Podocyte and endothelial-specific elimination of BAMBI identifies differential transforming growth factor-ß pathways contributing to diabetic glomerulopathy.

Transforming growth factor-ß (TGF-ß) is a central mediator of diabetic nephropathy. The effect of TGF-ß, mediated by the type I TGF-ß receptor, ALK5, and subsequent Smad2/3 activation results in podocyte apoptosis and loss. Previously, we demonstrated that the genetic deletion of the BMP and Activin Membrane-Bound Inhibitor (BAMBI), a negative modulator TGF-ß signaling, accelerates diabetic nephropathy in mice. This was associated with heightened ALK1-mediated activation of Smad1/5 in the glomerular endothelial cells (ECs). Therefore, to evaluate the glomerular cell-specific effects of TGF-ß in diabetic nephropathy we examined the effects of the podocyte- or EC-specific loss of Bambi (Pod-Bambi-/- or EC-Bambi-/-) in streptozotocin-induced diabetic mice with endothelial nitric oxide synthase deficiency. Interestingly, although hyperglycemia and body weight loss were similar in all groups of diabetic mice, significant hypertension was present only in the diabetic EC-Bambi-/- mice. While the podocyte or EC-specific loss of BAMBI both accelerated the progression of diabetic nephropathy, the worsened podocyte injury and loss observed in the diabetic Pod-Bambi-/- mice were associated with enhanced Smad3 activation. Increased Smad1/5 activation and EC proliferation were apparent only in the glomeruli of diabetic EC-Bambi-/- mice. The enhanced Smad1/5 activation in diabetic EC-Bambi-/- mice was associated with increased glomerular expression of plasmalemma vesicle-associated protein, pointing to the involvement of immature or dedifferentiated glomerular ECs in diabetic nephropathy. Notably, diabetic EC-Bambi-/- mice displayed podocyte injury and loss that were comparable to diabetic Pod-Bambi-/- mice. Thus, our results highlight the glomerular cell-specific contribution of TGF-ß signaling and the intricate cross-talk between injured glomerular cells in the progression of diabetic nephropathy.

Role of CD8+ T cells in crescentic glomerulonephritis.

Crescentic glomerulonephritis (cGN) comprises three main types according to the pathogenesis and immunofluorescence patterns: anti-glomerular basement membrane antibody cGN, vasculitis-associated cGN and post-infectious immune complex cGN. In this brief review of the immune-pathogenesis of cGN, the focus is mainly on the role of CD8+ T cells in the progression of cGN. Under control conditions, Bowman's capsule (BC) provides a protected immunological niche by preventing access of cytotoxic CD8+ T cells to Bowman's space and thereby podocytes. Even in experimental nephrotoxic nephritis, leukocytes accumulate around the glomeruli, but remain outside of BC, as long as the latter remains intact. However, when and where breaches in BC occur, the inflammatory cells can gain access to and destroy podocytes, thus converting cGN into rapidly progressive glomerulonephritis (RPGN). These conclusions also apply to human cGN, where biopsies show that loss of BC integrity is associated with RPGN and progression to end-stage kidney disease. We propose a two-hit hypothesis for the role of cytotoxic CD8+ T cells in the progression of cGN. The initial insult occurs in response to the immune complex formation or deposition, resulting in local capillary and podocyte injury (first hit). The injured podocytes release neo-epitopes, eventually causing T-cell activation and migration to the glomerulus. Upon generation of breaches in BC, macrophages and CD8+ T cells can now gain access to the glomerular space and destroy neo-epitope expressing podocytes (second hit), resulting in RPGN. While further investigation will be required to test this hypothesis, future therapeutic trials should consider targeting of CD8+ T cells in the therapy of progressive cGN.

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease.

BACKGROUND: Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited. METHODS: To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS-/-) mice and control eNOS-/- mice. RESULTS: We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury. CONCLUSIONS: Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-ß-Induced Angiogenesis.

