Gretchen S. Crary

Involvement of p21 and p27 in the regulation of CDK activity and cell cycle progression in the regenerating liver

In tissue culture systems, p21 and p27 inhibit cyclin-dependent kinase (CDK) activity and cell cycle progression in response to numerous stimuli, but little is known about their involvement in cell growth in vivo. We examined the modulation of CDK activity by these proteins after 70% partial hepatectomy (PH), an in vivo model of synchronous hepatocyte cell cycle progression. After PH in BALB/c mice, p21 was induced during the prereplicative (G1) phase and was maximally expressed after peak hepatocyte DNA synthesis. p27 was present in quiescent liver and was minimally induced after PH. p21 and p27 immunoprecipitated with CDK2, CDK4, and cyclin D1 in the regenerating liver. The activity of CDK2-, CDK4- and cyclin D1-associated kinases was upregulated after PH, and maximal activity of these enzyme complexes corresponded to peak DNA synthesis. Immunodepletion experiments suggested that p27 plays a role in downregulating CDK2 activity before and after peak DNA synthesis. Compared to cogenic wild-type mice, p21−/− mice demonstrated evidence of markedly accelerated hepatocyte progression through G1 phase after PH: DNA synthesis, upregulation of cyclin A and PCNA, induction of cyclin D1- and CDK2-associated kinase activity, and appearance of a phosphorylated retinoblastoma protein (Rb) species occurred earlier in the p21−/− mice. These results suggest that p21 and p27 modulate CDK activity in the regenerating liver, and that p21 regulates the rate of progression through G1 phase of the cell cycle in vivo.

Akt-mediated Liver Growth Promotes Induction of Cyclin E through a Novel Translational Mechanism and a p21-mediated Cell Cycle Arrest

The control of hepatocyte growth is relevant to the processes of liver regeneration, development, metabolic homeostasis, and cancer. A key component of growth control is the protein kinase Akt, which acts downstream of mitogens and nutrients to affect protein translation and cell cycle progression. In this study, we found that transient transfection of activated Akt triggered a 3-4-fold increase in liver size within days but only minimal hepatocyte proliferation. Akt-induced liver growth was associated with marked up-regulation of cyclin E but not cyclin D1. Analysis of liver polyribosomes demonstrated that the post-transcriptional induction of cyclin E was associated with increased translational efficiency of this mRNA, suggesting that cell growth promotes expression of this protein through a translational mechanism that is distinct from the cyclin D-E2F pathway. Treatment of Akt-transfected mice with rapamycin only partially inhibited liver growth and did not prevent the induction of cyclin E protein, indicating that target of rapamycin activity is not necessary for this response. In the enlarged livers, cyclin E-Cdk2 complexes were present in high abundance but were inactive due to increased binding of p21 to these complexes. Akt transfection of p21-/- mice promoted liver growth, activation of Cdk2, and enhanced hepatocyte proliferation. In conclusion, growth promotes cyclin E expression through a novel translational mechanism in the liver, suggesting a new link between cell growth and the cell cycle machinery. Furthermore, p21 suppresses proliferation in the overgrown livers and may play a role in preventing cell cycle progression in response to organ size homeostatic mechanisms.

Interobserver Variability in Thyroid Fine-Needle Aspiration Interpretation of Lesions Showing Predominantly Colloid and Follicular Groups

We studied interobserver variability (IV) in the assessment of thyroid fine-needle aspiration (FNA). We limited our cases to those showing predominantly colloid and follicular cell groups. Twenty cases of thyroid FNA diagnosed by 1 experienced cytopathologist were reviewed by 4 other cytopathologists who made their own diagnoses while unaware of the original diagnoses. Two cytopathologists then assessed the cytologic features of the 20 cases. IV was calculated for noncollapsed and collapsed diagnoses. Diagnoses and observer agreement were compared with cytologic features. There was little correlation among observers regarding the diagnosis of follicular lesion vs neoplasm. IV was somewhat poor before data were collapsed to treatment recommendations (kappa = 0.35) but was relatively good when data were collapsed (kappa = 0.65). Cellularity, cyst change, and amount of colloid correlated with treatment recommendations; no specific features correlated with poor performance. Thyroid FNA shows good interobserver agreement in the diagnoses of lesions showing predominantly colloid or follicular cells (when collapsed). We speculate that IV is poor in some cases owing to difficulty assessing thin colloid, some lack of agreement regarding criteria for adequacy, and a possible gray zone that might exist with lesions showing colloid and abundant follicular cells.

