Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase-3ß-positive epithelium.

It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3ß (GSK-3ß). GSK-3ß immunoreactivity was associated with glomeruli, the thick ascending limb of Henle's loop, and collecting ducts in the developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3ß (pGSK-3ß) protein decreased during postnatal development. After feeding of dams with litters lithium [50 mmol Li/kg chow, postnatal (P) days 7-28], the offspring showed plasma lithium concentration of 1.0 mmol/l. Kidneys from lithium-treated rat pups exhibited dilated distal nephron segments with microcysts. Stereological analysis showed reduced cortex and outer medullary volumes. Lithium increased pGSK-3ß and the proliferation marker proliferating cell nuclear antigen (PCNA) protein abundances in the cortex and medulla. After lithium treatment, pGSK-3ß-immunopositive cells exhibited restricted distribution and were associated primarily with subsets of cells in dilated and microcystic segments of cortical collecting ducts. After 6 wk of lithium discontinuation, adult rats exhibited attenuated urine concentration capacity and diminished outer medullary volume. Histological sections of two nephrectomy samples and a biopsy from three long-term lithium-treated patients showed multiple cortical microcysts that originated from normally appearing tubules. Microcysts were lined by a cuboidal PCNA-, GSK-3ß-, and pGSK-3ß-immunopositive epithelium. The postnatal rat kidney may serve as an experimental model for the study of lithium-induced human kidney injury. The data are compatible with a causal relationship between epithelial entry of lithium into cells of the aldosterone-sensitive distal nephron, inactivation of GSK-3ß, proliferation, and microcysts.

Angiotensin II promotes development of the renal microcirculation through AT1 receptors.

Pharmacologic or genetic deletion of components of the renin-angiotensin system leads to postnatal kidney injury, but the roles of these components in kidney development are unknown. To test the hypothesis that angiotensin II supports angiogenesis during postnatal kidney development, we quantified CD31(+) postglomerular microvessels, performed quantitative PCR analysis of vascular growth factor expression, and measured renal blood flow by magnetic resonance. Treating rats with the angiotensin II type 1 receptor antagonist candesartan for 2 weeks after birth reduced the total length, volume, and surface area of capillaries in both the cortex and the medulla and inhibited the organization of vasa recta bundles. In addition, angiotensin II type 1 antagonism inhibited the transcription of angiogenic growth factors vascular endothelial growth factor, angiopoietin-1, angiopoietin-2, and the angiopoietin receptor Tie-2 in cortex and medulla. Similarly, Agtr1a(-/-);Agtr1b(-/-) mouse kidneys had decreased angiopoietin-1, angiopoietin-2, and Tie-2 mRNAs at postnatal day 14. To test whether increased urinary flow leads to microvascular injury, we induced postnatal polyuria with either lithium or adrenalectomy, but these did not alter vascular endothelial growth factor expression or vasa recta organization. Compared with vehicle-treated rats, renal blood flow was significantly (approximately 20%) lower in candesartan-treated rats even 14 days after candesartan withdrawal. Taken together, these data demonstrate that angiotensin II promotes postnatal expansion of postglomerular capillaries and organization of vasa recta bundles, which are necessary for development of normal renal blood flow.

Gene Signal Distribution and HER2 Amplification in Gastroesophageal Cancer.

Background: HER2 serves as an important therapeutic target in gastroesophageal cancer. Differences in HER2 gene signal distribution patterns can be observed at the tissue level, but how it influences the HER2 amplification status has not been studied so far. Here, we investigated the link between HER2 amplification and the different types of gene signal distribution. Methods: Tumor samples from 140 patients with gastroesophageal adenocarcinoma where analyzed using the HER2 IQFISH pharmDx™ assay. Specimens covered non-amplified and amplified cases with a preselected high proportion of HER2 amplified cases. Based on the HER2/CEN-17 ratio, specimens were categorized into amplified or non-amplified. The signal distribution patterns were divided into homogeneous, heterogeneous focal or heterogeneous mosaic. The study was conducted based on anonymized specimens with limited access to clinicopathological data. Results: Among the 140 analyzed specimens 83 had a heterogeneous HER2 signal distribution, with 62 being focal and 21 of the mosaic type. The remaining 57 specimens had a homogeneous signal distribution. HER2 amplification was observed in 63 of the 140 specimens, and nearly all (93.7%) were found among specimens with a heterogeneous focal signal distribution (p<0.0001). The mean HER2/CEN-17 ratio for the focal heterogeneous group was 8.75 (CI95%: 6.87 - 10.63), compared to 1.53 (CI95%: 1.45 - 1.61) and 1.70 (CI95%: 1.22 - 2.18) for the heterogeneous mosaic and homogeneous groups, respectively, (p<0.0001). Conclusions: A clear relationship between HER2 amplification and the focal heterogeneous signal distribution was demonstrated in tumor specimens from patients with gastroesophageal cancer. Furthermore, we raise the hypothesis that the signal distribution patterns observed with FISH might be related to different subpopulations of HER2 positive tumor cells.

