Mitochondrial changes in cidofovir therapy for BK virus nephropathy.

Polyoma (BK) virus nephropathy (BKVN) is often treated with the nucleotide analog cidofovir. An adverse effect of this drug class is proximal tubular toxicity, and ultrastructural abnormalities in proximal tubular mitochondria have been observed in patients treated with similar drugs for other viral infections. We report similar changes in biopsies from BKVN treated with cidofovir. Renal allograft biopsies showing BKVN, on which electron microscopy was performed, were categorized into 3 groups: initial diagnosis (BD), postcidofovir treatment (CT), and posttreatment with immunosuppression reduction (IR). Nineteen cases from each group were randomly selected. Mitochondrial changes were present in 6 biopsies from patients receiving CT therapy (31.5%), ranging from diffuse mitochondrial swelling to profound morphologic changes. No similar abnormalities were seen in other groups. In those with atypical mitochondria, the mean number of cidofovir doses was 2.67, with an average interval between last dose and biopsy of 2.17 weeks. CT patients without mitochondrial changes had a mean of 4.6 doses and an average interval between last dose and biopsy of 27.2 weeks. Some renal transplant patients treated with cidofovir display alterations in proximal tubular mitochondria akin to those seen with similar drugs. The findings support the mitochondrial toxicity of nucleotide analogs.

Identifying specific causes of kidney allograft loss.

The causes of kidney allograft loss remain unclear. Herein we investigated these causes in 1317 conventional kidney recipients. The cause of graft loss was determined by reviewing clinical and histologic information the latter available in 98% of cases. During 50.3 +/- 32.6 months of follow-up, 330 grafts were lost (25.0%), 138 (10.4%) due to death with function, 39 (2.9%) due to primary nonfunction and 153 (11.6%) due to graft failure censored for death. The latter group was subdivided by cause into: glomerular diseases (n = 56, 36.6%); fibrosis/atrophy (n = 47, 30.7%); medical/surgical conditions (n = 25, 16.3%); acute rejection (n = 18, 11.8%); and unclassifiable (n = 7, 4.6%). Glomerular pathologies leading to failure included recurrent disease (n = 23), transplant glomerulopathy (n = 23) and presumed nonrecurrent disease (n = 10). In cases with fibrosis/atrophy a specific cause(s) was identified in 81% and it was rarely attributable to calcineurin inhibitor (CNI) toxicity alone (n = 1, 0.7%). Contrary to current concepts, most cases of kidney graft loss have an identifiable cause that is not idiopathic fibrosis/atrophy or CNI toxicity. Glomerular pathologies cause the largest proportion of graft loss and alloinmunity remains the most common mechanism leading to failure. This study identifies targets for investigation and intervention that may result in improved kidney transplantation outcomes.

The spectrum of antibody-mediated renal allograft injury: implications for treatment.

Improvements in anti-HLA antibody detection and diagnostic criteria have increased recognition of antibody-mediated rejection (AMR) following renal transplantation. Therapy of acute AMR is directed toward rapidly lowering circulating donor-specific antibody (DSA) activity. Despite reversal of acute renal dysfunction, however, antibody-secreting plasma cells in spleen and bone marrow are not depleted by treatment and circulating DSA commonly remains detectable in peripheral blood. Sequential ultrastructural studies of renal allografts during acute AMR show progression of microvascular endothelial abnormalities from necrosis and apoptosis to glomerular and peritubular capillary basement membrane duplication, termed transplant glomerulopathy (TG), a manifestation of chronic AMR. Additionally, long-term exposure to anti-HLA antibodies (particularly against class II antigens) is associated with shortened allograft survival and TG even in the absence of documented acute AMR. The association of TG with prior acute AMR and with circulating DSA provides evidence that antibody-mediated allograft injury exists as a spectrum of renal injury. Although effective therapy is available for acute AMR, allografts remain at risk for chronic AMR and shortened survival. The optimum approach to treatment for chronic AMR remains to be determined.

A mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1(del2/del2) mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1(del2/del2) mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD.

Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation.

