T-cell prolymphocytic leukemia (T-PLL) is a rare and deadly mature leukemia with few treatment options and adverse prognosis. Currently there is no effective treatment therapy for patients with relapse disease, with an overall survival of less than 6 months. Our group demonstrated a strong T-PLL dependency on the BCL2 family of antiapoptotic proteins using BH3 profiling (2023, ASH Annual Meeting, #4192). We generated an in vivo patient derived T-PLL xenograft (PDX) model by engrafting a relapsed/refractory T-PLL patient sample into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. This model reproduces key human clinicopathological features of T-PLL. Mice develop enlarged spleens, immunohistochemical and flow cytometry studies show engrafted T-PLL tumor cells are CD3+, CD4+ and can be detected in liver, spleen, and peripheral blood (PB). Tumor cells have TCL1 chromosomal rearrangements detected by FISH. The model was expanded and utilized in a pre-clinical trial to evaluate a novel therapeutic combination using Fadraciclib (a CDK2/9 inhibitor) and Venetoclax (BCL2 inhibitor). In vitro evidence from our team has demonstrated an additive/synergistic effect of Venetoclax in combination with Fadraciclib to cause mitochondrial dysfunction and cell death of T-PLL. These drugs inhibit separate, but partially overlapping signaling pathways covering BCL2, and MCL1. In our first set of experiments, we evaluated the effect of Venetoclax and Fadraciclib independently and in combination on the overall survival of T-PLL PDX mice. Mice were enrolled for treatment once human/Total CD45 positive cells reached 5% in PB. Fadraciclib (10mg/kg) and Venetoclax (25mg/kg) administered via gavage once daily for 5 days, 2 days of rest, and repeat for a total of 2 weeks was well tolerated. We demonstrate that therapy with Fadraciclib plus Venetoclax significantly prolonged survival compared with vehicle (p=0.0267, Log-rank (Mantel-Cox) test), and Fadraciclib alone (p=0.0103). We also evaluated the effects of these drugs on the percentage of circulating T-PLL cells in peripheral blood (PB; hCD45+). The Venetoclax and Fadraciclib combination maintained a low percentage of circulating T-PLL cells in PB, (maximum average 4.2%), Fadraciclib (20.8%), Venetoclax (17.9%), and Vehicle (42.2%) during treatment. In conclusion, we have developed a T-PLL PDX model that can be used to reproducibly evaluate the effect of new drugs and combinations for T-PLL in a preclinical setting. Combination therapy with the CDK9 inhibitor Fadraciclib and the BCL2 inhibitor Venetoclax was well tolerated, controlled the burden of circulating T-PLL cells in PB better than independent drugs and vehicle, and improved survival of T-PLL mice that were treated with the combination. Such data can provide the rationale for a clinical trial exploring this combination in patients with relapsed or refractory T-PLL.

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive, mature T-cell leukemia with very few treatment options and adverse prognosis. There are currently no approved therapies for patients with relapsed T-PLL, where the median OS is less than 6 months. There are limited reports suggesting clinical activity of the BCL2 inhibitor venetoclax in patients with T-PLL. We aimed to characterize the dependencies of T-PLL on members of the BCL2-family of antiapoptotic proteins using BH3 profiling, a functional assay to assess the propensity of a cell to undergo apoptosis (priming) and the relative dependence of a cell on different antiapoptotic proteins. We then sought to experimentally exploit these dependencies with targeted therapies alone, and in combination, designed to inhibit the antiapoptotic proteins towards a therapeutic goal. Cell lines of mature T cell lymphomas/leukemias (TCL/L) including SUPT11 (T-cell leukemia with rearranged TCL1) (n=10) and bone marrow and peripheral blood samples from untreated patients with T-PLL (n=7) were analyzed by BH3 profiling. We also used a relapsed/refractory T-PLL patient sample that were able to engraft and expand in a PDX model. Finally, scRNA/ATAC seq has also been performed in a set of naïve-treated T-PLL patients. In contrast to TCL/L that demonstrated heterogeneity on BCL2 family member dependency, T-PLL samples were primed and consistently exhibited dependency on BCL2/BCL-xL and MCL1. Integration of scRNA/ATAC seq confirmed high expression of BCL2 and MCL1 in T-PLL cells from four patients supporting the premise that BCL2 and MCL1 could potentially be targeted for therapeutic benefit. Therefore, SUPT11 cells (used as a model of T-PLL) were exposed to venetoclax (25 and 50nM; BCL-2inh), fadraciclib (25 and 50nM; CDK2/9inh - indirect inhibitor of MCL-1 as it decreases mRNAs with high decay rates including MCL1 and MYC) or a combination of venetoclax and fadraciclib. We demonstrate that exposure to fadraciclib caused a rapid decline in the levels of phosphorylation on Serine 2 of RNA polymerase II C-terminal domain, as well as decline in the levels of MCL1 within 4 hours and completely depleted these proteins by 8-12 h of exposure. We also show that there was a synergistic increase both in mitochondrial dysfunction as measured by cytochrome C release by 18h and in cell death in cells exposed to the combination at 24h. These results suggest that combining venetoclax and fadraciclib might represent a potential therapeutic approach for T-PLL. In conclusion, preclinical evaluation of a T-PLL cell line and primary patient samples demonstrate BCL2-family dependency that can be effectively targeted with small molecule inhibitors of BCL2 and CDK2/9. In vivo studies in PDX models of T-PLL are underway to form the basis for clinical trials to study these combinations.

