Early-Onset albuminuria and Associated Renal Pathology in Leucine-Rich Repeat Kinase 2 Knockout Rats.

Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

Universal Accessible Biomarkers of Drug-Induced Tissue Injury and Systemic Inflammation in Rat: Performance Assessment of TIMP-1, A2M, AGP, NGAL, and Albumin.

The ability to monitor for general drug-induced tissue injury (DITI) or systemic inflammation in any tissue using blood-based accessible biomarkers would provide a valuable tool in early exploratory animal studies to understand potential drug liabilities. Here we describe the evaluation of 4 biomarkers of tissue remodeling and inflammation (α2-macroglobulin [A2M], α1-acid glycoprotein [AGP], neutrophil gelatinase-associated lipocalin [NGAL], and tissue inhibitor of metalloproteinases [TIMP-1]) as well as the traditional serum parameter albumin as potential blood-based biomarkers of DITI and systemic inflammatory response (SIR). Biomarker performance was assessed in 51 short-term rat in vivo studies with various end-organ toxicities or SIR and receiver operating characteristic curves were generated to compare relative performances. All 4 biomarkers performed well in their ability to detect DITI and SIR with an area under the curve (AUC) of 0.82-0.78, however TIMP-1 achieved the best sensitivity (at 95% specificity) of 61%; AGP, NGAL, and A2M sensitivity was 51%-52%. AUC for albumin was 0.72 with sensitivity of 39%. A2M was the best performer in studies with only SIR (AUC 0.91). In the subset of studies with drug-induced vascular injury, TIMP-1 performed best with an AUC of 0.96. Poor performance of all tested biomarkers was observed in samples with CNS toxicity. In summary, TIMP-1, A2M, AGP, and NGAL demonstrated performance as sensitive accessible biomarkers of DITI and SIR, supporting their potential application as universal accessible tissue toxicity biomarkers to quickly identify dose levels associated with drug-induced injury in early exploratory rat safety and other studies.

Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability.

Systemic inflammation co-activates coagulation, which unchecked culminates in a lethal syndrome of multi-organ microvascular thrombosis known as disseminated intravascular coagulation (DIC). We studied an endotoxin-induced inflammatory state in rats to identify biomarkers of hemostatic imbalance favoring hypercoagulability. Intraperitoneal injection of LPS at 15 mg/kg body weight resulted in peripheral leukopenia and widespread neutrophilic sequestration characteristic of an acute systemic inflammatory response. Early indicators of hemostatic pathway activation developed within 4 hours, including increased circulating concentrations of procoagulant extracellular vesicles (EVs), EVs expressing endothelial cell and platelet membrane markers, and high concentration of soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor-1 (PAI-1), and D-dimers. Inflammation persisted throughout the 48-hour observation period; however, increases were found in a subset of serum microRNA (miRNA) that coincided with gradual resolution of hemostatic protein abnormalities and reduction in EV counts. Dose-adjusted LPS treatment in rats provides a time-course model to develop biomarker profiles reflecting procoagulant imbalance and rebalance under inflammatory conditions.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or diethylstilbestrol (DES) cause similar hematopoietic hypocellularity and hepatocellular changes in murine fetal liver, but differentially affect gene expression.

TCDD and DES have immunotoxic effects, including selective diminution of T lymphocyte progenitors in the fetal liver. The histologic presentation of fetal liver after exposure to either chemical has not been described. Similarly, limited information exists regarding mechanisms by which TCDD or DES may alter fetal hematopoiesis. Treatment of pregnant C57BL/6 mice with either 10 micro g/kg/day TCDD or 48 micro g/kg/day DES on gestation days (gd) 14 and 16 led to increased fetal liver weight on gd 18. Moderate anisocytosis and anisokaryosis with increased cytoplasmic and nuclear sizes, and increased cytoplasmic basophilia were present within hepatocytes after TCDD or DES. Both chemicals also decreased the presence of hematopoietic cells, however megakaryocyte numbers were unaffected. In contrast to these similar outcomes, real time quantitative PCR using a preliminary panel of 4 genes suggested that the chemicals act through different gene targets. TCDD increased c-jun gene expression in fetal liver, and decreased p53 without alteration in bcl-2 expression, indicating possible pro-proliferative and antiapoptotic effects. DES decreased c-jun and bcl-2, without altering p53, suggesting a shift away from proliferation. Both agents decreased PKCalpha expression, which may suggest shared decreased phosphorylation of substrates required for normal cell cycle progression.

Diethylstilbestrol (DES)-induced fetal thymic atrophy in C57BL/6 mice: inhibited thymocyte differentiation and increased apoptotic cell death.

Treatment of pregnant C57Bl/6 mice with 48 mu g/kg diethylstilbestrol (DES) on gestation days (GDs) 14 and 16 resulted in both decreased day 18 fetal thymic cellularity as well as alterations in thymocyte phenotype. Histopathologic examination of GD 18 fetal thymi from DES-exposed dams demonstrated a decrease in thymic size and cellularity and an increase in pyknotic nuclei, indicative of apoptosis, relative to control thymi. Thymic architecture was also altered by DES treatment with a decrease in the distinction between the cortical and medullary regions. Flow cytometric staining of day 18 thymocyte suspensions with the apoptotic marker 7-aminoactinomycin D showed a decrease in thymocyte viability after DES, and a concomitant increase of thymocytes in early apoptosis. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, trends toward increased apoptosis were present in the CD4+CD8+ and CD4+CD8- subpopulations, and significantly increased apoptosis occurred in the CD4-CD8- and CD4-CD8+ subpopulations. These histopathologic and flow cytometric findings support enhanced apoptosis of thymocytes as a contributing factor to fetal thymic atrophy caused by DES.