Analysis of HER2 status in gastroesophageal tumor specimens using a new automated HER2 IQFISH pharmDx™ (Dako Omnis) assay.

The human epidermal growth factor receptor 2 (HER2) is an important target for treatment of gastroesophageal cancer. Different slide-based assays are available for assessment of HER2 status. Overexpression of the HER2 protein is assessed by immunohistochemistry (IHC) whereas amplification of the HER2 gene is assessed by fluorescence in situ hybridization (FISH) or other in situ hybridization (ISH) methods. Here we report a summary of the validation data on HER2 IQFISH pharmDx™ (Dako Omnis), a newly developed assay for the automated staining platform Dako Omnis. This assay uses a non-toxic buffer that significantly reduces the hybridization time, which results in a total turnaround time of less than 4 hours from deparaffinization to counting of the gene and centromere signals. The data reported in the current summary cover method comparison, assessment of staining quality, observer-to-observer reproducibility as well as reproducibility within and between laboratories. Based on data from the different studies it was concluded that HER2 IQFISH pharmDx (Dako Omnis) is a reliable and robust assay, with high precision and at least comparable to the manual HER2 IQFISH pharmDx™ assay. The HER2 IQFISH pharmDx (Dako Omnis) assay is currently not commercially available outside the Europe Union.

Assessment of HER2 amplification status in breast cancer using a new automated HER2 IQFISH pharmDx™ (Dako Omnis) assay.

In breast cancer the human epidermal growth factor receptor 2 (HER2) is an important target for a number of different HER2 inhibitors. Different slide-based assays are available for assessment of treatment eligibility, which include fluorescence in situ hybridization (FISH) or other in situ hybridization (ISH) methods for assessment of the HER2 gene status. Here we report a summary of the validation data on HER2 IQFISH pharmDx™ (Dako Omnis), a newly developed assay for the automated staining platform Dako Omnis. The assay uses a non-toxic buffer that significantly reduces the hybridization time, which results in a total turnaround time of 3½ to 4h from deparaffinization to counting of the gene and centromere signals. The data reported in the current summary covers method comparison, assessment of staining quality, observer-to-observer reproducibility as well as reproducibility within and between laboratories. Based on data from the different studies it was concluded that HER2 IQFISH pharmDx (Dako Omnis) is a reliable and robust assay with a high precision that is at least comparable to the manual HER2 IQFISH pharmDx™ assay and the PathVysion(®)HER-2 DNA Probe Kit.

Lithium induces microcysts and polyuria in adolescent rat kidney independent of cyclooxygenase-2.

In patients, chronic treatment with lithium leads to renal microcysts and nephrogenic diabetes insipidus (NDI). It was hypothesized that renal cyclooxygenase-2 (COX-2) activity promotes microcyst formation and NDI. Kidney microcysts were induced in male adolescent rats by feeding dams with lithium (50 mmol/kg chow) from postnatal days 7-34. Lithium treatment induced somatic growth retardation, renal microcysts and dilatations in cortical collecting duct; it increased cortical cell proliferation and inactive pGSK-3ß abundance; it lowered aquaporin-2 (AQP2) protein abundance and induced polyuria with decreased ability to concentrate the urine; and it increased COX-2 protein level in thick ascending limb. Concomitant treatment with lithium and a specific COX-2 inhibitor, parecoxib (5 mg/kg per day, P10-P34), did not prevent lithium-induced microcysts and polyuria, but improved urine concentrating ability transiently after a 1-desamino-8-D-arginine vasopressin challenge. COX-2 inhibition did not reduce cortical lithium-induced cell proliferation and phosphorylation of glycogen synthase kinase-3ß (GSK-3ß). COX-1 protein abundance increased in rat kidney cortex in response to lithium. COX-1 immunoreactivity was found in microcyst epithelium in rat kidney. A human nephrectomy specimen from a patient treated for 28 years with lithium displayed multiple, COX-1-immunopositive, microcysts. In chronic lithium-treated adolescent rats, COX-2 is not colocalized with microcystic epithelium, mitotic activity, and inactive pGSK-3ß in collecting duct; a blocker of COX-2 does not prevent cell proliferation, cyst formation, or GSK-3ß inactivation. It is concluded that COX-2 activity is not the primary cause for microcysts and polyuria in a NaCl-substituted rat model of lithium nephropathy. COX-1 is a relevant candidate to affect the injured epithelium.