Email: rtorra@clinic.ub.es

Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest hereditary diseases in man, affecting 1/1000 individuals. It is characterized by the progressive development and enlargement of multiple cysts in the kidney that may ultimately lead to end stage renal disease (ESRD).

At least three different genes are involved in this disease: polycystic kidney disease 1 (PKD1), localized at 16p13.3; PKD2 localized at 4q13-23 and a third locus still unmapped. At present, several mutations have been described in the PKD1 and PKD2 genes, most of them are expected to produce truncated proteins (http://www.uwcm.ac.uk/uwcm/mg). These truncating mutations suggest that ADPKD is caused by inadequate levels of polycystin (i.e. haploinsufficiency), by a model of dominant/negative function, in which a mutated form of polycystin would inactivate the normal polycystin produced by the normal allele, or by a cellular recessive mechanism. The latter hypothesis has been recently studied due to the fact that less than 1% of nephrons become cystic. Evidence of loss of heterozygosity (LOH) at the PKD1 and PKD2 loci in cystic epithelia has been reported suggesting that cystogenesis in ADPKD results from the inactivation of the normal copy of the gene by a second somatic mutation.

It has been demonstrated that epithelial cells isolated from individual cysts are monoclonal and may follow the Knudson’s two-hit tumor suppressor gene model. The loss of the wild type allele would constitute a second hit which would be necessary for disease expression. Thus, a germ-line and a somatic inactivation of both copies of a PKD gene confers growth advantages for an individual cell to clonally expand into a cyst. It has been proven that a percentage of cells from different cysts show LOH or inactivating mutations in PKD1 or PKD2 (Table 1) both in renal and hepatic cystic epithelia in PKD1 or PKD2 patients, respectively. Renal epithelial cells have been demonstrated to be a frequent target tissue for somatic mutation. Martin et al demonstrated that somatic mutation in these cells constitute a frequent event. There are several reasons why renal epithelial cells are so prone to suffer from somatic mutations: they are metabolically active and consume large amounts of oxygen, they perform the majority of the secretory and resorptive work to obtain the renal filtrate, they are mitotically competent, specially when an injury to the kidney has occurred and eventually they are the cells from which the primary renal tumor and adenocarcinoma arise. Although it has been suggested that simple cysts in adults could also arise from LOH in both ADPKD alleles this hypothesis has yet to be proven, and we think that these cysts may basically be a consequence of tubular obstruction. In that case these cysts would not show clonality. It remains to be answered whether other extrarenal features of ADPKD, such as intracranial aneurysms, also arise from a second-hit mechanism. This hypothesis is quite feasible as all extrarenal features of ADPKD are focal and may arise from a single cell.

In order to demonstrate whether the process of cystogenesis occurs through a molecular recessive mechanism we have searched for somatic mutations and LOH in PKD1 and PKD2 kidneys and also in a PKD1 liver. Moreover we have looked for somatic mutations within the PKD2 gene. We have analyzed 9 PKD1 kidneys, 2 PKD2 kidneys and 1 PKD1 liver. We obtained cells from the interior of each cyst following Qian’s procedure, performed a preamplification and looked for loss of heterozygosity. For LOH studies the microsatellites studied were: KG8-3’UTR, D16S291 (AC2.5), D16S663 (CW2), D4S1563 and D3S1478. Moreover in PKD2 cysts we studied the presence of somatic mutations by SSCA.

The PKD1 gene covers 52 kb of genomic DNA, contains 46 exons and encodes a 14 kb mRNA. The predicted protein, polycystin, is a glycoprotein with 4302 amino acids. It contains 11 transmembrane domains, a large N extracellular tail that it is supposed to be involved in cell-cell or cell-matrix interactions, and a cytoplasmic C terminus. It contains a coiled-coil domain which interacts with PKD2. It is supposed that both proteins may function through a common signaling pathway that is necessary for normal tubulogenesis.

