Michael J. Mihatsch

Risk Factors for Cyclosporine-Induced Nephropathy in Patients with Autoimmune Diseases

BACKGROUND Cyclosporine is an immunosuppressive drug that is used to treat patients with autoimmune disease as well as patients who have received allografts. The drug can cause renal damage, but the incidence of and risk factors for nephropathy in patients treated with cyclosporine for autoimmune or inflammatory diseases are not known. METHODS We analyzed data from renal biopsies performed in 192 patients (129 adults and 63 children) who had been treated with cyclosporine for insulin-dependent diabetes mellitus of recent onset, uveitis, psoriasis, Sjögrens syndrome, or polychondritis. The mean (+/- SD) initial dose of cyclosporine was 8.2 +/- 2.8 mg per kilogram of body weight per day, and the duration of treatment was 4 to 39 months (median, 13). RESULTS Forty-one patients (37 adults and 4 children) had cyclosporine-induced nephropathy, defined as at least moderate focal interstitial fibrosis with tubular atrophy, arteriolar alterations, or both. As compared with patients in whom nephropathy did not develop, these patients received a larger initial dose of cyclosporine (9.3 +/- 2.8 vs. 8.0 +/- 2.8 mg per kilogram per day), had a larger maximal increase in the serum creatinine concentration above base-line values (101 +/- 77 percent vs. 50 +/- 33 percent), and were older (31 +/- 13 vs. 23 +/- 12 years). These three variables were shown by multivariate logistic-regression analysis to be significant risk factors. The duration of the elevation in the serum creatinine concentration and the occurrence of elevated blood pressure were not additional risk factors. CONCLUSIONS Nephropathy is an important potential effect of cyclosporine therapy. The risk of its development in patients with autoimmune diseases who are treated with cyclosporine can be minimized by allowing a dose no higher than 5 mg per kilogram per day and avoiding increases in serum creatinine of more than 30 percent above the patients base-line value.

Osmotic Nephrosis: Acute Kidney Injury With Accumulation of Proximal Tubular Lysosomes Due to Administration of Exogenous Solutes

Osmotic nephrosis describes a morphological pattern with vacuolization and swelling of the renal proximal tubular cells. The term refers to a nonspecific histopathologic finding rather than defining a specific entity. Osmotic nephrosis can be induced by many different compounds, such as sucrose, hydroxyethyl starch, dextrans, and contrast media. It has a broad clinical spectrum that includes acute kidney injury and chronic kidney failure in rare cases. This article discusses the pathological characteristics, pathogenesis, and various clinical entities of osmotic nephrosis.

Amplification pattern of 12q13-q15 genes (MDM2, CDK4, GLI) in urinary bladder cancer.

The chromosomal region 12q13-q15 is recurrently amplified in bladder cancer. Putative target genes located in this region include MDM2, CDK4, and GLI. To evaluate the involvement of these genes in bladder cancer, we screened a tissue microarray (TMA) containing 2317 samples by fluorescence in situ hybridization (FISH). Amplification was found for MDM2 in 5.1%, for CDK4 in 1.1%, and for GLI in 0.4% of interpretable tumors. Among tumors having amplification of at least one of these 12q13-q15 genes, 76.6% had amplification of MDM2 alone and 6.4% had amplification of CDK4 alone. Coamplifications were seen of MDM2 and CDK4 in 10.6%, and of CDK4 and GLI in 6.4%. Neither coamplifications of all three genes nor isolated GLI amplifications were found. These data suggest a prominent role of MDM2 as a 12q13-q15 amplification target in bladder cancer. However, independent CDK4 amplifications do also occur suggesting either two non-overlapping amplification sites or else a minimal overlapping region between MDM2 and CDK4 perhaps containing another yet unknown oncogene. The frequency of amplification increased significantly from stage pTa to pT1-4 (P<0.04) and from low to high grade (P<0.005). These data are consistent with a high level of genetic instability in invasively growing and high-grade bladder tumors.

E2F3 amplification and overexpression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer

E2F3 is located in the 6p22 bladder amplicon and encodes a transcription factor important for cell cycle regulation and DNA replication. To further investigate the role of E2F3 in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for gene copy number and expression analysis by means of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). E2F3 amplification was strongly associated with invasive tumor phenotype and high tumor grade (P<0.0001 each). None of 272 pTaG1/G2 tumors, but 35 of 311 pT1-4 carcinomas (11.3%), had E2F3 amplification. A high E2F3 expression level was associated with high grade, advanced stage, and E2F3 gene amplification (P<0.0001 each). To evaluate whether E2F3 expression correlates with tumor proliferation, the Ki67 labeling index (LI) was analysed for each tumor. There was a strong association between a high Ki67 LI and E2F3 expression (P<0.0001), which was independent of grade and stage. We conclude that E2F3 is frequently amplified and overexpressed in invasively growing bladder cancer (stage pT1-4). E2F3 expression appears to provide a growth advantage to tumor cells by activating cell proliferation in a subset of bladder tumors.