The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generated diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model to investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that these conditional mice exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping proteins in linking NDUFS4 with improved cristae morphology. Taken together, we discover the central role of NDUFS4 as a powerful regulator of cristae remodeling, respiratory supercomplexes assembly, and mitochondrial ultrastructure in vitro and in vivo. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.

The kidney is one of the most metabolically active organs and contains several different cell types that work in varied and dynamic macro- and microenvironments. This high compartmentalization reflects the organ's structural characteristics, which are dictated by the important functions of blood filtration, electrolyte regulation, and fluid balance. As a result, energy demands within different renal cells are extremely tailored to the specific cellular functions. Cellular energy demands are largely met by mitochondria and, as such, the mitochondrion is uniquely intertwined with kidney function. This organelle is the powerhouse of the cell, and it provides for the high demand of adenosine triphosphate in the kidney, but it has become increasingly apparent that energy production is only one of the many important functions that the mitochondria play within different renal cells. Placement of mitochondria as the central hub of a multitude of cellular processes underlies why there has been so much current interest in this organelle's role in health and disease. Indeed, mitochondrial dysfunction is associated with almost all types of kidney diseases and is a growing area of current research. In this chapter, we attempt to cover this very broad topic by highlighting the most recent findings describing the role of mitochondria in kidney homeostasis and disease condition. In particular, we describe how mitochondria are distributed in the kidney and influence cellular specification, as well as the different functions within specific renal cell types. Additionally, we describe the main process regulating mitochondrial homeostasis, including mitochondrial biogenesis, dynamics, trafficking, and disposal, and how these processes may be involved in the initiation and progression of kidney diseases. Lastly, we have mentioned how noncoding RNA molecules and one-carbon metabolism may impact mitochondria.

Long non-coding RNA Tug1 is emerging as a novel therapeutic target in the progression of diabetic nephropathy (DN), but the molecular basis of its protection in vivo remains poorly understood. Here we generated a triple mutant diabetic mouse model coupled with metabolomic profiling data to interrogate whether Tug1 interaction with PGC1α is required for the mitochondrial remodeling and progression of DN in vivo . Using an established diabetic mouse model with conditional inducible deletion of Pgc1α in podocytes, alone ( db/db;Pgc1α Pod-f/f ), or in combination with podocyte-specific Tug1 overexpression ( db/db;TugPodTg ; Pgc1αPod-f/f ), we found that the protective phenotype of Tug1 overexpression in diabetic db/db mice was reversed with conditional knockout of Pgc1α in podocytes, suggesting the requirement of PGC1α for the modulatory effects of lncRNA Tug1 in DN progression. Mechanistically, our findings highlight the multifaceted contributions of lncRNA Tug1 on mitochondrial bioenergetics, biogenesis and dynamics in podocytes of diabetic mice. Using an unbiased metabolomics profiling, we unexpectedly discovered that altered urea cycle metabolites and mitochondrial arginase 2 play an important role in Tug1/PGC1α-induced mitochondrial remodeling. Our work identifies an important but previously unappreciated functional role of lncRNA Tug1/PGC1α axis on mitochondrial metabolic homeostasis and urea cycle metabolites in experimental models of diabetes.