BACKGROUND: Glomerular endothelial dysfunction and neoangiogenesis have long been implicated in the pathogenesis of diabetic kidney disease (DKD). However, the specific molecular pathways contributing to these processes in the early stages of DKD are not well understood. Our recent transcriptomic profiling of glomerular endothelial cells identified a number of proangiogenic genes that were upregulated in diabetic mice, including leucine-rich α-2-glycoprotein 1 (LRG1). LRG1 was previously shown to promote neovascularization in mouse models of ocular disease by potentiating endothelial TGF-ß/activin receptor-like kinase 1 (ALK1) signaling. However, LRG1's role in the kidney, particularly in the setting of DKD, has been unclear. METHODS: We analyzed expression of LRG1 mRNA in glomeruli of diabetic kidneys and assessed its localization by RNA in situ hybridization. We examined the effects of genetic ablation of Lrg1 on DKD progression in unilaterally nephrectomized, streptozotocin-induced diabetic mice at 12 and 20 weeks after diabetes induction. We also assessed whether plasma LRG1 was associated with renal outcome in patients with type 2 diabetes. RESULTS: LRG1 localized predominantly to glomerular endothelial cells, and its expression was elevated in the diabetic kidneys. LRG1 ablation markedly attenuated diabetes-induced glomerular angiogenesis, podocyte loss, and the development of diabetic glomerulopathy. These improvements were associated with reduced ALK1-Smad1/5/8 activation in glomeruli of diabetic mice. Moreover, increased plasma LRG1 was associated with worse renal outcome in patients with type 2 diabetes. CONCLUSIONS: These findings identify LRG1 as a potential novel pathogenic mediator of diabetic glomerular neoangiogenesis and a risk factor in DKD progression.

Bowman's capsule provides a protective niche for podocytes from cytotoxic CD8+ T cells.

T cells play a key role in immune-mediated glomerulonephritis, but how cytotoxic T cells interact with podocytes remains unclear. To address this, we injected EGFP-specific CD8+ T cells from just EGFP death inducing (Jedi) mice into transgenic mice with podocyte-specific expression of EGFP. In healthy mice, Jedi T cells could not access EGFP+ podocytes. Conversely, when we induced nephrotoxic serum nephritis (NTSN) and injected Jedi T cells, EGFP+ podocyte transgenic mice showed enhanced proteinuria and higher blood urea levels. Morphometric analysis showed greater loss of EGFP+ podocytes, which was associated with severe crescentic and necrotizing glomerulonephritis. Notably, only glomeruli with disrupted Bowman's capsule displayed massive CD8+ T cell infiltrates that were in direct contact with EGFP+ podocytes, causing their apoptosis. Thus, under control conditions with intact Bowman's capsule, podocytes are not accessible to CD8+ T cells. However, breaches in Bowman's capsule, as also noted in human crescentic glomerulonephritis, allow access of CD8+ T cells to the glomerular tuft and podocytes, resulting in their destruction. Through these mechanisms, a potentially reversible glomerulonephritis undergoes an augmentation process to a rapidly progressive glomerulonephritis, leading to end-stage kidney disease. Translating these mechanistic insights to human crescentic nephritis should direct future therapeutic interventions at blocking CD8+ T cells, especially in progressive stages of rapidly progressive glomerulonephritis.

Gene expression profiles of glomerular endothelial cells support their role in the glomerulopathy of diabetic mice.

Endothelial dysfunction promotes the pathogenesis of diabetic nephropathy (DN), which is considered to be an early event in disease progression. However, the molecular changes associated with glomerular endothelial cell (GEC) injury in early DN are not well defined. Most gene expression studies have relied on the indirect assessment of GEC injury from isolated glomeruli or renal cortices. Here, we present transcriptomic analysis of isolated GECs, using streptozotocin-induced diabetic wildtype (STZ-WT) and diabetic eNOS-null (STZ-eNOS-/-) mice as models of mild and advanced DN, respectively. GECs of both models in comparison to their respective nondiabetic controls showed significant alterations in the regulation of apoptosis, oxidative stress, and proliferation. The extent of these changes was greater in STZ-eNOS-/- than in STZ-WT GECs. Additionally, genes in STZ-eNOS-/- GECs indicated further dysregulation in angiogenesis and epigenetic regulation. Moreover, a biphasic change in the number of GECs, characterized by an initial increase and subsequent decrease over time, was observed only in STZ-eNOS-/- mice. This is consistent with an early compensatory angiogenic process followed by increased apoptosis, leading to an overall decrease in GEC survival in DN progression. From the genes altered in angiogenesis in STZ-eNOS-/- GECs, we identified potential candidate genes, Lrg1 and Gpr56, whose function may augment diabetes-induced angiogenesis. Thus, our results support a role for GEC in DN by providing direct evidence for alterations of GEC gene expression and molecular pathways. Candidate genes of specific pathways, such as Lrg1 and Gpr56, can be further explored for potential therapeutic targeting to mitigate the initiation and progression of DN.

Revisiting the determinants of the glomerular filtration barrier: what goes round must come round.

The glomerular filtration barrier (GFB) is characterized by a very high hydraulic permeability, combined with a marked permselectivity that excludes macromolecules such as albumin. Thus, the GFB retains most of the plasma proteins, with only 0.06% of albumin getting across the basement membrane. The GFB consists of 3 layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes. Injury to any of these components can result in the development of proteinuria. The contribution of the major components of the GFB has recently been reexamined and is discussed in the context of our past and present understanding.

