Peter Y. Chuang

Down-regulation of NF-κB Transcriptional Activity in HIV-associated Kidney Disease by BRD4 Inhibition

Background: NF-κB and BRD4 control proinflammatory gene activation in HIV-associated kidney disease. Results: Small molecule inhibition of BRD4 binding to NF-κB blocks target gene activation. Conclusion: Targeting the proinflammatory activity of NF-κB may be a new therapeutic approach. Significance: This study has broad implications as NF-κB-mediated inflammation represents the major pathology in chronic kidney and non-kidney diseases. NF-κB-mediated inflammation is the major pathology in chronic kidney diseases, including HIV-associated nephropathy (HIVAN) that ultimately progresses to end stage renal disease. HIV infection in the kidney induces NF-κB activation, leading to the production of proinflammatory chemokines, cytokines, and adhesion molecules. In this study, we explored selective inhibition of NF-κB transcriptional activity by small molecule blocking NF-κB binding to the transcriptional cofactor BRD4, which is required for the assembly of the productive transcriptional complex comprising positive transcription elongation factor b and RNA polymerase II. We showed that our BET (Bromodomain and Extra-Terminal domain)-specific bromodomain inhibitor MS417, designed to block BRD4 binding to the acetylated NF-κB, effectively attenuates NF-κB transcriptional activation of proinflammatory genes in kidney cells treated with TNFα or infected by HIV. MS417 ameliorates inflammation and kidney injury in HIV-1 transgenic mice, an animal model for HIVAN. Our study suggests that BET bromodomain inhibition, targeting at the proinflammatory activity of NF-κB, represents a new therapeutic approach for treating NF-κB-mediated inflammation and kidney injury in HIVAN.

Dysregulated nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study.

Background Podocyte specific proteins are dysregulated in diabetic nephropathy, though the extent of their expression loss is not identical and may be subject to different regulatory factors. Quantifying the degree of loss may help identify the most useful protein to use as an early biomarker of diabetic nephropathy. Methodology/Principal Findings Protein expression of synaptopodin, podocin and nephrin were quantified in 15 Type 2 diabetic renal biopsies and 12 control patients. We found statistically significant downregulation of synaptopodin (P<0.0001), podocin (P = 0.0002), and nephrin (P<0.0001) in kidney biopsies of diabetic nephropathy as compared with controls. Urinary nephrin levels (nephrinuria) were then measured in 66 patients with Type 2 diabetes and 10 healthy controls by an enzyme-linked immunosorbent assay (Exocell, Philadelphia, PA). When divided into groups according to normo-, micro-, and macroalbuminuria, nephrinuria was found to be present in 100% of diabetic patients with micro- and macroalbuminuria, as well as 54% of patients with normoalbuminuria. Nephrinuria also correlated significantly with albuminuria (rho = 0.89, p<0.001), systolic blood pressure (rho = 0.32, p = 0.007), and correlated negatively with serum albumin (rho = −0.48, p<0.0001) and eGFR (rho = −0.33, p = 0.005). Conclusions/Significance These data suggest that key podocyte-specific protein expressions are significantly and differentially downregulated in diabetic nephropathy. The finding that nephrinuria is observed in a majority of these normoalbuminuric patients demonstrates that it may precede microalbuminuria. If further research confirms nephrinuria to be a biomarker of pre-clinical diabetic nephropathy, it would shed light on podocyte metabolism in disease, and raise the possibility of new and earlier therapeutic targets.

A systems approach identifies HIPK2 as a key regulator of kidney fibrosis.

Kidney fibrosis is a common process that leads to the progression of various types of kidney disease. We used an integrated computational and experimental systems biology approach to identify protein kinases that regulate gene expression changes in the kidneys of human immunodeficiency virus (HIV) transgenic mice (Tg26 mice), which have both tubulointerstitial fibrosis and glomerulosclerosis. We identified homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in the kidneys of Tg26 mice and in those of patients with various kidney diseases. HIV infection increased the protein concentrations of HIPK2 by promoting oxidative stress, which inhibited the seven in absentia homolog 1 (SIAH1)-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and the expression of epithelial-to-mesenchymal transition markers in kidney epithelial cells by activating the p53, transforming growth factor β (TGF-β)–SMAD family member 3 (Smad3) and Wnt-Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 mice, as well as in other murine models of kidney fibrosis. We therefore conclude that HIPK2 is a potential target for anti-fibrosis therapy.