The role and nature of mitochondrial dysfunction in diabetic kidney disease (DKD) has been extensively studied. Yet, the molecular drivers of mitochondrial remodeling in DKD are poorly understood. Diabetic kidney cells exhibit a cascade of mitochondrial dysfunction ranging from changes in mitochondrial morphology to significant alterations in mitochondrial biogenesis, biosynthetic, bioenergetics and production of reactive oxygen species (ROS). How these changes individually or in aggregate contribute to progression of DKD remain to be fully elucidated. Nevertheless, because of the remarkable progress in our basic understanding of the role of mitochondrial biology and its dysfunction in DKD, there is great excitement on future targeted therapies based on improving mitochondrial function in DKD. This review will highlight the latest advances in understanding the nature of mitochondria dysfunction and its role in progression of DKD, and the development of mitochondrial targets that could be potentially used to prevent its progression.

The role and nature of mitochondrial dysfunction in diabetic kidney disease (DKD) has been extensively studied. Yet, the molecular drivers of mitochondrial remodeling in DKD are poorly understood. Diabetic kidney cells exhibit a cascade of mitochondrial dysfunction ranging from changes in mitochondrial morphology to significant alterations in mitochondrial biogenesis, biosynthetic, bioenergetics and production of reactive oxygen species (ROS). How these changes individually or in aggregate contribute to progression of DKD remain to be fully elucidated. Nevertheless, because of the remarkable progress in our basic understanding of the role of mitochondrial biology and its dysfunction in DKD, there is great excitement on future targeted therapies based on improving mitochondrial function in DKD. This review will highlight the latest advances in understanding the nature of mitochondria dysfunction and its role in progression of DKD, and the development of mitochondrial targets that could be potentially used to prevent its progression.

Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.

Mitochondrial medicine has experienced significant progress in recent years and is expected to grow significantly in the near future, yielding many opportunities to translate novel bench discoveries into clinical medicine. Multiple lines of evidence have linked mitochondrial dysfunction to a variety of metabolic diseases, including diabetic nephropathy (DN). Mitochondrial dysfunction presumably precedes the emergence of key histologic and biochemical features of DN, which provides the rationale to explore mitochondrial fitness as a novel therapeutic target in patients with DN. Ultimately, the success of mitochondrial medicine is dependent on a better understanding of the underlying biology of mitochondrial fitness and function. To this end, recent advances in mitochondrial biology have led to new understandings of the potential effect of mitochondrial dysfunction in a myriad of human pathologies. We have proposed that molecular mechanisms that modulate mitochondrial dynamics contribute to the alterations of mitochondrial fitness and progression of DN. In this comprehensive review, we highlight the possible effects of mitochondrial dysfunction in DN, with the hope that targeting specific mitochondrial signaling pathways may lead to the development of new drugs that mitigate DN progression. We will outline potential tools to improve mitochondrial fitness in DN as a novel therapeutic strategy. These emerging views suggest that the modulation of mitochondrial fitness could serve as a key target in ameliorating progression of kidney disease in patients with diabetes.

One-carbon metabolism plays a central role in a broad array of metabolic processes required for the survival and growth of tumor cells. However, the molecular basis of how one-carbon metabolism may influence RNA methylation and tumorigenesis remains largely unknown. Here we show MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, contributes to the progression of renal cell carcinoma (RCC) via a novel epitranscriptomic mechanism that involves HIF-2α. We found that expression of MTHFD2 was significantly elevated in human RCC tissues, and MTHFD2 knockdown strongly reduced xenograft tumor growth. Mechanistically, using an unbiased methylated RNA immunoprecipitation sequencing (meRIP-Seq) approach, we found that MTHFD2 plays a critical role in controlling global N6-methyladenosine (m6A) methylation levels, including the m6A methylation of HIF-2α mRNA, which results in enhanced translation of HIF-2α. Enhanced HIF-2α translation, in turn, promotes the aerobic glycolysis, linking one-carbon metabolism to HIF-2α-dependent metabolic reprogramming through RNA methylation. Our findings also suggest that MTHFD2 and HIF-2α form a positive feedforward loop in RCC, promoting metabolic reprograming and tumor growth. Taken together, our results suggest that MTHFD2 links RNA methylation status to the metabolic state of tumor cells in RCC.

