In this study, we integrated experimental and computational approaches to identify upstream protein kinases responsible for observed gene regulation changes and connected it to a disease state. With this approach, we identified MAPK1 (ERK2), MAPK3 (ERK1), and HIPK2 as the top three protein kinases mediating gene expression in the Tg26 kidney. We previously showed that ERK mediates HIV-induced podocyte proliferation and RTEC apoptosis10,39
. Here, we identified HIPK2 as a critical regulator of EMT in vitro and kidney fibrosis in vivo. Our data suggest that both ERK and HIPK2 are critical for HIV-induced kidney disease. Further studies are required to determine the crosstalk of ERK and HIPK2 pathways.
We confirmed that HIPK2 expression is increased not only in kidneys of Tg26 mice and patients with HIVAN, but also in kidneys of patients with FSGS, DN and IgAN. This supports a more general role for HIPK2 in human kidney diseases, which was corroborated by the fact that HIPK2 mediates renal fibrosis in three established animal models of renal fibrosis (Tg26, UUO and FA).
We found that HIPK2 mediates HIV-induced apoptosis and expression of EMT markers in cultured hRTEC. Apoptosis and EMT of RTEC leading to renal fibrosis have been described in many kidney diseases including HIVAN40,41,42,43
. Recent studies suggest that complete EMT of RTEC may not occur in vivo. However, RTEC can gain EMT markers (epithelial plasticity in fibrogenesis) after injury44,45
. Our data suggest that HIPK2 mediates expression of EMT markers in RTEC both in vitro and in vivo.
Our data indicate that HIPK2 is increased in RTEC as well as glomeruli of diseased kidney. It is possible that fibroblasts and/or pericytes also express HIPK2. We found that HIPK2 also mediates HIV-induced dedifferentiation of podocytes and the expression of EMT markers. Consistent with this, knockout of HIPK2 significantly reduced proteinuria, podocyte hyperplasia, and glomerulosclerosis in Tg26 mice. Future studies are needed to determine whether HIPK2 is required for the activation of fibroblasts and/or pericytes or if HIPK2 mediates RTEC-fibroblast crosstalk.
In this study we focused on the intracellular mechanisms of HIPK2 regulation in diseased kidneys. We found that HIV decreases SIAH1 expression through DNA damage and oxidative stress, leading to the accumulation of HIPK2. In addition, we confirmed that inhibition of ROS by NAC restores Siah1 expression, reduces HIPK2 protein level, and attenuates kidney injury/fibrosis in Tg26 mice. Although renal protective effects of NAC in human kidney disease remain controversial46
, our data indicate that anti-ROS therapy could be a potential approach to treat patients with kidney fibrosis. Our findings are consistent with prior studies that poly-ubiquitination promotes HIPK2 degradation47
and DNA-damage and oxidative stress activate HIPK248
We confirmed that HIPK2 mediates TGFβ- and HIV-induced pro-apoptotic and pro-fibrotic pathways in RTEC including p53, Smad3, and Notch/Wnt. Previous study showed that HIPK2 can either repress or promote Wnt-β-catenin-mediated transcriptional activation 34,14
. Consistent with this, recent studies suggest that HIPK2 could affect Wnt pathway in a context-dependent manner28
. HIPK2 up-regulates Wnt-mediated transcription by phosphorylating TCF3, a transcriptional repressor, but inhibites Wnt-mediated transcription by phosphorylating LEF1, a transcriptional activator28
. Consistent with this, we found that overexpression of WT-HIPK2 increases TCF3 phosphorylation, but not LEF1 in RTEC. NFkB-mediated inflammation is known to cause tissue injury/fibrosis. Our studies also suggest that HIPK is a key regulator of the NFkB pathway, which is a novel finding that has never been explored previously. These findings, summarized in , demonstrate that signals from initial insults, such as DNA damage or oxidative stress, increase HIPK2 expression to activate multiple signaling pathways that are involved in kidney fibrosis. HIPK2 is a logical therapeutic drug target for kidney fibrosis and protein kinases are “drug-able” targets.
In conclusion, we presented here a systems approach to identifying upstream protein kinases based on genome-wide mRNA expression microarrays and DNA/protein arrays. We identified and confirmed that HIPK2, a protein kinase previously unrecognized in kidney disease, plays a critical role in renal fibrosis. We elucidated the regulation of HIPK2 in kidney disease and downstream signaling pathways that mediate HIPK2-induced apoptosis and the expression of EMT markers in kidney cells. We believe that HIPK2 could be a novel therapeutic target for kidney disease.