Role of Transcription Factor Acetylation in Diabetic Kidney Disease

Nuclear factor (NF)-κB and signal transducer and activator of transcription 3 (STAT3) play a critical role in diabetic nephropathy (DN). Sirtuin-1 (SIRT1) regulates transcriptional activation of target genes through protein deacetylation. Here, we determined the roles of Sirt1 and the effect of NF-κB (p65) and STAT3 acetylation in DN. We found that acetylation of p65 and STAT3 was increased in both mouse and human diabetic kidneys. In human podocytes, advanced glycation end products (AGEs) induced p65 and STAT3 acetylation and overexpression of acetylation-incompetent mutants of p65 and STAT3 abrogated AGE-induced expression of NF-κB and STAT3 target genes. Inhibition of AGE formation in db/db mice by pyridoxamine treatment attenuated proteinuria and podocyte injury, restored SIRT1 expression, and reduced p65 and STAT3 acetylation. Diabetic db/db mice with conditional deletion of SIRT1 in podocytes developed more proteinuria, kidney injury, and acetylation of p65 and STAT3 compared with db/db mice without SIRT1 deletion. Treatment of db/db mice with a bromodomain and extraterminal (BET)-specific bromodomain inhibitor (MS417) which blocks acetylation-mediated association of p65 and STAT3 with BET proteins, attenuated proteinuria, and kidney injury. Our findings strongly support a critical role for p65 and STAT3 acetylation in DN. Targeting protein acetylation could be a potential new therapy for DN.

Alteration of forkhead box O (foxo4) acetylation mediates apoptosis of podocytes in diabetes mellitus.

The number of kidney podocytes is reduced in diabetic nephropathy. Advanced glycation end products (AGEs) accumulate in patients with diabetes and promote the apoptosis of podocyte by activating the forkhead box O4 (Foxo4) transcription factor to increase the expression of a pro-apoptosis gene, Bcl2l11. Using chromatin immunoprecipitation we demonstrate that AGE-modified bovine serum albumin (AGE-BSA) enhances Foxo4 binding to a forkhead binding element in the promoter of Bcl2lll. AGE-BSA also increases the acetylation of Foxo4. Lysine acetylation of Foxo4 is required for Foxo4 binding and transcription of Bcl2l11 in podocytes treated with AGE-BSA. The expression of a protein deacetylase that targets Foxo4 for deacetylation, sirtuin (Sirt1), is down regulated in cultured podocytes by AGE-BSA treatment and in glomeruli of diabetic patients. SIRT1 over expression in cultured murine podocytes prevents AGE-induced apoptosis. Glomeruli isolated from diabetic db/db mice have increased acetylation of Foxo4, suppressed expression of Sirt1, and increased expression of Bcl2l11 compared to non-diabetic littermates. Together, our data provide evidence that alteration of Foxo4 acetylation and down regulation of Sirt1 expression in diabetes promote podocyte apoptosis. Strategies to preserve Sirt1 expression or reduce Foxo4 acetylation could be used to prevent podocyte loss in diabetes.

Systems biology of kidney diseases

Kidney diseases manifest in progressive loss of renal function, which ultimately leads to complete kidney failure. The mechanisms underlying the origins and progression of kidney diseases are not fully understood. Multiple factors involved in the pathogenesis of kidney diseases have made the traditional candidate gene approach of limited value toward full understanding of the molecular mechanisms of these diseases. A systems biology approach that integrates computational modeling with large-scale data gathering of the molecular changes could be useful in identifying the multiple interacting genes and their products that drive kidney diseases. Advances in biotechnology now make it possible to gather large data sets to characterize the role of the genome, epigenome, transcriptome, proteome, and metabolome in kidney diseases. When combined with computational analyses, these experimental approaches will provide a comprehensive understanding of the underlying biological processes. Multiscale analysis that connects the molecular interactions and cell biology of different kidney cells to renal physiology and pathology can be utilized to identify modules of biological and clinical importance that are perturbed in disease processes. This integration of experimental approaches and computational modeling is expected to generate new knowledge that can help to identify marker sets to guide the diagnosis, monitor disease progression, and identify new therapeutic targets.

Glomerular endothelial cell injury and cross talk in diabetic kidney disease.