Recent protocols have allowed successful positive crossmatch (+XM) and ABO incompatible (ABOI) kidney transplantation, although their long-term outcome is not clear. To begin to assess this issue we compared protocol biopsies performed 12 months posttransplant in 37 +XM, 24 ABOI and 198 conventional allografts. Although the majority in all three groups had only minimal histologic changes, transplant glomerulopathy (TG) was significantly increased in +XM (22% vs. 13% ABOI vs. 8% conventional, p = 0.015), and correlated with prior humoral rejection (HR) by multivariate analysis (odds ratio 17.5, p < or = 0.0001). Patients with a prior history of HR also had a significant increase in interstitial fibrosis (No HR 54% vs. HR 86%, p = 0.045). In the absence of HR no difference in histologic changes was seen between groups, although all three groups had a demonstrable mild increase in interstitial fibrosis from biopsies performed at the time of transplant. Thus, although HR is associated with an increase in TG, in its absence allograft histology is similar in +XM, ABOI and conventional allografts 1 year posttransplant.

Kidney transplant function and histological clearance of virus following diagnosis of polyomavirus-associated nephropathy (PVAN).

Polyomavirus-associated nephropathy (PVAN) is managed by reduced immunosuppression with or without antiviral therapy. Data from 55 patients with biopsy-proven PVAN were analyzed for adverse outcomes and influence of baseline variables and interventions. During 20+/-11 months follow-up, the frequencies of graft loss, major and any functional decline were 15%, 24% and 38%, respectively. Repeat biopsies were performed in 45 patients with persistent PVAN in 47%. Low-dose cidofovir, IVIG and cyclosporine conversion were used in 55%, 20% and 55% of patients. No single intervention was associated with improved outcome. Of the variables examined, only degree of interstitial fibrosis at diagnosis was associated with kidney function decline. In contrast, donor source, interstitial fibrosis, proportion of BKV positive tubules and plasma viral load at diagnosis were all associated with failure of histological viral clearance. This retrospective, nonrandomized analysis suggests that: (i) Graft loss within 2 years of PVAN diagnosis is now uncommon, but ongoing functional decline and persistent infection occur frequently. (ii) Low-dose cidofovir, IVIG and conversion to cyclosporine do not abrogate adverse outcomes following diagnosis. (iii) Fibrosis at the time of diagnosis predicts subsequent functional decline. Further elucidation of the natural history of PVAN and its response to individual interventions will require prospective clinical trials.

Metanephric adenoma, nephrogenic rests, and Wilms' tumor: a histologic and immunophenotypic comparison.

Metanephric adenoma (MA) is a renal tumor that is generally detected in adults and occasionally in children. These tumors usually behave in a benign fashion. Although the histogenesis of MA is unclear, a morphologic similarity to Wilms' tumor (WT) complex exists. Six cases of MA, five cases of childhood WT (CWT), two cases of adult WT (AWT), and four cases of treated MWT and/or nephrogenic rests (MWT/NR), with paraffin blocks available for use, were retrieved from the surgical pathology files of the Mayo Clinic. Clinical information was extracted from the medical record. Immunoperoxidase stains for WT1, AE1, CK7, CD57, CD56, and desmin were performed on paraffin sections from all cases. All six cases of MA were strongly and diffusely positive with antibodies to WT1 and CD57 and focally positive with antibodies to CK7. Three cases showed focal faint staining in <5% of the cells with keratin AE1. Stains for CD56 and desmin were negative. All seven cases of WT, including five CWT and two AWT, were strongly and diffusely positive with WT1 in the blastema and epithelium but showed only weak focal positivity in stromal cells. Six cases were diffusely positive for CD56 and one case showed focal positivity. Keratin AE1 was positive in one case of AWT and focally positive in the other AWT. The blastema of all cases of WT were negative for desmin, CK7, and CD57, although staining for keratin AE1, CD56, and CD57 was seen in maturing tubules of CWT cases. Of the five CWT cases, two had associated NR and two showed maturing WT after treatment. The areas of NR and maturing WT were histologically similar to MA and were composed of small tubules with uniform nuclei with no mitotic activity, scant cytoplasm, and focal calcifications. All four cases of maturing WT/NR were positive for WT1 and focally positive for CD57, CK7, and AE1. Stains for CD56 and desmin were negative except in foci of residual blastema, which stained for CD56 but lacked CD57 and CK7 staining. Five cases each of renal cell carcinoma, papillary renal cell carcinoma, and oncocytoma were negative for WT1. Two of five cases of chromophobe carcinoma showed very weak staining present in <10% of tumor nuclei. Metanephric adenoma is histogenetically related to WT and is morphologically and immunophenotypically identical to maturing WT and nephrogenic rests.

