John Cijiang He

Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference-mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm-mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling to preserve glomerular function and podocyte viability in adult mice.

BAMBI elimination enhances alternative TGF-β signaling and glomerular dysfunction in diabetic mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI+/+ or BAMBI−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.

In Vivo RNA Interference Models of Inducible and Reversible Sirt1 Knockdown in Kidney Cells

The silent mating type information regulation 2 homolog 1 gene (Sirt1) encodes an NAD-dependent deacetylase that modifies the activity of well-known transcriptional regulators affected in kidney diseases. Sirt1 is expressed in the kidney podocyte, but its function in the podocyte is not clear. Genetically engineered mice with inducible and reversible Sirt1 knockdown in widespread, podocyte-specific, or tubular-specific patterns were generated. We found that mice with 80% knockdown of renal Sirt1 expression have normal glomerular function under the basal condition. When challenged with doxorubicin (Adriamycin), these mice develop marked albuminuria, glomerulosclerosis, mitochondrial injury, and impaired autophagy of damaged mitochondria. Reversal of Sirt1 knockdown during the early phase of Adriamycin-induced nephropathy prevented the progression of glomerular injury and reduced the accumulation of dysmorphic mitochondria in podocytes but did not reverse the progression of albuminuria and glomerulosclerosis. Sirt1 knockdown mice with diabetes mellitus, which is known to cause mitochondrial dysfunction in the kidney, developed more albuminuria and mitochondrial dysfunction compared with diabetic mice without Sirt1 knockdown. In conclusion, these results demonstrate that our RNA interference-mediated Sirt1 knockdown models are valid and versatile tools for characterizing the function of Sirt1 in the kidney; Sirt1 plays a role in homeostatic maintenance of podocytes under the condition of mitochondrial stress/injury.

HIPK2 is a new drug target for anti-fibrosis therapy in kidney disease

In vitro and animal studies continue to elucidate the mechanisms of fibrosis and have led to advancements in treatment for idiopathic pulmonary fibrosis and cirrhosis, but the search for treatments for renal fibrosis has been more disappointing. Here, we will discuss homeodomain-interacting-protein kinase 2 (HIPK2), a novel regulator of fibrosis that acts upstream of major fibrosis signaling pathways. Its key role in renal fibrosis has been validated in vitro and in several murine models of chronic kidney diseases (CKD).

Genetics and Epigenetics of Diabetic Nephropathy.

Background: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) in the USA and worldwide, contributing to significant morbidity and mortality in diabetic patients. A genetic factor for the development of DN is strongly implicated, as only one third of diabetic patients eventually develop kidney disease. Growing evidence also supports an important role of epigenetic modifications in DN. Summary: Multiple studies have been performed to identify risk genes and loci associated with DN. So far, only several genes and loci have been identified, none of which showed a strong association with DN. Therefore, a better study design with a larger sample size to identify rare variants and a clinically defined patient population to identify genes and loci associated with progressive DN are still needed. In addition to genetic factors, epigenetic modifications, such as DNA methylation, histone modifications and microRNAs, also play a major role in the pathogenesis of DN through a second layer of gene regulation. Although a major progress has been made in this field, epigenetic studies in DN are still in the early phase and have been limited mostly due to the heterogeneity of kidney tissue samples with multiple cells. However, rapid development of high-throughput genome-wide techniques will help us to better identify genetic variants and epigenetic changes in DN. Key Message: Understanding of genetic and epigenetic changes in DN is needed for the development of new biomarkers and better drug targets against DN. Summarized in this review are important recent findings on genetic and epigenetic studies in the field of DN.

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.

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.

Podocyte Injury in HIV-Associated Nephropathy

Prior to the widespread use of antiretroviral therapy, HIV-associated nephropathy (HIVAN) was the leading cause of end-stage renal disease among individuals of African descent. HIVAN, a rapidly progressing glomerulopathy, is characterized by collapsing focal segmental glomerulosclerosis, podocyte hypertrophy, and proliferation of epithelial cells overlying the Bowmans space. Although the origin of these proliferating cells is debatable, a large body of evidence suggests that podocyte dysfunction occurs in HIVAN. In the last decade, several groups have demonstrated that the podocyte serves as a reservoir for the virus and podocyte dysfunction is a direct result of specific viral protein expression. The viral protein, nef, directly activates several signaling pathways, resulting in podocyte dedifferentiation and proliferation. Today, with the widespread use of antiretroviral therapy, classical HIVAN is rare. Nonetheless, as patients with chronically infected HIV live longer, many still succumb to chronic kidney disease due to coexisting illnesses such as diabetes and hypertension. However, it remains unclear whether this viral reservoir serves a medium for development of non-HIVAN-related kidney diseases such as diabetic nephropathy. We also provide some insights into potential areas of research in characterizing podocyte biology in this changing spectrum of HIV-related kidney disease.

Retinoic Acid: A Potential Pharmacologic Approach in the Treatment of Podocytopathy

In the setting of injury, podocytes can undergo dedifferentiation and proliferation, apoptosis, or cell detachment, resulting in proteinuria. Significant injury resulting in a loss of podocytes contributes to progressive kidney disease. Consequently, therapies that prevent podocyte injury and promote their regeneration will have a major clinical impact on glomerular disease. All-trans-retinoic acid (RA), which is a derivative of vitamin A, has many cellular functions including induction of cell differentiation, regulation of apoptosis, and inhibition of inflammation and proliferation. In addition to its established role in the treatment of variety of cancers, RA has been demonstrated to provide protection in various experimental models of kidney diseases with podocyte injury. RA produces its beneficial effects in the kidney due to its anti-inflammatory and antiproliferative effects. Also, RA helps maintain the normal morphology and preserves the differentiation markers of the podocytes. RA produces its effects in the podocytes via retinoic acid receptor-α (RAR-α). Treatment with RAR-α agonist, Am580, has a similar efficacy as RA in a mouse model of HIVAN. However, due to its systemic toxicity, clinical studies evaluating the use of RA have been challenging. Therefore, novel RAR-α agonist such as BD4 has been synthesized, which has a similar efficacy as RA and Am580, with lower toxicity.