Faculty Bibliography

Background: Activation of kinases and the downstream signaling pathways they activate is central to the pathogenesis of cyst growth in ADPKD. However, while the human kinome consists of more than 500 kinases, only a fraction of these kinases have been tested to determine if they play a role in ADPKD pathogenesis. As a result, there are likely many kinases that are more active in ADPKD kidneys that play prominent roles in disease that are yet-to-be discovered and may be good therapeutic targets. We have now adapted a novel approach to broadly screen PKD kidneys in an unbiased manner for kinases that are more active in PKD kidneys compared with wild type kidneys.
Method(s): Active kinases were affinity captured by passing wild type and PKD kidney lysates through columns containing multiplexed kinase inhibitor beads. Bound kinases were then identified by LC separation followed by tandem mass spectrometry. Increase in kinase expression and/or activity was validated by Western Blot and the specific kidney cells expressing the kinase was determined by Immunohistochemistry. The relevance of a kinase to cyst growth in vivo was assessed by treating PKD mutant mice with specific kinase inhibitors and/or genetically by generating kinase knockouts using CRISPR/Cas9.
Result(s): We identified a number of both known and unknown kinases specifically upregulated or downregulated in mouse PKD kidneys. Focal adhesion Kinase (FAK) is one of the promising kinase identified in the screen. We found that FAK and phospho-FAK expression was upregulated in cyst lining epithelium in PKD kidneys. Consistent with FAK playing an important role in cyst growth, treatment of Pkhd1-Cre;Pkd1^fl/fl mice with the FAK inhibitor VS-4718 slowed cyst growth, preserved renal function, and prolonged survival. VS-4718 treatment led to the inhibition of multiple signaling pathways that could account for the therapeutic benefit including paxillin, p130cas, AKT and Stat3.
Conclusion(s): This is the first time PKD kidneys have been probed proteomically in an unbiased manner to identify the full range of kinases that are more active in PKD kidneys with the goal of identifying new therapeutic targets. So far we have identified FAK as a potential drug target that can slow cyst growth and preserve renal function and are currently in the process of working up several other promising kinases

African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.

CONTEXT: Manganese-associated parkinsonism is well described in occupational settings, in chronic methcathinone users, and in patients receiving long-term total parenteral nutrition. We present a unique case of acute intravenous manganese poisoning with a systematic evaluation of hemodialysis efficacy. CASE DETAILS: A 52-year-old woman was inadvertently administered a single intravenous dose of 800 mg compounded manganese chloride at an outpatient chelation center. In an attempt to minimize central nervous system (CNS) manganese deposition, she underwent urgent hemodialysis followed by five days of therapy with calcium disodium EDTA (1 g/m2 over eight hours daily). Her initial whole blood manganese concentration, obtained six hours after exposure and prior to treatment, was 120 mcg/L (2.19 micromol/L); normal <5 mcg/L (< 0.09 micromol/L). Following the first four-hour hemodialysis session her blood manganese concentration decreased to 20 mcg/L (0.36 micromol/L). Despite the fall in her blood manganese concentration, analysis of dialysate revealed a total elimination of only 604 mcg (11 micromol) manganese ( approximately 1.4% of manganese burden). Although she remained asymptomatic, an MRI on hospital day two revealed T1 hyperintensities within the bilateral globus pallidi, consistent with manganese exposure. DISCUSSION: Manganese poisoning is associated with irreversible neurologic toxicity. Hemodialysis did not appear to significantly enhance elimination in this case of acute intravenous manganese toxicity, beyond supportive care and calcium disodium EDTA chelation.