Renal amyloidosis: a new time for a complete diagnosis.

Amyloidoses are a group of disorders in which soluble proteins aggregate and deposit extracellularly in tissues as insoluble fibrils, causing organ dysfunction. Clinical management depends on the subtype of the protein deposited and the affected organs. Systemic amyloidosis may stem from anomalous proteins, such as immunoglobulin light chains or serum amyloid proteins in chronic inflammation or may arise from hereditary disorders. Hereditary amyloidosis consists of a group of rare conditions that do not respond to chemotherapy, hence the identification of the amyloid subtype is essential for diagnosis, prognosis, and treatment. The kidney is the organ most frequently involved in systemic amyloidosis. Renal amyloidosis is characterized by acellular pathologic Congo red-positive deposition of amyloid fibrils in glomeruli, vessels, and/or interstitium. This disease manifests with heavy proteinuria, nephrotic syndrome, and progression to end-stage kidney failure. In some situations, it is not possible to identify the amyloid subtype using immunodetection methods, so the diagnosis remains indeterminate. In cases where hereditary amyloidosis is suspected or cannot be excluded, genetic testing should be considered. Of note, laser microdissection/mass spectrometry is currently the gold standard for accurate diagnosis of amyloidosis, especially in inconclusive cases. This article reviews the clinical manifestations and the current diagnostic landscape of renal amyloidosis.

Renal amyloidosis: a new time for a complete diagnosis

Amyloidoses are a group of disorders in which soluble proteins aggregate and deposit extracellularly in tissues as insoluble fibrils, causing organ dysfunction. Clinical management depends on the subtype of the protein deposited and the affected organs. Systemic amyloidosis may stem from anomalous proteins, such as immunoglobulin light chains or serum amyloid proteins in chronic inflammation or may arise from hereditary disorders. Hereditary amyloidosis consists of a group of rare conditions that do not respond to chemotherapy, hence the identification of the amyloid subtype is essential for diagnosis, prognosis, and treatment. The kidney is the organ most frequently involved in systemic amyloidosis. Renal amyloidosis is characterized by acellular pathologic Congo red-positive deposition of amyloid fibrils in glomeruli, vessels, and/or interstitium. This disease manifests with heavy proteinuria, nephrotic syndrome, and progression to end-stage kidney failure. In some situations, it is not possible to identify the amyloid subtype using immunodetection methods, so the diagnosis remains indeterminate. In cases where hereditary amyloidosis is suspected or cannot be excluded, genetic testing should be considered. Of note, laser microdissection/mass spectrometry is currently the gold standard for accurate diagnosis of amyloidosis, especially in inconclusive cases. This article reviews the clinical manifestations and the current diagnostic landscape of renal amyloidosis.

Prevalence of autosomal dominant polycystic kidney disease in Persian and Persian-related cats in Brazil.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.

Prevalence of autosomal dominant polycystic kidney disease in Persian and Persian-related cats in Brazil / Prevalência da doença renal policística autossômica dominante em gatos Persas e raças relacionadas no Brasil

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.

A doença renal policística autossômica dominante (DRPAD) é a doença genética mais comum em gatos. No entanto, poucos dados sobre sua prevalência estão disponíveis no Brasil. Gatos Persas e de raças relacionadas foram avaliados por genotipagem molecular para a transversão C→A no exon 29 do gene PKD1 felino para determinar a prevalência de DRPAD. DNA genômico extraído de sangue total periférico ou amostras de swabs orais foram utilizados para amplificar o exon 29 do gene PKD1 pela técnica de PCR-RFLP. De um total de 616 gatos, 27/537 Persas e 1/17 Himalaia mostraram a variante de nucleotídeo único (C→A) na posição 3284 no exon 29 do gene PKD1. Esta variante patogênica foi identificada apenas em heterozigose. A prevalência de DRPAD em gatos Persas e raças relacionadas foram de 5,03% e 1,6%, respectivamente. Não houve associações significativas entre raça, gênero ou idade dos felinos e incidência de DRPAD. A prevalência de DRPAD em gatos Persas e raças relacionadas no Brasil foi menor do que em outras partes do mundo, o que pode estar relacionado ao aconselhamento genético e consequente seleção de gatos sem ADPKD para reprodução.

Identification of housekeeping genes for microRNA expression analysis in kidney tissues of Pkd1 deficient mouse models.

Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1-deficient mouse models from a panel of five candidates genes (miR-20a, miR-25, miR-26a, miR-191 and U6) and 3 target genes (miR-17, miR-21 and let-7a) using samples from kidneys of cystic mice (Pkd1flox/flox:Nestincre, CY), non-cystic controls (Pkd1flox/flox, NC), Pkd1-haploinsufficient (Pkd1+/-, HT), wild-type controls (Pkd1+/+, WT), severely cystic mice (Pkd1V/V, SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative ΔCt method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR-25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a, miR-25 and miR-26a. Our findings established miR-20a, miR-25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1-deficient mouse models.

Prevalence of autosomal dominant polycystic kidney disease in Persian and Persian-related cats in Brazil

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.

Resumo A doença renal policística autossômica dominante (DRPAD) é a doença genética mais comum em gatos. No entanto, poucos dados sobre sua prevalência estão disponíveis no Brasil. Gatos Persas e de raças relacionadas foram avaliados por genotipagem molecular para a transversão CA no exon 29 do gene PKD1 felino para determinar a prevalência de DRPAD. DNA genômico extraído de sangue total periférico ou amostras de swabs orais foram utilizados para amplificar o exon 29 do gene PKD1 pela técnica de PCR-RFLP. De um total de 616 gatos, 27/537 Persas e 1/17 Himalaia mostraram a variante de nucleotídeo único (CA) na posição 3284 no exon 29 do gene PKD1. Esta variante patogênica foi identificada apenas em heterozigose. A prevalência de DRPAD em gatos Persas e raças relacionadas foram de 5,03% e 1,6%, respectivamente. Não houve associações significativas entre raça, gênero ou idade dos felinos e incidência de DRPAD. A prevalência de DRPAD em gatos Persas e raças relacionadas no Brasil foi menor do que em outras partes do mundo, o que pode estar relacionado ao aconselhamento genético e consequente seleção de gatos sem ADPKD para reprodução.

Associations between OPG and RANKL polymorphisms, vertebral fractures, and abdominal aortic calcification in community-dwelling older subjects: the Sao Paulo Ageing & Health Study (SPAH).

This is the first study analyzing concomitantly osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) polymorphisms and OPG/RANKL serum levels and their association with bone mineral density (BMD), vertebral fractures, and vascular aortic calcification in a cohort of 800 subjects in community-dwelling older individuals. INTRODUCTION: Osteoprotegerin (OPG) and RANKL play an important role in osteoclast activation and differentiation as well as in vascular calcification. At present, there are no studies of OPG or RANKL gene polymorphisms in Brazilian older populations. The aim of this study was to evaluate OPG/RANKL polymorphism and their association with vertebral fractures (VFs) and aortic calcification. METHODS: Eight hundred subjects (497 women/303 men) were genotyped for the OPG 1181G>C (rs2073618), 163C>T (rs3102735), 245T>G (rs3134069), and 209G>A (rs3134070) and RANKL A>G (rs2277438) single-nucleotide polymorphisms (SNPs). VFs were evaluated by spine radiography (Genant's method). Aortic calcification was quantified using Kauppila's method. RESULTS: The isolated genotype analyses and single-allele frequency data showed association of OPG 163C, 245G, and 209A alleles with presence of VFs (P < 0.05). Multiple logistic regression of subjects with absence of VFs vs. those with VFs (grades II/III) revealed only OPG 209A homozygosity as a risk factor for higher-grade VFs (odds ratio (OR) = 4.17, 95 % CI 1.03-16.93, P = 0.046). Regarding aortic calcification, the isolated genotype analysis frequency data revealed a significant association of OPG 1181G, 163C, 245G, and 209A alleles with absent aortic calcification (P < 0.05). Multiple logistic regression data confirmed that the OPG 209A allele was protective for aortic calcification (OR = 0.63, 95 % CI 0.45-0.88, P = 0.007) and the OPG 1181C allele was a risk factor for aortic calcification (OR = 1.26, 95 % CI 1.00-1.58, P = 0.046). CONCLUSION: This study showed that the OPG 209AA genotype was a risk factor for higher-grade VFs, the OPG 209A allele was protective for aortic calcification, and the OPG 1181C was a risk factor for aortic calcification, supporting the involvement of OPG polymorphisms in the analyzed phenotypes and the concept that the related pathogenesis is multifactorial.