Phosphorylation of dynamin-related protein 1 (Drp1) represents an important regulatory mechanism for mitochondrial fission. Here, we established the role of Drp1 serine 600 (Drp1S600) phosphorylation in mitochondrial fission in vivo and assessed the functional consequences of targeted elimination of the Drp1S600 phosphorylation site in the progression of diabetic nephropathy (DN). We generated a knockin mouse in which S600 was mutated to alanine (Drp1S600A). We found that diabetic Drp1S600A mice exhibited improved biochemical and histological features of DN along with reduced mitochondrial fission and diminished mitochondrial ROS in vivo. Importantly, we observed that the effect of Drp1S600 phosphorylation on mitochondrial fission in the diabetic milieu was stimulus dependent but not cell type dependent. Mechanistically, we show that mitochondrial fission in high-glucose conditions occurs through concomitant binding of phosphorylated Drp1S600 with mitochondrial fission factor (MFF) and actin-related protein 3 (Arp3), ultimately leading to accumulation of F-actin and Drp1 on the mitochondria. Taken together, these findings establish the idea that a single phosphorylation site in Drp1 can regulate mitochondrial fission and progression of DN in vivo and highlight the stimulus-specific consequences of Drp1S600 phosphorylation in mitochondrial dynamics.

This chapter presents an overview of zinc finger proteins (ZFPs), the various methods available to researchers for engineering them and options for genomic modifications. Modular assembly for engineering ZFPs utilizes a combination of validated ZF modules that each targets a separate DNA triplet. The chapter discusses the methods for validation of genomic modifications and also presents an overview of options for delivery to cells. The delivery methodology use for ZF nuclease (ZFN)-mediated genome modification will depend on the cell type targeted and whether or not the cells will be modified in vitro or in vivo. The chapter explores some novel ways that ZFs are being used for genomic alteration. It mainly focuses on the use of engineered ZFPs called ZFNs for site-directed genomic modification through targeted DNA cleavage. Fusion of ZF DNA-binding domains to recombinases and transposases has resulted in successful redirection of the integration events to varying degrees.

The outcome for advanced stage hepatocellular carcinoma (HCC) remains poor, highlighting the need for novel therapies. Genetically modified mesenchymal stem cells (MSCs) are actively being explored as cancer therapeutics due to their inherent ability to migrate to tumor sites. We reasoned that MSCs can be genetically modified to redirect T cells to Glypican-3 (GPC3)+ HCC, and genetically modified these with viral vectors encoding a GPC3/CD3 bispecific T cell engager (GPC3-ENG), a bispecifc T cell engager specific for an irrelevant antigen (EGFRvIII), and/or costimulatory molecules (CD80 and 41BBL). Coculture of GPC3+ cells, GPC3-ENG MSCs, and T cells resulted in T cell activation, as judged by interferon γ (IFNγ) production and killing of tumor cells by T cells. Modification of GPC3-ENG MSCs with CD80 and 41BBL was required for antigen-dependent interleukin-2 (IL-2) production by T cells and resulted in faster tumor cell killing by redirected T cells. In vivo, GPC3-ENG MSCs ± costimulatory molecules had antitumor activity in the HUH7 HCC xenograft model, resulting in a survival advantage. In conclusion, MSCs genetically modified to express GPC3-ENG ± costimulatory molecules redirect T cells to GPC3+ tumor cells and have potent antitumor activity. Thus, further preclinical exploration of our modified approach to GPC3-targeted immunotherapy for HCC is warranted.

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.

While increased mitochondrial reactive oxygen species have been commonly implicated in a variety of disease states, their in vivo role in the pathogenesis of diabetic nephropathy remains controversial. Using a two-photon imaging approach with a genetically encoded redox biosensor, we monitored mitochondrial redox state in the kidneys of experimental models of diabetes in real-time in vivo. Diabetic (db/db) mice that express a redox-sensitive Green Fluorescent Protein biosensor (roGFP) specifically in the mitochondrial matrix (db/dbmt-roGFP) were generated, allowing dynamic monitoring of redox changes in the kidneys. These db/dbmt-roGFP mice exhibited a marked increase in mitochondrial reactive oxygen species in the kidneys. Yeast NADH-dehydrogenase, a mammalian Complex I homolog, was ectopically expressed in cultured podocytes, and this forced expression in roGFP-expressing podocytes prevented high glucose-induced increases in mitochondrial reactive oxygen species. Thus, in vivo monitoring of mitochondrial roGFP in diabetic mice confirms increased production of mitochondrial reactive oxygen species in the kidneys.