Polyomavirus nephropathy in native kidneys and renal allografts : an update on an escalating threat

Polyomavirus nephropathy, also termed BK‐virus nephropathy (BKN) after the main causative agent, the polyoma‐BK‐virus strain, is a significant complication after kidney transplantation. BKN is the most common viral infection that affects renal allografts with a prevalence of 1–9% on average 8–13 months post surgery. It can also occur sporadically in native kidneys. Viral nephropathy is caused by the (re)activation of latent BK viruses that enter into a replicative cycle under sustained and intensive immunosuppression. Pure productive kidney infections with JC‐ and SV‐40 polyomaviruses are exceptionally rare. BKN is morphologically defined by the presence of intranuclear viral inclusion bodies in epithelial cells and tubular injury, which is the morphological correlate for renal dysfunction. Renal disease can progress through different histologic stages (from early BKN stage A to late fibrotic stage C) that carry prognostic significance; disease stages B and C often result in chronic kidney (allograft) dysfunction and end‐stage renal disease. The clinical goal is to diagnose viral nephropathy in disease stage A and to limit chronic renal injury. Strategies to recognize, classify, and manage BKN are critically discussed including ancillary techniques for risk assessment and patient monitoring: (i) urine cytology and the search for so‐called ‘decoy cells’; (ii) PCR analyses for viral load measurements in the plasma and urine; and (iii) negative staining urine electron microscopy to identify viral particles.

Multi-target fluorescence in situ hybridization in bladder washings for prediction of recurrent bladder cancer

The objective of this study was to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for predicting recurrence of urothelial carcinoma (UC) after transurethral resection. One hundred and thirty‐eight patients (median age 68.5 years) with a history of UC were eligible for this prospective study. FISH was applied to cytospin specimens prepared from bladder washings taken during a negative control cystoscopy. The multi‐target FISH test UroVysion® (Abbott/Vysis) containing probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus was used. UC recurrence was defined as a positive biopsy during follow‐up. The median follow‐up time was 19.2 (4–52) months. FISH was positive in 50 (36%) patients and negative in 88 (64%) patients. A recurrence occurred in 39% of the patients with a positive FISH test and in 21% of patients with a negative FISH test. FISH positivity according to manufacturers criteria, at the time of a negative cystoscopy, was not significantly associated with the risk of recurrence (p = 0.12). However, the sensitivity of the FISH test to predict recurrence was significantly improved by considering specimens with rare (≤10) tetraploid cells as negative (p < 0.006). In addition, presence of 9p21 deletion was significantly associated with recurrence (p < 0.01). Notably, positive standard cytology was an independent factor for subsequent recurrence in this study (p < 0.001). Taken together, multi‐target FISH may help to stratify the risk of recurrence of UC at the time of a negative follow‐up cystoscopy. Defining the optimal threshold for FISH positivity requires consideration of tetraploid pattern and 9p21 deletion. Our results also emphasize the paramount importance of conventional cytology for UC surveillance.

Patterns of Metastasis in Muscle-Invasive Bladder Cancer (pT2–4): An Autopsy Study on 367 Patients

Background: Metastatic status is an essential determinant of prognosis of patients with muscle-invasive bladder cancer treated by cystectomy, and preoperative metastases detection is crucial for treatment selection in these patients. To better understand the metastatic behavior of bladder tumors, autopsies of patients with muscle-invasive bladder carcinomas (pT2–4) were evaluated. Methods: Protocols and histologic sections from autopsies of 367 patients with pT2–4 bladder cancer were reviewed. Results: Metastases were found in 251 of 367 patients (68%). The most frequent sites of metastases were regional lymph nodes (90%), liver (47%), lung (45%), bone (32%), peritoneum (19%), pleura (16%), kidney (14%), adrenal gland (14%), and the intestine (13%). There was no difference in the frequency and location of metastases between 308 transitional cell carcinomas and 38 squamous cell carcinomas. The frequency of metastases increased with local tumor extension (patients with cystectomy: pT2, 36%; pT3a, 45%; pT3b, 69%; pT4, 79%; p < 0.0001). For all pT classifications, the frequency of metastases was slightly higher in patients treated by cystectomy (metastases in 45% of 29 pT2 and 89% of 28 pT4 tumors) than in patients without cystectomy (36% of pT2 and 79% of pT4 tumors). Conclusions: These results argue against relevant clinical differences between histologic tumor types. The high frequency of metastases in patients having undergone cystectomy indicates that metastasis often occurs before the time of diagnosis. This emphasizes the need for a better prediction of the metastatic capability of these tumors at the time of diagnosis.