We have studied 9 kidneys and 1 liver from PKD1 patients and detected a rate of LOH of 13.3 %. This represents the largest number of cysts studied to date. This prevalence of LOH is a little lower than in other PKD1 studies, but higher than in PKD2 (Table 1). The difference with other PKD1 studies could be due to the large amount of cysts analyzed in this study (211) higher than in any other, and to the different percentage of LOH detected between kidneys (our range is between 6.7 and 20%). LOH is higher in PKD1 than in PKD2 cysts, this could be the result of a different mutational mechanism, gross deletions being more frequent in PKD1 than in PKD2. PKD2 mutational screening in cysts has been performed and a high percentage of cysts showing somatic point mutations has been detected. If we take into account both mutational mechanisms, we can see that there is no wild-type PKD2 protein in at least 30% of PKD2 cysts. On the other hand, PKD1 mutational screening is very difficult: it is a very large gene, with all but 3.5kb reiterated more proximally on the same chromosome and a small number of mutations being found in more than one family. Up to now, only 60 mutations have been identified, most of them resulting in a truncated protein ( http://www.uwcm.ac.uk/uwcm/mg ). In the literature mutational screening has been performed in cysts from only one PKD1 kidney. The authors searched for microsatellite LOH and a 9.5% rate was detected. They also screened from exon 15 to 34 of PKD1 and detected six different point mutations (that represent 28.6%). Thus, the average of second hit mutations is at least 38% (Table 1). There are several reasons why not every cyst shows LOH: point mutations could account for the second step and secondly, although we performed a strict washing of the cyst cavity, we cannot exclude contamination from cells other than the cyst epithelial cells and thus a false negative result would appear.

The PKD2 gene consists of 15 exons with an open reading frame of 2,904 bp. Polycystin-2, the PKD2 gene product, is predicted to be a 968-amino acid integral membrane protein with six membrane spanning domains and intracellular amino- and carboxyl-termini. At present, several germline mutations have been described in the PKD2 gene, including nonsense, splice-site, frameshift, and two putative missense mutations. Most PKD2 mutations are expected to produce truncated proteins. Recent studies have demonstrated that polycystin 2 interacts with polycystin 1 through its cytoplasmic carboxyl-end, thus truncating mutations would interfere with this interaction.

It has been suggested that, as polycystin 1 interacts with polycystin 2, somatic mutations in PKD2 could cooperate with the germline mutation in PKD1. Wu et al showed results that support a two hit hypothesis in mice. Their studies on a mouse model for PKD2 found that mice carrying a null allele and an allele that can undergo genomic rearrangements leading to a null allele, develop renal cysts at a higher frequency than those heterozygous for any of these alleles. More recently Koptides et al showed evidence of somatic point mutations for human PKD2 cysts, but did not find evidence of LOH. In human PKD2 polycystic kidneys, a variable percentage of cysts are not immunoreactive for polycystin 2, suggesting the presence of a second PKD2-somatic mutation therein. In contrast, polycystin 2 is strongly expressed in other PKD2 renal cysts.

One of the PKD2 kidneys we studied was from a patient in which the germline mutation consists of a deletion encompassing most of the PKD2 gene. Loss-of-heterozygosity (LOH) studies with microsatellite markers closely flanking the gene demonstrated loss of the wild-type PKD2 gene in 10% of cysts (3/30). No LOH for PKD1-linked markers was observed in any cyst. As the rate of LOH detected here was rather low (10%) to fully explain the two-hit theory, we decided to search for point mutations within the PKD2 gene that could account for a second hit. Since about 70% of the germline mutations in PKD2 have been found in exons 2, 4, 5, 6, 11, and 12, SSCP analysis was performed for these exons. We analyzed the remaining 27 cysts where we did not find LOH and detected eight with an abnormal SSCP pattern, which upon sequencing corresponded to eight different mutations.

All of them should result in a truncated PKD2 protein, six being frame-shift mutations due to the insertion or deletion of one or several nucleotides, one creates a premature stop codon and the last one affects the splicing of intron 4. In most cases sequencing revealed only the somatic-mutated allele, as the germline mutation was a deletion; however, since a small amount of non-cystic cells are frequently present, in some sequences we could detect traces of sequence that corresponded to the wild type allele. Overall, at least 37% of the PKD2 cysts from this kidney presented somatic mutations.

The other PKD2 kidney showed truncating somatic mutations in 60% of cysts, after analyzing all PKD2 exons, but no cyst showed LOH. It is remarkable that we did not know that this kidney was PKD2 until after not finding any LOH for PKD1, we then searched for the germline mutation within the PKD2 gene. Once we knew it was a PKD2 kidney we searched for PKD2 mutations in all PKD2 exons and found that almost 60% of cysts had a somatic mutation.

The fact that the rate of cyst formation in PKD2 is lower, may be explained because the rate of somatic mutation at this locus may be lower than at PKD1. One can speculate that PKD1 suffers from a higher rate of somatic mutations due to the presence of 3 polypyrimidine tracts within introns 1, 21 and 22. These polypyrimidine tracts may predispose to triple helix formation, erroneous repair, and mutations during transcription.

