Chronic Kidney Disease (CKD) is a major global health burden. In the United States alone, roughly 30 million Americans have CKD. Each year in the United States, thousands of CKD patients progress toward the most severe form of CKD, End-Stage Renal Disease (ESRD). Once patients reach ESRD, costly renal replacement therapy is needed to sustain life.Striking disparities also exist within the CKD patient population. In fact, Native Americans, Americans of African descent, Native Hawaiians, and Asians are several times more likely to advance to ESRD relative to Americans of European descent. To decrease the burden of CKD, effective early interventions and accessible therapeutics are desperately needed. Bone Morphogenetic Protein (BMP) signaling is critical in renal development and disease. In animal models of CKD or kidney fibrosis, re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires high and harmful doses to achieve efficacy. BMPs are also expensive therapeutic agents to use clinically due to the complexity of their synthesis. Consequently, alternative strategies are needed to harness the beneficial properties of BMP signaling in fibrotic kidneys. The first aim of the work in this dissertation was to identify simpler to synthesize small-molecules that could potentially be developed further to treat kidney disease. Therefore, current and powerful high-throughput screening (HTS) methodologies used for drug discovery were implemented to assess approximately 64,000 small-molecules in human kidney cells. These cells have a genomically integrated BRE-Luc reporter making them highly responsive to BMPs. The HTS campaign identified twelve small-molecules that activated the BRE-Luc reporter in a dose-dependent manner. Of these compounds, sb4, displayed the lowest EC50 (74 nM) and further investigations revealed its capacity to induce key downstream BMP signaling events. For instance, in the presence of sb4 levels of key second messengers in BMP signaling, p-SMADs-1/5/9, rapidly increased. Additional studies suggested that the increase in p-SMADs-1/5/9 was likely due to stabilization by sb4, which resulted in activation of BMP target genes, ID1 and ID3. These results are significant as they demonstrate the feasibility of identifying small-molecules using HTS that mimic key downstream BMP signaling events. Accordingly, compounds like sb4 could prove useful in clinically treating fibrosis by activating the BMP signaling pathway. Furthermore, sb4 also resists inhibition by endogenous BMP inhibitors, such as Noggin. This mechanistic feature may prove advantageous in clinical settings where BMPs alone are ineffective due to elevated levels of Noggin, which suppress BMP signaling. Proliferative kidney diseases such as cystic kidney disease and kidney cancer are subtypes of CKD. Pax2, a critical transcription factor required for kidney development is generally undetectable in mature nephrons. However, Pax2 is reactivated in proliferative kidney diseases and correlates with disease progression. Inhibiting Pax2 arrests cystogenesis and decelerates kidney cancer, which makes Pax2 a potential therapeutic target. Thus, the second aim of this dissertation was to identify small-molecule inhibitors of Pax2. Re-mining efforts lead to the discovery of an emerging lead series of promising Pax2 inhibitors. These Pax2 inhibitors can potentially be further developed into therapeutics that could reverse kidney disease or manage it with less side effects than current FDA-approved agents. Ultimately, the BMP signaling agonists and Pax2 inhibitors identified can be further enhanced and used as tools to advance biomedical knowledge of pathophysiological processes of various forms of CKD.
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Developing Novel Therapeutics for Chronic Kidney Disease
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