Zhengzhe Li

RTN1 mediates progression of kidney disease by inducing ER stress

Identification of new biomarkers and drug targets for chronic kidney disease (CKD) is required for the development of more effective therapy. Here we report an association between expression of reticulon 1 (RTN1) and severity of CKD. An isoform-specific increase in the expression of RTN1A is detected in the diseased kidneys from mice and humans, and correlates inversely with renal function in patients with diabetic nephropathy. RTN1 overexpression in renal cells induces ER stress and apoptosis, whereas RTN1 knockdown attenuates tunicamycin-induced and hyperglycaemia-induced ER stress and apoptosis. RTN1A interacts with PERK through its N-terminal and C-terminal domains, and mutation of these domains prevents this effect on ER stress. Knockdown of Rtn1a expression in vivo attenuates ER stress and renal fibrosis in mice with unilateral ureteral obstruction, and also attenuates ER stress, proteinuria, glomerular hypertrophy and mesangial expansion in diabetic mice. Together, these data indicate that RTN1A contributes to progression of kidney disease by inducing ER stress.

Intronic locus determines SHROOM3 expression and potentiates renal allograft fibrosis

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7-like 2-dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2-mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell-specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

Novel retinoic acid receptor alpha agonists for treatment of kidney disease.

Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

Reduced Krüppel-like factor 2 expression may aggravate the endothelial injury of diabetic nephropathy.

Kruppel-like Factor 2 (KLF2), a shear-stress inducible transcription factor, has endoprotective effects. In streptozotocin-induced diabetic rats, we found that glomerular Klf2 expression was reduced in comparison to non-diabetic rats. However, normalization of hyperglycemia by insulin treatment increased Klf2 expression to a level higher than that of non-diabetic rats. Consistent with this, we found that Klf2 expression was suppressed by high glucose but increased by insulin in cultured endothelial cells. To determine the role of KLF2 in streptozotocin-induced diabetic nephropathy, we used endothelial cell-specific Klf2 heterozygous knockout mice and found that diabetic knockout mice developed more kidney/glomerular hypertrophy and proteinuria than diabetic wide type mice. Glomerular expression of Vegfa, Flk1, and angiopoietin 2 increased but expression of Flt1, Tie2, and angiopoietin 1 decreased in diabetic knockout compared to diabetic wide type mice. Glomerular expression of ZO-1, glycocalyx, and eNOS was also decreased in diabetic knockout compared to diabetic wide type mice. These data suggest knockdown of Klf2 expression in the endothelial cells induced more endothelial cell injury. Interestingly, podocyte injury was also more prominent in diabetic knockout compared to diabetic wide type mice, indicating a crosstalk between these two cell types. Thus, KLF2 may play a role in glomerular endothelial cell injury in early diabetic nephropathy.

Basic ResearchReduced Krüppel-like factor 2 expression may aggravate the endothelial injury of diabetic nephropathy

Kruppel-like Factor 2 (KLF2), a shear-stress inducible transcription factor, has endoprotective effects. In streptozotocin-induced diabetic rats, we found that glomerular Klf2 expression was reduced in comparison to non-diabetic rats. However, normalization of hyperglycemia by insulin treatment increased Klf2 expression to a level higher than that of non-diabetic rats. Consistent with this, we found that Klf2 expression was suppressed by high glucose but increased by insulin in cultured endothelial cells. To determine the role of KLF2 in streptozotocin-induced diabetic nephropathy, we used endothelial cell-specific Klf2 heterozygous knockout mice and found that diabetic knockout mice developed more kidney/glomerular hypertrophy and proteinuria than diabetic wide type mice. Glomerular expression of Vegfa, Flk1, and angiopoietin 2 increased but expression of Flt1, Tie2, and angiopoietin 1 decreased in diabetic knockout compared to diabetic wide type mice. Glomerular expression of ZO-1, glycocalyx, and eNOS was also decreased in diabetic knockout compared to diabetic wide type mice. These data suggest knockdown of Klf2 expression in the endothelial cells induced more endothelial cell injury. Interestingly, podocyte injury was also more prominent in diabetic knockout compared to diabetic wide type mice, indicating a crosstalk between these two cell types. Thus, KLF2 may play a role in glomerular endothelial cell injury in early diabetic nephropathy.

Rtn1a-Mediated Endoplasmic Reticulum Stress in Podocyte Injury and Diabetic Nephropathy

We previously reported a critical role of reticulon (RTN) 1A in mediating endoplasmic reticulum (ER) stress in kidney tubular cells and the expression of RTN1A correlates with the renal function and the severity of kidney injury in patients with diabetic nephropathy (DN). Here, we determined the roles of RTN1A and ER stress in podocyte injury and DN. We used db/db mice with early unilateral nephrectomy (Unx) as a murine model of progressive DN and treated mice with tauroursodeoxycholic acid (TUDCA), a specific inhibitor of ER stress. We found increased expression of RTN1A and ER stress markers in the kidney of db/db-Unx mice. Treatment of TUDCA not only attenuated proteinuria and kidney histological changes, but also ameliorated podocyte and glomeruli injury in diabetic mice, which were associated with reduction of RTN1A and ER stress marker expression in the podocytes of TUDCA-treated mice. In vitro, we showed RTN1A mediates albumin-induced ER stress and apoptosis in human podocytes. A positive feedback loop between RTN1A and CHOP was found leading to an enhanced ER stress in podocytes. Our data suggest that ER stress plays a major role in podocyte injury in DN and RTN1A might be a key regulator of ER stress in podocytes.

