A "two-hit" hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD) A gene mutation can result in disease through direct or indirect mechanisms. For instance, in the gain-offunction mutation, a germline mutant allele confers new or enhanced protein activity with a pathologic function, whereas a dominant-negative mutation produces an aberrant protein that interferes with the function of the normal protein..In haploinsufficiency, a loss of 50% of normal protein as a result of a mutation in one of its alleles is sufficient to cause disease. In the two-hit mechanism, the disease results from a germline mutation in one allele, followed by the subsequent acquisition of a somatic mutation in the second normal allele with no remaining functional protein.Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. PKD1 and PKD2 are the genes that encode for the polycystin-1 (PC1) and polycystin-2 (PC2) proteins, respectively. Although patients with ADPKD carry heterozygous mutations in either PKD1 or PKD2 and present 100% penetrance of cystic kidney phenotypes, fewer than 5% of nephrons form cysts. These fluid-filled cysts are lined by a single layer of epithelial cells and can occur at any site along the nephron. The presence of renal cysts in ADPKD, despite the low number, results in a gradual decline in renal function. To explain the focal nature of renal cyst formation in ADPKD, Reeders (1) proposed a "two-hit" hypothesis suggesting that a second somatic alteration to the gene, in addition to a germline mutation, is a prerequisite to the disease phenotype. Although a mechanism based on haploinsufficiency has not been excluded, somatic mutations in either PKD1 or PKD2 indeed have been found in several ADPKD cyst-lining epithelia (2-8), even though a somatic loss of other chromosomes or mutations in other loci also are found (2). These data provided hints that ADPKD is a recessive disease at the cellular level. The lack of a cellular assay for PC1 function has prevented an experimental demonstration of loss of function in cyst-lining epithelia in ADPKD.We and others have shown previously that PC1 and PC2 are localized to the primary cilia (9). The mechanosensation function of polycystins can be assayed in cultured mouse kidney epithelial cells by monitoring changes in the intracellular calcium concentration in response to fluid-flow shear stress (10). To test the loss-of-function hypothesis in ADPKD with regard to mechanosensory ability, we used the flow assay to examine shear stress-induced calcium responses in cells that were derived from a heterozygous Pkd1 mouse model. Furthermore, we

Environmental and industrial lead exposures continue to pose major public health problems in children and in adults. Acute exposure to high concentrations of lead can result in proximal tubular damage with characteristic histologic features and manifested by glycosuria and aminoaciduria. Chronic occupational exposure to lead, or consumption of illicit alcohol adulterated with lead, has also been linked to a high incidence of renal dysfunction, which is characterized by glomerular and tubulointerstitial changes resulting in chronic renal failure, hypertension, hyperuricemia, and gout. A high incidence of nephropathy was reported during the early part of this century from Queensland, Australia, in persons with a history of childhood lead poisoning. No such sequela has been found in studies of three cohorts of lead-poisoned children from the United States. Studies in individuals with low-level lead exposure have shown a correlation between blood lead levels and serum creatinine or creatinine clearance. Chronic low-level exposure to lead is also associated with increased urinary excretion of low molecular weight proteins and lysosomal enzymes. The relationship between renal dysfunction detected by these sensitive tests and the future development of chronic renal disease remains uncertain. Epidemiologic studies have shown an association between blood lead levels and blood pressure, and hypertension is a cardinal feature of lead nephropathy. Evidence for increased body lead burden is a prerequisite for the diagnosis of lead nephropathy. Blood lead levels are a poor indicator of body lead burden and reflect recent exposure. The EDTA lead mobilization test has been used extensively in the past to assess body lead burden. It is now replaced by the less invasive in vivo X-ray fluorescence for determination of bone lead content.Imagesp928-aFigure 1.Figure 2.Figure 2.Figure 3.

Hypertension is a common complication of autosomal dominant polycystic kidney disease (ADPKD), often present before the onset of renal failure. A role for the renin-angiotensin system (RAS) has been proposed, but studies of systemic RAS have failed to show a correlation between plasma renin activity and blood pressure in ADPKD. Ectopic renin expression by cyst epithelium was first reported in 1992 (Torres VE, Donovan KA, Sicli G, Holley KE, Thibodeau ST, Carretero OA, Inagami T, McAteer JA, and Johnson CM. Kidney Int 42: 364-373, 1992). It is not known, however, whether other RAS components are also expressed by cysts in ADPKD. We show that, in addition to renin, angiotensinogen (AGT) is produced by some cysts and dilated tubules. Angiotensin-converting enzyme, ANG II type 1 receptor, and ANG II peptide are also present within cysts and in many tubules; and some cyst fluids contain high ANG II concentrations. Additionally, cyst-derived cells in culture continue to express the components of the RAS at both the protein and mRNA levels. We further show that renin is expressed primarily in cysts of distal tubule origin and in cyst-derived cells with distal tubule characteristics, whereas AGT is expressed primarily in cysts of proximal tubule origin and in cyst-derived cells with proximal tubule characteristics. Renin production by cyst-derived cells appears to be regulated by extracellular Na+ concentration. Based on these observations, we propose a model of an autocrine/paracrine RAS in polycystic kidney disease, whereby overactivity of the intrarenal system results in sustained increases in intratubular ANG II concentrations.

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by "second hit" somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37 degrees C in the WT cell lines and at 33 degrees C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39 degrees C. The majority (29) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.