump inhibition by cAMP agonists. Several P450 monoxygenase arachidonic acid metabolites (5,6-epoxyeicosatrienoic acid; 11,12-epoxyeicosatrienoic acid; 11,12-dihydroxyeicosatrienoic acid; and 12(R)-hydroxyeicosatetraenoic acid) as well as PGE2 inhibited the Na:K pump in dose-dependent manner, but the effect of PGE2 was blocked when Na availability was altered, whereas that of 12(R)-HETE remained unchanged. We conclude that the cytochrome P450-monooxygenase pathway of the arachidonic acid cascade plays a major role in the modulation of Na:K pump activity by eicosanoids in the rat cortical collecting duct, and that products of the cyclooxygenase pathway may contribute to pump inhibition indirectly, by decreasing intracellular Na.

Dopamine inhibits Na/K-ATPase in single tubules and cultured cells from distal nephron.

Dopamine decreases tubular sodium reabsorption, attributed in part to Na/K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where we have demonstrated specific dopamine DA1 binding sites, we examined the effects of dopamine, and of DA1 and DA2 receptor agonists on the Na/K pump in the microdissected rat cortical collecting duct (CCD) and in Madin-Darby canine kidney (MDCK) cells, a line derived from the dog distal nephron. Dopamine inhibited pump activity in CCD by approximately 40%-50%, an effect proportionally larger than in the PCT. Unlike in the latter, the effect of dopamine was reproduced by the DA1 agonist fenoldopam, which inhibited the CCD pump in dose-dependent manner (maximum, 10 microM). The DA2 agonist quinpirole was without effect, either alone or in combination with fenoldopam. These actions on Na/K-ATPase paralleled in reciprocal fashion effects on adenylate cyclase: dopamine or fenoldopam, but not quinpirole, produced a significant increase in cAMP content, and the stimulation by dopamine was blocked by SCH 23390. Inhibitors of cAMP phosphodiesterase (3-isobutyl-1-methyl-xanthine and theophylline), as well as forskolin and dibutyryl-cAMP, mimicked the effect of dopamine on the pump, underscoring the role of increased cAMP in this phenomenon. Both dopamine and fenoldopam inhibited Na/K-ATPase activity in MDCK cells. The results indicate that besides the PCT dopamine inhibits Na/K-ATPase activity in cells of the distal nephron, where its effect on the pump appears to be more pronounced and is mediated by activation of the DA1 receptor. The natriuretic effect of dopamine is probably exerted at both proximal and distal nephron sites.

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron.

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular signaling in the regulation of renal Na-K-ATPase. I. Role of cyclic AMP and phospholipase A2.

We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. This pathway is shared by other agonists that increase cell cAMP and thus stimulate PKA activity.

Localization of dopamine-1 receptors along the microdissected rat nephron.

Dopamine exerts numerous actions on the kidney but the precise location of its receptor subtypes along the nephron is unknown. Using a microassay we determined the specific binding of 125I-Sch 23982, a specific and selective dopamine-1 (DA1) receptor antagonist, to microdissected glomeruli and tubule segments. Binding of 125I-Sch 23982 in the proximal convoluted tubule (PCT) was time- and concentration dependent, saturable and reversible. The linear Scatchard plot of saturation experiments suggested binding to a single site with an apparent Kd of 16.7 nM and Bmax of 0.4 fmol.mm-1 in the PCT, and 6.2 nM and 0.1 fmol.mm-1 in the cortical collecting tubule (CCT). Mapping of DA1 binding sites along the nephron revealed their presence in each of the segments examined, albeit in markedly different concentrations: the highest specific binding was measured in PCT followed by the pars recta. Binding was less in the distal nephron, and least in the medullary and cortical thick ascending limb. Modest binding was also detected in glomeruli. In cortical collecting tubules competition studies with unlabeled dopamine and probes for DA1 (Sch 23390, fenoldopam), DA2 (domperidone, S-sulpiride), serotonergic (serotonin, ketanserin, mianserin), and alpha-(phentolamine) and beta-(propranolol) adrenergic receptors indicated a rank-order potency for displacement of 125I-Sch 23982 binding, consistent with labeling of DA1 receptors. Dopamine inhibited Na/K-ATPase both in PCT and CCT, an effect duplicated in the latter segment by the DA1 agonist fenoldopam, and blocked by the DA1 antagonist Sch23390.(ABSTRACT TRUNCATED AT 250 WORDS)