The pck rat: a new model that resembles human autosomal dominant polycystic kidney and liver disease.

BACKGROUND: The pck rat is a recently identified model of polycystic kidney disease (PKD) and liver disease (PLD) that developed spontaneously in the rat strain Crj:CD/SD. Its pattern of inheritance is autosomal recessive. METHODS: To characterize this new model, we studied pck rats derived from F9 breeding pairs from Charles River Japan and control Sprague-Dawley rats. Blood and tissues (kidneys, liver, and pancreas), obtained from these rats at 1, 7, 21, 70, and 182 days of age, were used for biochemical determinations, light and electron microscopy, and immunohistochemistry. RESULTS: The pck rats develop progressive cystic enlargement of the kidneys after the first week of age, and liver cysts are evident by day 1. The renal cysts developed as a focal process from thick ascending loops of Henle, distal tubules, and collecting ducts in the corticomedullary region and outer medulla. Flat and polypoid epithelial hyperplasia were common in dilated tubules and cysts. Apoptosis was common and affected normal, as well as dilated tubules, but less frequently cysts lined by flat epithelium. The basement membranes of the cyst walls exhibited a variety of alterations, including thinning, lamellation, and thickening. Focal interstitial fibrosis and inflammation were evident by 70 days of age. Segmental glomerulosclerosis and segmental thickening of the basement membrane with associated effacement of the podocyte foot processes were noted in some rats at 70 days of age. The PKD was more severe in male than in female pck rats, as reflected by the higher kidney weights, while there was no gender difference in the severity of the PLD. Mild bile duct dilation was present as early as one day of age. With age, it became more severe, and the livers became markedly enlarged. Even then, however, there was only a mild increase in portal fibrosis, without formation of fibrous septae. Slight elevations of plasma blood urea nitrogen levels were detected at 70 and 182 days of age. CONCLUSIONS: The pck rat is a new inherited model of PKD and PLD with a natural history and renal and hepatic histologic abnormalities that resemble human autosomal dominant PKD. This model may be useful for studying the pathogenesis and evaluating the potential therapies for PKD and PLD. The identification of the pck gene may provide further insight into the pathogenesis of autosomal dominant PKD.

Juxtaglomerular cell tumor: a clinicopathologic study of four cases and review of the literature.

We studied 4 new cases of juxtaglomerular cell tumor and compared their morphologic and immunohistochemicalfeatures with 2 renal hemangiopericytomas and 5 cutaneous glomus tumors. The juxtaglomerular tumors were resectedfrom 2 males and 2 females (mean age at diagnosis, 23 years). Three patients manifested with severe hypertension. Tumors ranged from 2.2 to 8.0 cm and were well circumscribed. The tumors consisted of solid sheets and nodules of variably sized tumor cells with round, oval, and spindled nuclei alternating with edematous microcystic foci. Nuclear atypia, present in all tumors, was a prominent feature in 2. Mitotic activity was not identified. All cases showed hemorrhage, numerous mast cells, and thick-walled blood vessels. Unusual features included coagulative tumor necrosis, a hemangiopericytoma-like vascular pattern, and hyalinized stroma. All tumors were immunoreactive for CD34 and actin. Ultrastructural analysis revealed the presence of rhomboid-shaped renin protogranules. Patients were treated by partial or radical nephrectomy and followed up for 14 to 48 months. There were no recurrences or metastases. The characteristic clinical and morphologic features of juxtaglomerular cell tumor permit distinction from renal hemangiopericytoma and other renal tumors.

Acute granulomatous interstitial nephritis and colitis in anticonvulsant hypersensitivity syndrome associated with lamotrigine treatment.

We present the case of a 17-year-old woman with a history of bipolar disorder, who developed a clinical syndrome manifested by fever, lymphadenopathy, skin rash, diarrhea, and acute renal failure requiring dialysis after the use of lamotrigine. Renal biopsy showed acute interstitial nephritis (AIN) with focal granulomas. Similarly, colonic biopsy specimens showed colitis and ileitis with non-necrotizing epithelioid granulomas. The patient had a complete recovery after withdrawal of the medication and steroid treatment. Although lamotrigine has been previously implicated in the development of anticonvulsant hypersensitivity syndrome, there have been no previous reports of acute granulomatous interstitial nephritis or colitis associated with the use of this drug.

Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds.

Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. Using immunohistochemistry, we examined thrombomodulin expression during healing of partial-thickness wounds in human skin and full-thickness wounds in mouse skin. We also examined thrombomodulin expression and wound healing in heterozygous thrombomodulin-deficient mice, compound heterozygous mice that have <1% of normal thrombomodulin anticoagulant activity, and chimeric mice derived from homozygous thrombomodulin-deficient embryonic stem cells. In both human and murine wounds, thrombomodulin was absent in keratinocytes at the leading edge of the neoepidermis, but it was expressed strongly by stratifying keratinocytes within the neoepidermis. No differences in rate or extent of reepithelialization were observed between wild-type and thrombomodulin-deficient mice. In chimeric mice, both thrombomodulin-positive and thrombomodulin-negative keratinocytes were detected within the neoepidermis. Compared with wild-type mice, heterozygous and compound heterozygous thrombomodulin-deficient mice exhibited foci of increased collagen deposition in the wound matrix. These findings demonstrate that expression of thrombomodulin in keratinocytes is regulated during cutaneous wound healing. Severe deficiency of thrombomodulin anticoagulant activity does not appear to alter reepithelialization but may influence collagen production by fibroblasts in the wound matrix.

Malignant cervical schwannoma: An unusual pelvic tumor.

BACKGROUND: Primary malignant cervical schwannomas (malignant peripheral nerve sheath tumors) are extremely rare tumors that grossly may resemble many other more common lesions. The diagnosis, management, and follow-up of a malignant cervical schwannoma are presented with a review of the literature. CASE: A 51-year-old female presented with a 3-year history of perimenopausal vaginal bleeding. A 3 x 3 cm friable, spongy lesion was noted on the posterior aspect of the cervix. Frozen-section analysis could not rule out a malignant smooth muscle tumor. The patient underwent an exploratory laparotomy, total abdominal hysterectomy, and bilateral salpingo-oophorectomy. Immunohistochemistry and electron microscopy aided in the final diagnosis of a malignant cervical schwannoma. The patient is alive and well 1 year from her definitive surgery. Another patient with the same tumor received the same surgical management. This patient is now 10 years from her surgery and is alive with no evidence of disease. CONCLUSION: Immunohistochemistry and electron microscopy are useful in the diagnosis of a malignant cervical schwannoma. This case and long-term follow-up from another case provide evidence that simple hysterectomy may be sufficient therapy for this uncommon lesion.

Leydig cell tumor of the testis: a clinicopathologic, DNA content, and MIB-1 comparison of nonmetastasizing and metastasizing tumors.

Leydig cell tumors of the testis are rare and account for a small proportion of testicular neoplasms. The objective of this study was to identify clinical and morphologic features predictive of metastasis in a large series of Leydig cell tumors, and to determine whether ploidy or proliferative activity were predictive of malignancy. Thirty cases of Leydig cell tumor of the testis (23 tumors that had not metastasized and 7 that had metastasized) were studied. Clinical history and follow-up were collected in all cases. The morphologic features examined included tumor size, mitotic index (mitotic figures/10 high-power fields), necrosis, angiolymphatic invasion, cell type, tumor-testicle interface, presence of extension beyond the testicular parenchyma, and presence of lipochrome and Reinke crystals. Most patients (93%) had a testicular mass. Patients with Leydig cell tumors that metastasized were diagnosed at a mean age of 62 years (range, 39-70 years) compared with 48 years (range, 9-79 years) in patients with nonmetastasizing tumors (p = 0.25). Leydig cell tumors that metastasized were significantly larger than nonmetastasizing tumors (mean, 4.7 versus 2.6 cm, respectively; p = 0.008), and had a significantly higher mitotic index (mean, 13.9 versus 1.9, respectively; p < 0.0001). Metastasizing Leydig cell tumors were significantly associated with atypical mitotic figures (p < 0.0001), nuclear variation (p = 0.0025), necrosis (p < 0.0001), angiolymphatic invasion (p = 0.009), infiltrative margins (p < 0.0001), high grade (p = 0.0004), and invasion into rete testis, epididymis, or tunica (p = 0.001) when compared with nonmetastasizing tumors. There was no significant difference between metastasizing and nonmetastasizing tumors in regard to cell type, lipochrome content, presence of Reinke crystals, or nuclear inclusions. All Leydig cell tumors that metastasized and 7 of 18 (38.9%) nonmetastasizing tumors were DNA aneuploid by static image analysis (p = 0.02). Metastasizing Leydig cell tumors had a significantly higher mean MIB-1 activity of 18.6% (range, 5.8-33.6) compared with 1.2% (range, 0.04-8.2) in nonmetastasizing tumors (p = 0.001). In this study, the presence of cytologic atypia, necrosis, angiolymphatic invasion, increased mitotic activity, atypical mitotic figures, infiltrative margins, extension beyond the testicular parenchyma, DNA aneuploidy, and increased MIB-1 activity were significantly associated with metastatic behavior in Leydig cell tumors.