There is the possibility that the loss of one allele is the consequence of the process of hyperplasia that a cell undergoes and the consequent state of dedifferentiation that cells arising from this first cell achieve. In order to prove whether LOH was specific for ADPKD genes, we tested one microsatellite from a region in chromosome 3 (D3S1478) ,which is frequently deleted in human tumors, but we did not find any LOH for this marker in the studied cysts. Moreover no PKD1 cysts lost any PKD2 allele and vice versa. Brasier and Henske also studied PKD2 markers in PKD1 cysts and did not observe LOH. These findings support the theory that LOH constitutes an initial process during cyst formation.

Although the two-hit mechanism in PKD1 cystogenesis has been demonstrated, PKD1 immunoreactivity has been observed in the majority of cysts. Similarly, Ong et al. , working with material from one of the PKD2 kidneys studied by us, have found staining for polycystin 2 in about 85% of cysts, although 25% of them were partial positives. Perhaps the only way to reconcile these findings with the two-hit hypothesis would be if the somatic mutations were predominantly missense changes that maintain the epitope to which the antibody is directed against. However, the mutations detected by us cause either a loss of the gene product or truncated proteins lacking the carboxyl-end. Moreover, the germline mutation in one of the PKD2 families is a large deletion, which probably fails to produce a target for the antibody. Unfortunately, our results do not offer a satisfactory explanation as to the immunoreactivity for polycystin 2 observed in those cysts. Alternatively, it can be speculated that somatic mutations are not necessary for cyst formation but they arise later conferring an advantage to the mutant cell such that it repopulates the growing cyst. Further studies will be necessary to provide an explanation for this paradox.

The data presented herein reveals that a molecular recessive mechanism is implicated in the process of cystogenesis for PKD2 and PKD1. Polycystin 1 and 2 seems to be a member of a superfamily of proteins involved in regulation of renal epithelial cell growth. Thus, the loss of both copies of an ADPKD gene disrupts this cascade and causes hyperplasia of a given cell resulting in a cyst. This fact has important implications for the understanding of the pathophysiology of the disease and the development of new strategies for prevention or for retarding the progressive formation and enlargement of cysts. However many questions remain to be answered. Once the function of polycystins becomes elucidated the conciliation between the molecular and the immunohistochemical studies will be feasible.

REFERENCES RELATED TO THE TWO-HIT HYPOTHESIS IN ADPKD

Qian F, Watnick TJ, Onuchic L, Germino GG: The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I. Cell 87: 979-987, 1996.

Brasier JL, Henske EP: Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis. J Clin Invest 99: 194-199, 1997.

Koptides M, Constantinides R, Kyriakides G, Hadjigavriel M, Patsalis PC, Pierides A, Constantinou Deltas C: Loss of heterozygosity in polycystic kidney disease with a missense mutation in the repeated region of PKD1. Hum Genet 103: 709-717, 1998.

Wu G, D´Agati V, Cai Y, Markawitz G, Hoon Park J, Reynolds DM, Maeda Y, Le TC, Hou H, Kucherlapati R, Edelmann W, Somlo S: Somatic inactivation of PKD2 results in polycystic kidney disease. Cell 93: 177-188, 1998.

Watnick TJ, Torres VE, Gandolph A, Qian F, Onuchic L. Klinger KW, Landes G, and Germino GG: Somatic mutation in individual liver cysts supports a two-hit model of cystogenesis in autosomal dominant polycystic kidney disease. Molecular Cell 2: 247-251, 1998.

Koptides M, Hadjimichael C. Koupepidou P, Pierides A, Constantinou-Deltas C: Germinal and somatic mutations in the PKD2 gene of renal cysts in autosomal dominant polycystic kidney disease. Hum Molec Genet 8:509-513, 1999.

Martin GM, Ogburn CE, Colgin LM, Gown AM, Edland SD, Monnat RJ: Somatic mutations are frequent and increase with age in human kidney epithelial cells. Hum Molec Genet 5: 215-221, 1996.

Torra R, Badenas C, San Millán JL, Pérez-Oller L, Estivill X, Darnell A: A loss-of-function model for cystogenesis in human autosomal dominant polycystic kidney disease type 2. Am J Hum Genet 1999; 65: 345-352.

Pei Y, Watnick T, He N et al: Somatic PKD2 mutations in individual kidney and liver cysts support a “two-hit” model of cystogenesis in type 2 autosomal dominant polycystic kidney disease. J Am Soc Nephrol 1999; 10: 1524-1529.

Badenas C, Torra R , Pérez-Oller L, Mallolas J, Talbot-Wright R, Torregrosa V, Darnell A. Loss of heterozygosity in renal and hepatic epithelial cystic cells from PKD1 patients. Eur J Hum Genet (in press)