The Role of Procurement Biopsies in Acceptance Decisions for Kidneys Retrieved for Transplant

BACKGROUND AND OBJECTIVESnThere is a shortage of kidneys for transplant, and many patients on the deceased donor kidney transplant waiting list would likely benefit from kidneys that are currently being discarded. In the United States, the most common reason given for discarding kidneys retrieved for transplant is procurement biopsy results. This study aimed to compare biopsy results from discarded kidneys with discard attributed to biopsy findings, with biopsy results from comparable kidneys that were successfully transplanted.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnIn this retrospective, observational, case-control study, biopsy reports were examined from 83 kidneys discarded in 2010 due to biopsy findings (cases), 83 contralateral transplanted kidneys from the same donor (contralateral controls), and 83 deceased donors randomly matched to cases by donor risk profile (randomly matched controls). A second procurement biopsy was obtained in 64 of 332 kidneys (19.3%).nnnRESULTSnThe quality of biopsy reports was low, with amounts of tubular atrophy, interstitial inflammation, arteriolar hyalinosis, and acute tubular necrosis often not indicated; 69% were wedge biopsies and 94% used frozen tissue. The correlation between first and second procurement biopsies was poor; only 25% of the variability (R(2)) in glomerulosclerosis was explained by biopsies being from the same kidney. The percentages of glomerulosclerosis overlapped substantially between cases, contralateral controls, and randomly matched controls: 17.1%±15.3%, 9.0%±6.6%, and 5.0%±5.9%, respectively. Of all biopsy findings, only glomerulosclerosis>20% was independently correlated with discard (cases versus contralateral controls; odds ratio, 15.09; 95% confidence interval, 2.47 to 92.41; P=0.003), suggesting that only this biopsy result was used in acceptance decisions. One-year graft survival was 79.5% and 90.7% in contralateral and randomly matched controls, respectively, versus 91.6% among all deceased donor transplants in the Scientific Registry of Transplant Recipients.nnnCONCLUSIONSnRoutine use of biopsies could lead to unnecessary kidney discards.

Cdk2 plays a critical role in hepatocyte cell cycle progression and survival in the setting of cyclin D1 expression in vivo.

Cdk2 was once believed to play an essential role in cell cycle progression, but cdk2-/- mice have minimal phenotypic abnormalities. In this study, we examined the role of cdk2 in hepatocyte proliferation, centrosome duplication, and survival. Cdk2-/- hepatocytes underwent mitosis and had normal centrosome content after mitogen stimulation. Unlike wild-type cells, cdk2-/- liver cells failed to undergo centrosome overduplication in response to ectopic cyclin D1 expression. After mitogen stimulation in culture or partial hepatectomy in vivo, cdk2-/- hepatocytes demonstrated diminished proliferation. Cyclin D1 is a key mediator of cell cycle progression in hepatocytes, and transient expression of this protein is sufficient to promote robust proliferation of these cells in vivo. In cdk2-/- mice and animals treated with the cdk2 inhibitor seliciclib, cyclin D1 failed to induce hepatocyte cell cycle progression. Surprisingly, cdk2 ablation or inhibition led to massive hepatocyte and animal death following cyclin D1 transfection. In a transgenic model of chronic hepatic cyclin D1 expression, seliciclib induced hepatocyte injury and animal death, suggesting that cdk2 is required for survival of cyclin D1-expressing cells even in the absence of substantial proliferation. In conclusion, our studies demonstrate that cdk2 plays a role in liver regeneration. Furthermore, it is essential for centrosome overduplication, proliferation, and survival of hepatocytes that aberrantly express cyclin D1 in vivo. These studies suggest that cdk2 may warrant further investigation as a target for therapy of liver tumors with constitutive cyclin D1 expression.

Optimal cutoff point for immunoperoxidase detection of C4d in the renal allograft: results from a multicenter study.

