Leucine-Rich Repeat Kinase 2 (LRRK2)-Deficient Rats Exhibit Renal Tubule Injury and Perturbations in Metabolic and Immunological Homeostasis

<div><p>Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson’s disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV) and <i>Streptococcus pneumoniae</i>. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.</p></div

Flow cytometric immunophenotyping of male LRRK2 wild type and deficient spleenocytes.

<p>The average percentage of splenocyte cells staining for (A) CD11b, (B) CD4, (C) CD8 and (D) CD3 in uninfected and infected male rats is presented. B-cell data is from uninfected animals only (N = 4 male LRRK2 wild type and deficient rats in uninfected animals and N = 5 in infected animals; a two-tailed Student’s T-test was used to assess significance between genotypes where * represents a P-value <0.05 and ** represents a P-value <0.01; standard deviations of the mean are provided).</p

Metabolic analysis of LRRK2 wild type and knockout male rats.

<p>The average levels of peripheral blood metabolic parameters are presented (N = 5 male LRRK2 wild type and deficient rats; a two-tailed Student’s T-test was used to assess significance between genotypes; standard deviations of the mean are provided).</p

Toxic pathology biomarker categories that are over-represented for LRRK2 in the local protein interaction network.

<p>P-values (from the hypergeometric distribution) for enrichment of the 50-node <i>Autoexpand</i> network around LRRK2 with biomarkers of toxic pathologies from the MetaCore Systems Toxicology Module database are presented.</p

Wet tissue and relative wet tissue weights for LRRK2 wild type and knockout male rats.

<p>The average wet tissue weights and relative wet tissue weights are presented (N = 5 male LRRK2 wild type and deficient rats; a two-tailed Student’s T-test was used to assess significance between genotypes; standard deviations of the mean are provided).</p><p>GI: Gastrointestinal; Testes: Testicles.</p

Clinical chemical analysis of LRRK2 wild type and knockout male rats.

<p>The average plasma clinical chemistry levels are presented (N = 5 male LRRK2 wild type and deficient rats; a two-tailed Student’s T-test was used to assess significance between genotypes; standard deviations of the mean are provided).</p><p>ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; TP: Total protein; CHOL: Cholesterol; TRIG: Triglycerides; CRT: Creatinine: GGT: Gamma-glutyamyl transpeptidase; TBIL: Total Bilirubin; PHOSPH: Phosphate; ALB: Albumin; K: Potassium; CL: Chloride; NA: Sodium; CA: Calcium.</p

Autofluorescent hyaline droplets are observed in LRRK2 KO, but not wild type rat kidneys.

<p>Fluorescent microscopic analysis of kidney proximal tubules reveal no autofluorescence in LRRK2 wild type male kidneys (A) whereas large levels of autofluorescence is readily apparent in LRRK2 deficient kidneys (B).</p

Direct interaction network of LRRK2 with genes associated in the MetaCore disease ontology with nutritional and metabolic disorders.

<p>Compounds are represented by hexagons, proteins by solid shapes representing different classes of compound, and enzymatic reactions by gray rectangles. Protein-protein, compound-protein and compound-reaction interactions are shown as unidirectional arrows, and a mechanism of interaction represented by letters in hexagonal boxes over the arrows.</p

Complete blood count (CBC) analysis of LRRK2 wild type and knockout male rats.

<p>The average complete blood count levels are presented (N = 5 male LRRK2 wild type and deficient rats; a two-tailed Student’s T-test was used to assess significance between genotypes; standard deviations of the mean are provided).</p><p>WBC: White blood cells; RBC: Red blood cells; HGB: Hemoglobin; HCT: Hematocrits; MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; NRBC: Nucleated red blood cells.</p

Urinary and serum kidney biomarker analysis of LRRK2 wild type and knockout male rats.

<p>The average levels of urinary and serum kidney biomarkers are presented (N = 5 male LRRK2 wild type and deficient rats; a two-tailed Student’s T-test was used to assess significance between genotypes; standard deviations of the mean are provided).</p><p>NGAL: Neutrophil gelatinase-associated lipocalin; KIM1: Kidney injury molecule 1; TIM1: T-cell immunoglobulin mucin 1; GST-α & -µ: Glutathione S-transferase-α & -µ; TIMP-1: Tissue inhibitor of metalloproteinase 1; VEGF-α: Vascular epilethilial growth factor-α.</p