Detection of subclinical tubular injury after renal transplantation: comparison of urine protein analysis with allograft histopathology.

Background. Tubulointerstitial injury due to rejection leads to tubular atrophy (TA)/interstitial fibrosis (IF) followed by deterioration of allograft function. This study investigated whether urinary tubular injury biomarkers can detect subclinical tubulitis found in protocol biopsies allowing for a noninvasive screening procedure. Methods. Four rigidly defined groups (stable transplants with normal tubular histology [n=24], stable transplants with subclinical tubulitis [n=38], patients with clinical tubulitis Ia/Ib [n=18], and patients with other clinical tubular pathologies [n=20]) were compared for differences in urinary intact/cleaved β2-microglobulin (i/cβ2m), retinol-binding protein (RBP), neutrophil-gelatinase-associated lipocalin (NGAL), and α1-microglobulin (α1m). Results. Tubular proteinuria was present in 38% (RBP) to 79% (α1m) of patients in the stable transplant with normal tubular histology group. The stable transplant with subclinical tubulitis group had slightly higher levels of i/cβ2m (P=0.11), RBP (P=0.17), α1m (P=0.09), and NGAL (P=0.06) than the stable transplant with normal tubular histology group with a substantial overlap. The clinical tubulitis Ia/Ib and the other clinical tubular pathology groups had significantly higher levels of RBP, NGAL, and α1m than stable transplants with normal tubular histology or stable transplants with subclinical tubulitis (P<0.002). Conclusions. None of the investigated biomarkers allow for clear differentiation between stable transplants with normal tubular histology and stable transplants with subclinical tubulitis. Therefore, the protocol allograft biopsy currently remains the preferred tool to screen for subclinical tubulitis. Further longitudinal studies should determine whether tubular proteinuria in stable transplants with normal tubular histology indicates a clear risk for early development of TA/IF.

Cytomegalovirus infection and graft rejection in renal transplantation.

Background. Cytomegalovirus (CMV) infection and CMV disease have been associated with acute and chronic graft rejection. The introduction of the sensitive CMV antigenemia pp65 assay for detection of CMV infection allowed us to study the time course of CMV infection and acute rejection and the long-term outcome in renal transplant recipients with and without a CMV risk constellation. Methods. Prospective single center study including 48 renal transplant recipients at risk for CMV infection (donor and/or recipient CMV seropositive) and a control group of 36 CMV seronegative recipients of CMV seronegative kidney donors. Evidence of CMV infection was monitored by the CMV antigenemia pp65 assay every 1 to 2 weeks and compared with the occurrence of acute rejection in the posttransplant period and graft function at 5 years. Results. CMV infection developed in 83% (40/48) of patients of the CMV risk group within 4 months posttransplant. A total of 18 of patients experienced an acute rejection episode (control group 16/36;P =0.65). In 12/18 CMV infection followed rejection and in three patients antigenemia preceded the diagnosis of rejection. In three patients CMV antigenemia remained negative. Five-year follow up: Patient survival (44/48 vs. 31/36;P =0.48), graft survival (38/48 vs. 27/36;P =0.79), number of patients with at least one acute rejection episode: CMV risk group: 42.1%, control group 51% (P =0.46), serum creatinine: CMV risk group:130±66 &mgr;mol/liter, control group: 126±37 &mgr;mol/liter (P =0.56), proteinuria: CMV risk group: 0.02±0.02 g/mmol creatinine, control group: 0.02±0.02 g/mmol creatinine (P =1.0). Conclusion. CMV infection within 4 months posttransplant, as defined by a positive antigenemia assay was not found to be a risk factor for acute graft rejection or chronic graft dysfunction at 5 years.

KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

Molecular mechanisms of prostate cancer progression are poorly understood. Here, we studied gene amplification of the large conductance calcium-activated potassium channel alpha subunit (KCNMA1), which is located at the chromosomal region 10q22. Fluorescence in situ hybridization (FISH) revealed KCNMA1 amplification in 16% of 119 late-stage human prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3. In contrast, KCNMA1 amplification was absent in 33 benign controls, 32 precursor lesions and in 105 clinically organ-confined prostate cancers. Amplification was associated with mRNA and protein overexpression as well as increased density of BK channel protein and β-estradiol-insensitive BK currents in PC-3 cells as compared to non-amplified control cell lines. Specific blockade of BK channels by iberiotoxin or RNAi significantly inhibited K+ currents and growth of PC-3 cells. The data demonstrate that 10q22 amplification drives KCNMA1 expression and cell proliferation. Thus, KCNMA1 qualifies as a promising diagnostic and therapeutic target in patients with prostate cancer.