Background. Although C4d deposition in peritubular capillaries has been identified as a strong risk factor for subsequent renal allograft loss, the optimal cutoff for the fraction of peritubular capillaries needed to establish a positive stain in formalin-fixed, paraffin-embedded material has not been defined systematically. The objective of this study was to establish the threshold for positive staining that best predicts renal outcome in renal biopsies in a multicenter study in which local and central pathologic conditions were compared. Methods. Unstained renal biopsy slides were obtained from 296 patients. The percentage of peritubular capillaries staining positively for C4d was detected by immunoperoxidase staining. Results. The percentage C4d deposition ranged from 0% to 90% with 44% (129/296) having a positive percentage of C4d staining. The median for positive cases was 25%. Local C4d+ results were reported qualitatively, with 28% recorded as positive for C4d. Using a centrally determined cutoff of 10%, tests for agreement of local and central C4d staining were fair (&kgr; 0.40, 95% confidence interval 0.29–0.51). Raising the centrally determined cutoff to 25% or 50% did not change the &kgr; values (0.44 and 0.41, respectively). By Cox proportional hazards model, C4d positivity (centrally determined assessment) using a cutoff of 10% was the strongest predictor of time to graft loss (hazard ratio 2.66, 95% confidence interval 1.68–4.21). Centrally determined C4d positivity correlated with Banff scores indicative of acute inflammation but not with scores indicative of fibrosis/atrophy or transplant glomerulopathy. Conclusions. Our findings indicate that C4d positivity, defined as more than or equal to 10% by immunoperoxidase, is a strong predictor of graft loss.

Kidney Biopsy Yield: An Examination of Influencing Factors

Kidney biopsies are conducted under varying scenarios, presenting variables that could potentially influence yield and adequacy of tissue collected. We retrospectively reviewed 636 native and allograft kidney biopsies, and compared tissue collected between differing practitioners performing the biopsy (nephrologists or radiologists), imaging modalities for guidance (ultrasound or computed tomography), gauge needle used (18 or 16 G), and between on-site evaluators of biopsy adequacy conducted at the time of biopsy (general pathologists, renal pathologists, nephrologists). For radiologists using ultrasound guidance and 18 G needles, those using on-site evaluation of adequacy collected more glomeruli and glomeruli per length of tissue core than those not using on-site evaluation. Radiologists not using on-site evaluation but who used a larger bore needle (16 vs. 18 G) could generally collect comparable tissue as other biopsy performers who used on-site evaluation. Radiologists performing ultrasound-guided biopsies with 18 G needles without on-site evaluation consistently provided poorer tissue yield and had a higher rate of providing insufficient tissue so that a diagnosis could not be rendered. Nephrologists collected less total length of tissue cores, glomeruli, and arteries per case (whether performing the biopsy and/or performing on-site adequacy) compared with other groups using on-site evaluation, however, providing comparable density of glomeruli and arteries. Complication rates did not differ between compared groups using 18 G needles. It is our observation that the various conditions by which a kidney biopsy is obtained influences the yield of tissue collected and the subsequent ability for a pathologist to effectively provide a diagnosis.

BK Polyomavirus Tubulointerstitial Nephritis With Urothelial Hyperplasia in a Kidney Transplant

Polyomavirus nephropathy is characterized histopathologically by evidence of viral replication and acute tubular injury with interstitial inflammation, tubulitis, and intranuclear inclusions. Polyomavirus nephropathy typically develops in the kidney transplant as a combination of the unique nature of the transplanted tissue and the immunomodulated status of the patient. We present a case in which a patient had lingering BK viremia and declining kidney function following receipt of lung and kidney transplants. A kidney biopsy was performed, which demonstrated BK polyomavirus tubulointerstitial nephritis, resultant cytopathic changes and tubular/ductal injury, associated urothelial hyperplasia with foci of squamous metaplasia, suspected membranous glomerulopathy, and moderate arterial/arteriolar sclerosis. There was also evidence of more proximal nephron viral involvement, with glomerular parietal epithelium infection and injury present. This case shows impressive BK polyomavirus-associated urothelial hyperplasia in the kidney, which to our knowledge has not been previously illustrated in the literature. There have been numerous studies attempting to show the association of polyomaviruses with the development of carcinoma, and this case report is significant because it is an example of viral-induced changes that are concerning and hold potential for malignant transformation.

Unexpected Death from Hemorrhagic Pericarditis Complicating IgA Nephropathy

IgA nephropathy is the most common cause of glomerulonephritis in the developed world; however, it is usually an indolent or slowly progressive disease presenting initially as self-limited hematuria, typically following a viral or bacterial infection. We report a case of previously undiagnosed IgA nephropathy manifesting with end stage renal disease, fibrinous pericarditis, and hemorrhagic pericardial effusion in a 26-year-old white male. Clinical manifestations of IgA nephropathy, the presumed pathogenesis, and complications are reviewed. This case highlights the importance of interdisciplinary collaborations with other specialized pathology services—in this case renal pathology, including direct immunofluorescence and electron microscopy—that may not be routinely available or used in forensic pathology practices. In this instance, such collaboration provided the etiologically specific diagnosis for an unusual presentation of a common disease.