Cyst infection in hospital-admitted autosomal dominant polycystic kidney disease patients is predominantly multifocal and associated with kidney and liver volume

Positron-emission tomography/computed tomography (PET/CT) has improved cyst infection (CI) management in autosomal dominant polycystic kidney disease (ADPKD). The determinants of kidney and/or liver involvement, however, remain uncertain. In this study, we evaluated clinical and imaging factors associated with CI in kidney (KCI) and liver (LCI) in ADPKD. A retrospective cohort study was performed in hospital-admitted ADPKD patients with suspected CI. Clinical, imaging and surgical data were analyzed. Features of infected cysts were evaluated by PET/CT. Total kidney (TKV) and liver (TLV) volumes were measured by CT-derived multiplanar reconstruction. CI was detected in 18 patients who experienced 24 episodes during an interval of 30 months (LCI in 12, KCI in 10 and concomitant infection in 2). Sensitivities of CT, magnetic resonance imaging and PET/CT were 25.0, 71.4, and 95.0%. Dysuria (P<0.05), positive urine culture (P<0.01), and previous hematuria (P<0.05) were associated with KCI. Weight loss (P<0.01) and increased C-reactive protein levels (P<0.05) were associated with LCI. PET/CT revealed that three or more infected cysts were present in 70% of the episodes. TKV was higher in kidney-affected than in LCI patients (AUC=0.91, P<0.05), with a cut-off of 2502 mL (72.7% sensitivity, 100.0% specificity). TLV was higher in liver-affected than in KCI patients (AUC=0.89, P<0.01) with a cut-off of 2815 mL (80.0% sensitivity, 87.5% specificity). A greater need for invasive procedures was observed in LCI (P<0.01), and the overall mortality was 20.8%. This study supports PET/CT as the most sensitive imaging method for diagnosis of cyst infection, confirms the multifocal nature of most hospital-admitted episodes, and reveals an association of kidney and liver volumes with this complication.

Cyst infection in hospital-admitted autosomal dominant polycystic kidney disease patients is predominantly multifocal and associated with kidney and liver volume.

Positron-emission tomography/computed tomography (PET/CT) has improved cyst infection (CI) management in autosomal dominant polycystic kidney disease (ADPKD). The determinants of kidney and/or liver involvement, however, remain uncertain. In this study, we evaluated clinical and imaging factors associated with CI in kidney (KCI) and liver (LCI) in ADPKD. A retrospective cohort study was performed in hospital-admitted ADPKD patients with suspected CI. Clinical, imaging and surgical data were analyzed. Features of infected cysts were evaluated by PET/CT. Total kidney (TKV) and liver (TLV) volumes were measured by CT-derived multiplanar reconstruction. CI was detected in 18 patients who experienced 24 episodes during an interval of 30 months (LCI in 12, KCI in 10 and concomitant infection in 2). Sensitivities of CT, magnetic resonance imaging and PET/CT were 25.0, 71.4, and 95.0%. Dysuria (P<0.05), positive urine culture (P<0.01), and previous hematuria (P<0.05) were associated with KCI. Weight loss (P<0.01) and increased C-reactive protein levels (P<0.05) were associated with LCI. PET/CT revealed that three or more infected cysts were present in 70% of the episodes. TKV was higher in kidney-affected than in LCI patients (AUC=0.91, P<0.05), with a cut-off of 2502 mL (72.7% sensitivity, 100.0% specificity). TLV was higher in liver-affected than in KCI patients (AUC=0.89, P<0.01) with a cut-off of 2815 mL (80.0% sensitivity, 87.5% specificity). A greater need for invasive procedures was observed in LCI (P<0.01), and the overall mortality was 20.8%. This study supports PET/CT as the most sensitive imaging method for diagnosis of cyst infection, confirms the multifocal nature of most hospital-admitted episodes, and reveals an association of kidney and liver volumes with this complication.