A Novel Inhibitor of Homeodomain Interacting Protein Kinase 2 Mitigates Kidney Fibrosis through Inhibition of the TGF-β1/Smad3 Pathway

Homeodomain interacting protein kinase 2 (HIPK2) is a critical regulator of multiple profibrotic pathways, including that of TGF-β1/Smad3. Genetic ablation of HIPK2 was shown previously to significantly reduce renal fibrosis in the experimental unilateral ureteral obstruction model and Tg26 mice, a model of HIV-associated nephropathy. To develop specific pharmacologic inhibitors of HIPK2 for antifibrotic therapy, we designed and synthesized small molecule inhibitor compounds on the basis of the predicted structure of HIPK2. Among these compounds, we identified one, BT173, that strongly inhibited the ability of HIPK2 to potentiate the downstream transcriptional activity of Smad3 in kidney tubular cells. Notably, binding of BT173 to HIPK2 did not inhibit HIPK2 kinase activity but rather, interfered allosterically with the ability of HIPK2 to associate with Smad3. In vitro, treatment with BT173 inhibited TGF-β1-induced Smad3 phosphorylation and Smad3 target gene expression in human renal tubular epithelial cells. In vivo, administration of BT173 decreased Smad3 phosphorylation and mitigated renal fibrosis and deposition of extracellular matrix in unilateral ureteral obstruction and Tg26 mouse models of renal fibrosis. Our data indicate that BT173 is a novel HIPK2 inhibitor that attenuates renal fibrosis through suppression of the TGF-β1/Smad3 pathway and may be developed as an antifibrotic therapy in patients with kidney disease.

Expression of Glutamate Receptor Subtype 3 Is Epigenetically Regulated in Podocytes under Diabetic Conditions

Background: Recent studies suggest a role of epigenetics in the pathogenesis of diabetic kidney disease. However, epigenetic changes occurring specifically in kidney cells is poorly understood. Methods: To examine the epigenetic regulation of genes in podocytes under diabetic conditions, we performed DNA methylation and transcriptomic profiling in podocytes exposed to high glucose conditions. Results: Comparative analysis of genes with DNA methylation changes and correspondingly altered mRNA expression identified 337 hypomethylated genes with increased mRNA expression and only 2 hypermethyated genes (ESX1 and GRIA3) with decreased mRNA expression. Glutamate ionotropic receptor AMPA type subunit 3 (GRIA3) belongs to the ionotropic class of glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system. As podocytes have glutamate-containing vesicles and various glutamate receptors mediate important biological effects in podocytes, we further examined GRIA3 expression and its function in podocytes. Real-time PCR and western blots confirmed the suppression of GRIA3 expression in podocytes under high glucose conditions, which were abolished in the presence of a DNA methyltransferase inhibitor. Sites of DNA hypermethylation were also confirmed by bisulfite sequencing of the GRIA3 promoter region. GRIA3 mRNA and protein expression was suppressed in diabetic kidneys of human and mouse models, and knockdown of GRIA3 exacerbated high glucose-induced apoptosis in cultured podocytes. Conclusion: These results indicate that decreased GRIA3 expression in podocytes in diabetic condition heightens podocyte apoptosis and loss.

Protein S Protects against Podocyte Injury in Diabetic Nephropathy

Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.

Podocyte-Specific Induction of Krüppel-Like Factor 15 Restores Differentiation Markers and Attenuates Kidney Injury in Proteinuric Kidney Disease

BACKGROUND Podocyte injury is the hallmark of proteinuric kidney diseases, such as FSGS and minimal change disease, and destabilization of the podocytes actin cytoskeleton contributes to podocyte dysfunction in many of these conditions. Although agents, such as glucocorticoids and cyclosporin, stabilize the actin cytoskeleton, systemic toxicity hinders chronic use. We previously showed that loss of the kidney-enriched zinc finger transcription factor Krüppel-like factor 15 (KLF15) increases susceptibility to proteinuric kidney disease and attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte. METHODS We induced podocyte-specific KLF15 in two proteinuric murine models, HIV-1 transgenic (Tg26) mice and adriamycin (ADR)-induced nephropathy, and used RNA sequencing of isolated glomeruli and subsequent enrichment analysis to investigate pathways mediated by podocyte-specific KLF15 in Tg26 mice. We also explored in cultured human podocytes the potential mediating role of Wilms Tumor 1 (WT1), a transcription factor critical for podocyte differentiation. RESULTS In Tg26 mice, inducing podocyte-specific KLF15 attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis, and inflammation, while improving renal function and overall survival; it also attenuated podocyte injury in ADR-treated mice. Enrichment analysis of RNA sequencing from the Tg26 mouse model shows that KLF15 induction activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by WT1. CONCLUSIONS Inducing podocyte-specific KLF15 attenuates kidney injury by directly and indirectly upregulating genes critical for podocyte differentiation, suggesting that KLF15 induction might be a potential strategy for treating proteinuric kidney disease.