Diabetic kidney disease (DKD) remains a leading cause of new-onset end-stage renal disease (ESRD), and yet, at present, the treatment is still very limited. A better understanding of the pathogenesis of DKD is therefore necessary to develop more effective therapies. Increasing evidence suggests that glomerular endothelial cell (GEC) injury plays a major role in the development and progression of DKD. Alteration of the glomerular endothelial cell surface layer, including its major component, glycocalyx, is a leading cause of microalbuminuria observed in early DKD. Many studies suggest a presence of cross talk between glomerular cells, such as between GEC and mesangial cells or GEC and podocytes. PDGFB/PDGFRβ is a major mediator for GEC and mesangial cell cross talk, while vascular endothelial growth factor (VEGF), angiopoietins, and endothelin-1 are the major mediators for GEC and podocyte communication. In DKD, GEC injury may lead to podocyte damage, while podocyte loss further exacerbates GEC injury, forming a vicious cycle. Therefore, GEC injury may predispose to albuminuria in diabetes either directly or indirectly by communication with neighboring podocytes and mesangial cells via secreted mediators. Identification of novel mediators of glomerular cell cross talk, such as microRNAs, will lead to a better understanding of the pathogenesis of DKD. Targeting these mediators may be a novel approach to develop more effective therapy for DKD.

Therapeutic use of traditional Chinese herbal medications for chronic kidney diseases

Traditional Chinese herbal medications (TCHM) are frequently used in conjunction with western pharmacotherapy for treatment of chronic kidney diseases (CKD) in China and many other Asian countries. The practice of traditional Chinese medicine is guided by cumulative empiric experience. Recent in vitro and animal studies have confirmed the biological activity and therapeutic effects of several TCHM in CKD. However, the level of evidence supporting TCHM is limited to small, non-randomized trials. Due to variations in the prescription pattern of TCHM and the need for frequent dosage adjustment, which are inherent to the practice of traditional Chinese medicine, it has been challenging to design and implement large randomized clinical trials of TCHM. Several TCHM are associated with significant adverse effects, including nephrotoxicity. However, reporting of adverse effects associated with TCHM has been inadequate. To fully realize the therapeutic use of TCHM in CKD we need molecular studies to identify active ingredients of TCHM and their mechanism of action, rigorous pharmacologic studies to determine the safety and meet regulatory standards required for clinical therapeutic agents, and well-designed clinical trials to provide evidence-based support of their safety and efficacy.

Knockdown of Stat3 activity in vivo prevents diabetic glomerulopathy

Recent studies suggest that Stat3, a transcription factor that mediates cytokine signaling, plays a critical role in the pathogenesis of diabetic nephropathy. Complete Stat3 gene knockout is embryonic lethal; therefore, we crossed Stat3+/- mice with Stat3 mutant mice (SA/SA) that lack full Stat3 activity. This strategy generated Stat3SA/- mice (25% activity) and Stat3SA/+ mice (75% activity), which were made diabetic using streptozotocin in order to define the role of Stat3 in diabetic kidney disease. While the glomerular number was not different between these two groups of mice, the diabetic SA/- mice had significantly less proteinuria, mesangial expansion, glomerular cell proliferation, and macrophage infiltration than the diabetic SA/+ mice. The reduction in Stat3 activity did not affect glomerular hyperfiltration seen after the induction of diabetes, as it was increased to the same degree in both groups of mice. Phosphorylation of Stat3 was markedly increased in the glomeruli of diabetic SA/+ mice compared to diabetic SA/- mice. The expression of inflammatory markers, IL-6, MCP-1, and activated NF-kappaB; type IV collagen, TGF-beta, and ICAM-1 mRNA; or type IV collagen and TGF-beta protein, were all found to be significantly less in glomeruli isolated from diabetic SA/- mice, as compared with diabetic SA/+ mice. Our study shows that Stat3 plays a critical role in the regulation of inflammation and abnormal matrix synthesis at an early stage of DN.

Signaling in Regulation of Podocyte Phenotypes

The kidney podocyte is a terminally differentiated and highly specialized cell. The function of the glomerular filtration barrier depends on the integrity of the podocyte. Podocyte injury and loss have been observed in human and experimental models of glomerular diseases. Three major podocyte phenotypes have been described in glomerular diseases: effacement, apoptosis, and proliferation. Here, we highlight the signaling cascades that are responsible for the manifestation of these pathologic phenotypes. The integrity of the podocyte foot process is determined by the interaction of nephrin with proteins in the slit diaphragm complex, the regulation of actin dynamics by the Rho family of GTPases, and the transduction of extracellular signals through focal adhesion complexes. Activation of the p38 mitogen-activated protein kinase and transforming growth factor-β1 causes podocyte apoptosis. Phosphoinositide 3-kinase and its downstream target AKT protect podocytes from apoptosis. In human immunodeficiency virus-associated nephropathy, Src-dependent activation of Stat3, mitogen-activated protein kinase 1,2, and hypoxia-inducible factor 2α is an important driver of podocyte proliferation. At the level of intracellular signaling, it appears that different extracellular signals can converge onto a few pathways to induce changes in the phenotype of podocytes.