Molecular and cellular pathogenesis of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human life-threatening monogenic disorders. The disease is characterized by bilateral, progressive renal cystogenesis and cyst and kidney enlargement, often leading to end-stage renal disease, and may include extrarenal manifestations. ADPKD is caused by mutation in one of two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC2 is a non-selective cation channel permeable to Ca2+, while PC1 is thought to function as a membrane receptor. The cyst cell phenotype includes increased proliferation and apoptosis, dedifferentiation, defective planar polarity, and a secretory pattern associated with extracellular matrix remodeling. The two-hit model for cyst formation has been recently extended by the demonstration that early gene inactivation leads to rapid and diffuse development of renal cysts, while inactivation in adult life is followed by focal and late cyst formation. Renal ischemia/reperfusion, however, can function as a third hit, triggering rapid cyst development in kidneys with Pkd1 inactivation induced in adult life. The PC1-PC2 complex behaves as a sensor in the primary cilium, mediating signal transduction via Ca2+ signaling. The intracellular Ca2+ homeostasis is impaired in ADPKD, being apparently responsible for the cAMP accumulation and abnormal cell proliferative response to cAMP. Activated mammalian target for rapamycin (mTOR) and cell cycle dysregulation are also significant features of PKD. Based on the identification of pathways altered in PKD, a large number of preclinical studies have been performed and are underway, providing a basis for clinical trials in ADPKD and helping the design of future trials.

Molecular and cellular pathogenesis of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human life-threatening monogenic disorders. The disease is characterized by bilateral, progressive renal cystogenesis and cyst and kidney enlargement, often leading to end-stage renal disease, and may include extrarenal manifestations. ADPKD is caused by mutation in one of two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC2 is a non-selective cation channel permeable to Ca(2+), while PC1 is thought to function as a membrane receptor. The cyst cell phenotype includes increased proliferation and apoptosis, dedifferentiation, defective planar polarity, and a secretory pattern associated with extracellular matrix remodeling. The two-hit model for cyst formation has been recently extended by the demonstration that early gene inactivation leads to rapid and diffuse development of renal cysts, while inactivation in adult life is followed by focal and late cyst formation. Renal ischemia/reperfusion, however, can function as a third hit, triggering rapid cyst development in kidneys with Pkd1 inactivation induced in adult life. The PC1-PC2 complex behaves as a sensor in the primary cilium, mediating signal transduction via Ca(2+) signaling. The intracellular Ca(2+) homeostasis is impaired in ADPKD, being apparently responsible for the cAMP accumulation and abnormal cell proliferative response to cAMP. Activated mammalian target for rapamycin (mTOR) and cell cycle dysregulation are also significant features of PKD. Based on the identification of pathways altered in PKD, a large number of preclinical studies have been performed and are underway, providing a basis for clinical trials in ADPKD and helping the design of future trials.

Molecular and cellular pathogenesis of autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited disease characterized by a malformation complex which includes cystically dilated tubules in the kidneys and ductal plate malformation in the liver. The disorder is observed primarily in infancy and childhood, being responsible for significant pediatric morbidity and mortality. All typical forms of ARPKD are caused by mutations in a single gene, PKHD1 (polycystic kidney and hepatic disease 1). This gene has a minimum of 86 exons, assembled into multiple differentially spliced transcripts and has its highest level of expression in kidney, pancreas and liver. Mutational analyses revealed that all patients with both mutations associated with truncation of the longest open reading frame-encoded protein displayed the severe phenotype. This product, polyductin, is a 4,074-amino acid protein expressed in the cytoplasm, plasma membrane and primary apical cilia, a structure that has been implicated in the pathogenesis of different polycystic kidney diseases. In fact, cholangiocytes isolated from an ARPKD rat model develop shorter and dysmorphic cilia, suggesting polyductin to be important for normal ciliary morphology. Polyductin seems also to participate in tubule morphogenesis and cell mitotic orientation along the tubular axis. The recent advances in the understanding of in vitro and animal models of polycystic kidney diseases have shed light on the molecular and cellular mechanisms of cyst formation and progression, allowing the initiation of therapeutic strategy designing and promising perspectives for ARPKD patients. It is notable that vasopressin V2 receptor antagonists can inhibit/halt the renal cystic disease progression in an orthologous rat model of human ARPKD.