Recent advances have led to a greater appreciation of how mitochondrial dysfunction contributes to diverse acute and chronic pathologies. Indeed, mitochondria have received increasing attention as a therapeutic target in a variety of diseases because they serve as key regulatory hubs uniquely situated at crossroads between multiple cellular processes. This review provides an overview of the role of mitochondrial dysfunction in chronic kidney disease, with special emphasis on its role in the development of diabetic nephropathy. We examine the current understanding of the molecular mechanisms that cause mitochondrial dysfunction in the kidney and describe the impact of mitochondrial damage on kidney function. The new concept that mitochondrial shape and structure are closely linked with its function in the kidneys is discussed. Furthermore, the mechanisms that translate cellular cues and demands into mitochondrial remodeling and cellular damage, including the role of microRNAs and long noncoding RNAs, are examined with the final goal of identifying mitochondrial targets to improve treatment of patients with chronic kidney diseases.

The cross talk between the immune and nervous systems is critical not only for maintaining normal homeostasis but also for the progression of a variety of inflammatory diseases. Macrophage activation and beta(2)-adrenergic receptors are known to play important roles in facilitating this communication between these 2 systems. Using an integrated in vitro and in vivo study, Noh et al. reveal that beta(2)-adrenergic receptor agonists exhibit protective effects against the vascular complications of diabetes. The protective effects of beta(2)-adrenergic receptor agonists seemto be dependent on a beta-arrestin2/inhibitor of kappa B/nuclear factor-kappa B signaling pathway.

Diabetic nephropathy (DN) is the most common cause of ESRD in the United States. Current standard of care for patients with DN can slow the progression of DN, but there are no effective therapies to halt progression of established DN. The lack of more effective therapies in established DN is in part because the underlying mechanisms involved in its progression are not fully understood. Therefore, there is an urgent need for novel, effective, and safe approaches for the prevention and reversal of diabetic kidney disease. The IL-17 family is comprised of six structurally related ligands: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. The receptors for the IL-17 family members (IL-17RA–IL-17RE) form homo- and heterodimers leading to activation of downstream target gene expression. IL-17A, the most intensively studied family member, is produced by multiple cell types, such as CD4+αβ T cells, γδ T cells, natural killer cells, and neutrophils, and is principally implicated in neutrophil recruitment and migration. Increased IL-17A levels have been implicated in the pathogenesis of several diseases, including multiple sclerosis, rheumatoid arthritis, and cancer. Interestingly, loss of IL-17 has also been associated with disease susceptibility in part because it has been suggested that the absence of IL-17 results in enhanced production of other proinflammatory cytokines.1 However, more recent studies have challenged the traditional pathogenic role of IL-17 as a purely proinflammatory cytokine. For instance, increased levels of IL-17 were shown to protect against autoimmune mediated type 1 diabetes in non-obese diabetic mice.2 Furthermore, IL-17 knockout mice exhibited greater kidney damage in a deoxycorticosterone acetate and angiotensin II model of kidney injury.3 Our interpretation on the seemingly paradoxical effects of IL-17 is that the modulatory effects of IL-17 on inflammation may be dependent on disease context, tissue, isoform, and receptor-ligand interactions. Although inflammation is a key factor in the pathogenesis of diabetes and its major microvascular complications, including diabetic nephropathy (DN), less is known regarding the specific contributions of IL-17. On the basis of early work, IL-17 seemed to be implicated in type 1 diabetes progression; however, other studies have suggested opposite effects.4 For instance, in mice with streptozotocin-induced type 1 diabetes, inhibition of intrarenal IL-17A+ CD4+ T cells, which secrete IL-17A, leads to improvements in many features of DN, implying that reduced levels of IL-17 levels may be protective in DN.5,6 To address some of these discrepancies, Mohamed et al. set out to understand the role of IL-17 in DN pathogenesis and progression.7 Their study stems from their initial observations, indicating that IL-17 levels were elevated in the urine from microalbuminuric diabetic patients and mice, but interestingly, these levels were significantly reduced with DN progression. On the basis of these initial findings, they reasoned that maintenance of IL-17 levels could exert renoprotection in DN. To test this hypothesis, the authors used an integrated pharmaceutic and genetic approach by taking advantage of global IL-17 knockout mice, epithelial cell-specific IL-17 transgenic mice, and recombinant IL-17 administration. Importantly, they demonstrate that increased levels of IL-17 are able to exert a robust protective role on podocytes and tubular cells as measured by reduced markers of inflammation and fibrosis in kidneys of mouse models with DN. The authors conclude that low dose IL-17 therapy has considerable promise in preventing the progression of DN. Mechanistically, the