Keratinocyte-specific expression of human thrombomodulin in transgenic mice: effects on epidermal differentiation and cutaneous wound healing.

BACKGROUND: Thrombomodulin is a cell-surface glycoprotein that regulates coagulation and fibrinolysis. Expression of thrombomodulin by epidermal keratinocytes is tightly regulated during squamous differentiation and cutaneous wound healing. METHODS: To determine the consequences of overexpression of thrombomodulin on squamous differentiation and wound healing in vivo, we expressed full-length human thrombomodulin in transgenic mice using the human keratin 14 promoter. Human thrombomodulin was detected in keratinocytes of transgenic mice by immunohistochemistry and protein C activation assays. Full-thickness cutaneous wounds were created on the dorsum of transgenic mice and nontransgenic littermates, and allowed to heal for up to 35 days. RESULTS: Transgenic mice had normal viability and appeared healthy up to one year of age. In the skin, human thrombomodulin was expressed in basal and suprabasal keratinocytes, with variable expression in the outer root sheath of hair follicles. Thrombomodulin activity in neonatal epidermis was 2.5- to 3-fold higher in transgenic mice than in nontransgenic littermates (p < 0.01). In cutaneous wounds, human thrombomodulin was expressed in migrating neoepidermal keratinocytes. No differences in keratinocyte migration or re-epithelialization were observed between transgenic and nontransgenic mice, but transgenic mice exhibited delayed collagen bundle deposition within the wound matrix. CONCLUSIONS: These findings demonstrate that keratinocyte thrombomodulin supports activation of protein C, and that thrombomodulin activity in epidermis can be increased by keratinocyte-specific expression of human thrombomodulin in transgenic mice. Expression of human thrombomodulin in keratinocytes does not impair normal squamous differentiation or re-epithelialization of cutaneous wounds, but may modulate collagen reconstitution of the wound matrix.

Field cancerization: why late "recurrent" ovarian cancer is not recurrent.

OBJECTIVE: Late "recurrence" of ovarian cancer may result from either regrowth of dormant tumor cells or from development of a new cancer caused by the phenomenon of field cancerization. Clinically, some recurrent ovarian cancers show the same therapeutic sensitivities to chemotherapy and surgery as did the primary disease, whereas others are refractory to all therapy. We hypothesize that recurrent ovarian cancers are distinguishable on the basis of a molecular genetic fingerprint and that some are actually new primary cancers of the peritoneum rather than recurrent ovarian cancer. STUDY DESIGN: We constructed molecular genetic fingerprints of 13 paired primary and late recurrent ovarian cancers to study their clonal relationships. The tumor pairs were analyzed for p53 mutations and allelotypes, patterns of X-chromosome inactivation, loss of heterozygosity, and microsatellite instability at 12 different loci on 6 different chromosomes. Techniques used included single-strand conformational polymorphism mutation screening and polymerase chain reaction-based sequence analysis of the p53 locus, restriction digestion of the androgen receptor locus to determine X-chromosome inactivation, and polyacrylamide gel electrophoresis of highly polymorphic dinucleotide, trinucleotide, and tetranucleotide repeats. RESULTS: The average age at initial diagnosis for this cohort was 54.7 years (range 45.3 to 65.5). Mean interval to recurrence was 42.7 months (range 28 to 62). Molecular fingerprints were characterized for 4 to 8 informative loci per tumor pair. The fingerprints of 10 (77%) differed significantly, strongly suggesting that a second primary cancer had developed. The remaining 3 tumor pairs demonstrated identical allelotypes consistent with regrowth of dormant tumor cells. CONCLUSION: Our results are consistent with the "field cancerization" hypothesis of ovarian carcinogenesis but could also be explained by a polyclonal tumor origin, which contrasts with the currently accepted monoclonal theory of ovarian carcinogenesis. Late development of a new primary cancer may herald the proband as a member of a familial cancer phenotype. These studies provide a molecular genetic rationale that both explains and prognosticates the clinical course of recurrent ovarian cancer.