Molecular and cellular pathogenesis of autosomal recessive polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited disease characterized by a malformation complex which includes cystically dilated tubules in the kidneys and ductal plate malformation in the liver. The disorder is observed primarily in infancy and childhood, being responsible for significant pediatric morbidity and mortality. All typical forms of ARPKD are caused by mutations in a single gene, PKHD1 (polycystic kidney and hepatic disease 1). This gene has a minimum of 86 exons, assembled into multiple differentially spliced transcripts and has its highest level of expression in kidney, pancreas and liver. Mutational analyses revealed that all patients with both mutations associated with truncation of the longest open reading frame-encoded protein displayed the severe phenotype. This product, polyductin, is a 4,074-amino acid protein expressed in the cytoplasm, plasma membrane and primary apical cilia, a structure that has been implicated in the pathogenesis of different polycystic kidney diseases. In fact, cholangiocytes isolated from an ARPKD rat model develop shorter and dysmorphic cilia, suggesting polyductin to be important for normal ciliary morphology. Polyductin seems also to participate in tubule morphogenesis and cell mitotic orientation along the tubular axis. The recent advances in the understanding of in vitro and animal models of polycystic kidney diseases have shed light on the molecular and cellular mechanisms of cyst formation and progression, allowing the initiation of therapeutic strategy designing and promising perspectives for ARPKD patients. It is notable that vasopressin V2 receptor antagonists can inhibit/halt the renal cystic disease progression in an orthologous rat model of human ARPKD.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo.

The most common form of autosomal dominant polycystic kidney disease (PKD) results from mutation of the PKD1 gene on chromosome 16p13.3. The gene encodes a 14-kb messenger RNA that is predicted to express a 462-kd membrane protein. The gene product, polycystin-1, has a large extracellular portion composed of a novel combination of protein-protein interacting domains and is postulated to be a plasma membrane receptor involved in cell-cell/matrix interactions. However, slow progress has been made in the characterization of polycystin-1 or the determination of its function. In fact, the protein is expressed at very low levels in tissues and cell lines and previous efforts directed at expression of recombinant protein had been largely unsuccessful. We have recently developed constructs of full-length human PKD1 complementary (cDNA) that can be expressed in both a stable and transient fashion in mammalian cells. We used these systems to characterize our antibodies and to track the protein in vivo. We report here the first biochemical characterization of recombinant polycystin-1 and show that the protein is a 520-kd glycosylated polypeptide with an unglycosylated core of 460 kd. Subcellular fractionation as well as biotinylation studies confirmed that the protein is plasma-membrane associated. Furthermore, we show that the recombinant protein localizes to cell-cell junctions in polarized madin darby canine kidney cells as revealed by indirect immunofluorescence. Our data represent the first characterization of polycystin-1 performed under highly controlled conditions.

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma.

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4(+) T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 +/- 19.1 pg/ml; for IL-8, 1724.2 +/- 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 +/- 55.6 pg/ml; for IL-8, 4371.4 +/- 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 +/- 4.39 vs baseline, MFI = 12.26 +/- 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 +/- 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.

Polycystin-1, the gene product of PKD1, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells.

The major form of autosomal dominant polycystic kidney disease (ADPKD) results from mutation of a gene (PKD1) of unknown function that is essential for the later stages of renal tubular differentiation. In this report, we describe a novel cell culture system for studying how PKD1 regulates this process. We show that expression of human PKD1 in MDCK cells slows their growth and protects them from programmed cell death. MDCK cells expressing PKD1 also spontaneously form branching tubules while control cells form simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases. Our study suggests that PKD1 may function to regulate both pathways, allowing cells to enter a differentiation pathway that results in tubule formation.

Genomic structure of the gene for the human P1 protein (MCM3) and its exclusion as a candidate for autosomal recessive polycystic kidney disease.

The locus PKHD1 (polycystic kidney and hepatic disease 1) has been linked to all typical forms of the autosomal recessive polycystic kidney disease (ARPKD) and maps to chromosome 6p21.1-p12. We previously defined its genetic interval by the flanking markers D6S1714 and D6S1024. In our current work, we have fine-mapped the gene for the human P1 protein (MCM3), thought to be involved in the DNA replication process, to this critical region. We have also established its genomic structure. Mutation analyses using SSCP were performed in ARPKD patients' cDNA samples, leading to the exclusion of this gene as a candidate for this disorder. We also identified two intragenic polymorphisms that allowed families with critical recombination events to be evaluated. Although neither marker was informative in these individuals, they are the closest yet described for PKHD1 and may help to refine the candidate region.

A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6.

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.