authors demonstrate that IL-17A suppressed phosphorylation of signal transducer and activator of transcription 3, upregulated an anti-inflammatory protein microglia/macrophage WAP domain protein (AMWAP), and improved oxidative stress in the kidney. The findings of their study suggest that both IL-17A and IL-17F have similar renoprotection. This is important because current evidence suggests that each of these ligands has a different affinity for the IL-17RA subunit of their shared IL-17RA/C heterodimeric receptor, which may result in unique signaling and specific downstream gene targets.1,8 Therefore, it would be potentially interesting to consider investigating differentially expressed downstream target genes of IL-17A versus IL-17F in diabetic kidneys to identify unique signaling pathways, which could mediate the renoprotection of each specific ligand. Similarly, further studies are required to investigate whether changes in IL-17 levels lead to alterations in the levels of other cytokines, which could also explain the effects observed on IL-17 restoration. This is of paramount importance because it has been previously shown, as discussed previously, that changes in IL-17 may give rise to modulatory and compensatory changes in other cytokines (e.g., tumor necrosis factor alpha, interferon gamma)9. The study by Mohamed et al. does not establish which cell types in the kidney respond to IL-17 during diabetes; albeit, the authors provide evidence that IL-17 ultimately leads to increased survival in podocytes and tubular cells. One way to address the impact of IL-17 on specific cell types in the kidney would be to knockdown the IL-17A/F receptor specifically in podocytes and/or tubular cells in vivo. The expectation would be that IL-17A/F protective effects would be blunted in this mouse model. Future studies using conditional IL-17RA knockdown, in conjunction with current observations by Mohamed et al., would help to delineate the cell-type specific response to low-dose IL-17 therapy in DN. Among other important issues that remain unresolved in this study is the identification of regulatory mechanisms governing the temporal profile changes of IL-17 levels during DN progression. Early reports have suggested that high glucose can induce IL-17 expression in lymphocytes.10 It remains unknown, however, whether the source of changes in IL-17 levels is from infiltrated or resident immune cells. Appreciation of the exact sources of IL-17 in the diabetic kidney would help to further understand its cellular targets and define how diabetes modulate the expression of endogenous IL-17 in the kidney. Finally, although the findings of this study provide new evidence supporting the potential ability of IL-17A to protect against DN, the mechanisms underlying this protective impact are not completely understood. Mechanistically, reduced oxidative stress, phosphorylation of AMP-activated protein kinase, and inhibition of nuclear factor κB have been implicated as critical mediators of IL-17 protection. Importantly, the authors have identified AMWAP as a novel target of IL-17A. The AMWAP reduces proinflammatory responses, and in conjunction with AMP-activated protein kinase it acts to preserve cell survival. Similarly, AMWAP converts macrophages to a less inflammatory M2 phenotype. Although it may be expected to share similar signaling pathways between macrophage and epithelial cells, it would be interesting to assess whether macrophage specific knockdown of the AMWAP or transfer of macrophages lacking AMWAP could separate signaling in different cell types, expanding on our understanding on the protective role of IL-17 and its downstream target genes. In conclusion, the study by Mohamed et al. provides novel insights allowing development of tailor-made anti-inflammatory–based therapies for treatment and reversal of DN. However, it still warrants further investigation to fully clarify the role of IL-17 in this disease process. Nonetheless, this work represents a novel therapy for DN, which could be potentially translated to clinical applications. Disclosures None. This work was supported by NIH-NIDDK grants (RO1-DK091310 and RO1-DK078900).

Mitochondrial fission has been linked to the pathogenesis of diabetic nephropathy (DN). However, how mitochondrial fission affects progression of DN in vivo is unknown. Here, we report the effect of conditional podocyte-specific deletion of dynamin-related protein 1 (Drp1), an essential component of mitochondrial fission, on the pathogenesis and progression of DN. Inducible podocyte-specific deletion of Drp1 in diabetic mice decreased albuminuria and improved mesangial matrix expansion and podocyte morphology. Ultrastructure analysis revealed a significant increase in fragmented mitochondria in the podocytes of wild-type diabetic mice but a marked improvement in mitochondrial structure in Drp1-null podocytes of diabetic mice. When isolated from diabetic mice and cultured in high glucose, Drp1-null podocytes had more elongated mitochondria and better mitochondrial fitness associated with enhanced oxygen consumption and ATP production than wild-type podocytes. Furthermore, administration of a pharmacologic inhibitor of Drp1, Mdivi1, significantly blunted mitochondrial fission and rescued key pathologic features of DN in mice. Taken together, these results provide novel correlations between mitochondrial morphology and the progression of DN and point to Drp1 as a potential therapeutic target in DN.