Stage IIIC ovarian leiomyosarcoma in a premenopausal woman with multiple recurrences: prolonged survival with surgical therapy.

A case of Stage IIIC primary ovarian leiomyosarcoma in a premenopausal woman with multiple recurrences alive and well 7 years after diagnosis is presented. In addition to the typical light microscopic, immunohistochemical, and electron microscopic features of ovarian leiomyosarcoma, the tumor was progesterone receptor positive. This is 28th report of primary ovarian leiomyosarcoma and the first report of progesterone receptor in this tumor. This is the longest reported survival in a woman with this disease.

Complementation of reduced survival, hypotension, and renal abnormalities in angiotensinogen-deficient mice by the human renin and human angiotensinogen genes.

The aim of this study was to determine whether elements of the human renin-angiotensin system (RAS) could functionally replace elements of the mouse RAS by complementing the reduced survival and renal abnormalities observed in mice carrying a gene-targeted deletion of the mouse angiotensinogen gene (mAgt). Double transgenic mice containing the human renin (HREN) and human angiotensinogen (HAGT) genes were bred to mice heterozygous for the mAgt deletion and the compound heterozygotes were identified and intercrossed. The resulting progeny (n = 139) were genotyped at each locus and the population was stratified into two groups: the first containing both human transgenes (RA+) and the second containing zero or one, but not both human transgenes (RA-). Despite appropriate Mendelian ratios of RA- mice that were wildtype (+/+), heterozygous (+/-), and homozygous (-/-) for the deletion of mAgt at birth, there was reduced survival of RA- mAgt-/- mice to adulthood (P < 0.001 by chi2). In contrast, we observed appropriate Mendelian ratios of RA+ mAgt+/+, RA+ mAgt+/-, and RA+ mAgt-/- mice at birth and in adults (P > 0.05 by chi2). These results demonstrate that the presence of both human transgenes rescues the postnatal lethality in mAgt-/- mice. The renal histopathology exhibited by RA- mAgt-/- mice, including thickened arterial walls, severe fibrosis, lymphocytic infiltration, and atrophied parenchyma, was also rescued in the RA+ mAgt-/- mice. Direct arterial blood pressure recordings in conscious freely moving mice revealed that BP (in mmHg) varied proportionally to mAgt gene copy number in RA+ mice (approximately 20 mmHg per mAgt gene copy, P < 0.001). BP in RA+ mAgt-/- mice (132+/-3, n = 14) was intermediate between wild-type (RA- mAgt+/+, 105+/-2, n = 9) and RA+ mAgt+/+ (174+/-3, n = 10) mice. These studies establish that the human renin and angiotensinogen genes can functionally replace the mouse angiotensinogen gene, and provides proof in principle that we can examine the regulation of elements of the human RAS and test the significance of human RAS gene variants by a combined transgenic and gene targeting approach.

An abundance of p53 null mutations in ovarian carcinoma.

Ovarian cancers from 64 midwestern US women were screened for p53 dysfunction both by immunohistochemical staining (IHCS) and single strand conformation polymorphism (SSCP) analysis of the entire open reading frame (ORF). Forty SSCP abnormalities in 39 tumors included nine deletion, one insertion, two splice junction, two nonsense, one silent and 25 missense mutations were confirmed by direct genomic sequencing. Eight of the insertion/deletion defects may have occurred due to slippage during the course of DNA replication. This observation suggests that genomic instability may play an important role in ovarian carcinogenesis. Fifteen percent of the mutations encountered were located outside exons 5-9 and four of these were null. The sensitivity of IHCS was 96% for missense mutations but only 14% for null mutations. This contrasted with 100% sensitivity of the SSCP screening methodology. The 21% overall incidence of null mutations in the present study far exceeds the reported 6.8% incidence in the world literature (P=0.0003). Explanations for this difference include: (1) our complete analysis of the entire ORF of the p53 gene; (2) the tendency of others to rely upon IHCS to screen tumors prior to mutation analysis; and (3) environmental or endogenous genetic influences.