Glomerular Endothelial Mitochondrial Dysfunction Is Essential and Characteristic of Diabetic Kidney Disease Susceptibility.

The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.

Precision medicine comes of age in nephrology: identification of novel biomarkers and therapeutic targets for chronic kidney disease.

The goal of "precision medicine" is to characterize diseases based on the underlying molecular biology, in order to identify specific biomarkers and therapeutic targets that will ultimately improve clinical outcomes. The nephrology research community has developed a strong foundation for precision medicine, and recent publications demonstrate the feasibility of this approach to identify potential biomarkers and therapeutic targets in chronic kidney disease.

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

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-ß type I receptor family and a negative modulator of TGF-ß kinase signaling, and BAMBI(-/-) mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-ß 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-ß 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-ß 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-ß signaling may therefore be of interest in diabetic nephropathy.

Are serum suPAR determinations by current ELISA methodology reliable diagnostic biomarkers for FSGS?

A soluble proteinuric substance has been postulated as a cause of primary focal segmental glomerulosclerosis (FSGS), and one candidate soluble urokinase receptor (suPAR). Evaluation of published results shows that serum suPAR determinations by current methodology do not reliably distinguish FSGS from other proteinuric glomerular diseases and serum suPAR cannot currently be considered a valid biomarker for primary or secondary FSGS. However, this should not discourage further research on potential roles of suPAR in proteinuric renal disease, including FSGS.

Endothelial mitochondrial oxidative stress determines podocyte depletion in segmental glomerulosclerosis.

Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-ß activation in podocytes; however, it is not clear how TGF-ß signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-ß signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS.

Mpv17 in mitochondria protects podocytes against mitochondrial dysfunction and apoptosis in vivo and in vitro.

Mitochondrial dysfunction is increasingly recognized as contributing to glomerular diseases, including those secondary to mitochondrial DNA (mtDNA) mutations and deletions. Mitochondria maintain cellular redox and energy homeostasis and are a major source of intracellular reactive oxygen species (ROS) production. Mitochondrial ROS accumulation may contribute to stress-induced mitochondrial dysfunction and apoptosis and thereby to glomerulosclerosis. In mice, deletion of the gene encoding Mpv17 is associated with glomerulosclerosis, but the underlying mechanism remains poorly defined. Here we report that Mpv17 localizes to mitochondria of podocytes and its expression is reduced in several glomerular injury models and in human focal segmental glomerulosclerosis (FSGS) but not in minimal change disease. Using models of mild or severe nephrotoxic serum nephritis (NTSN) in Mpv17(+/+) wild-type (WT) and Mpv17(-/-) knockout mice, we found that Mpv17 deficiency resulted in increased proteinuria (mild NTSN) and renal insufficiency (severe NTSN) compared with WT. These lesions were associated with increased mitochondrial ROS generation and mitochondrial injury such as oxidative DNA damage. In vitro, podocytes with loss of Mpv17 function were characterized by increased susceptibility to apoptosis and ROS injury including decreased mitochondrial function, loss of mtDNA content, and change in mitochondrial configuration. In summary, the inner mitochondrial membrane protein Mpv17 in podocytes is essential for the maintenance of mitochondrial homeostasis and protects podocytes against oxidative stress-induced injury both in vitro and in vivo.

Epithelial cell TGFß signaling induces acute tubular injury and interstitial inflammation.

TGFß signaling plays a central role in the development of acute and chronic kidney diseases. Previous in vivo studies involved systemic alteration of TGFß signaling, however, limiting conclusions about the direct role of TGFß in tubular cell injury. Here, we generated a double transgenic mouse that inducibly expresses a ligand-independent constitutively active TGFß receptor type 1 (TßR1) kinase specifically in tubular epithelial cells, with expression restricted by the Pax8 promoter. In this model, activation of TGFß signaling in the tubular epithelium alone was sufficient to cause AKI characterized by marked tubular cell apoptosis and necrosis, oxidative stress, dedifferentiation and regenerative cell proliferation, reduced renal function, and interstitial accumulation of inflammatory cells. This tubular injury was associated with mitochondrial-derived generation of reactive oxygen species (ROS), but cell damage and apoptosis were partially independent of mitochondrial-derived ROS. TßR1 signaling-induced tubular injury also associated with significant leukocyte infiltration consisting of F4/80(+) macrophages, CD11c(+) F4/80(+) dendritic cells, CD11c(+) F4/80(-) Ly6C(high) dendritic cells/monocytes, and T cells. Inhibition of mitochondrial-derived ROS significantly reduced accumulation of CD11c(+) F4/80(+) dendritic cells and T cells, suggesting a role for ROS in the activation and recruitment of the adaptive immune response to tubular injury. Taken together, these results suggest that TGFß signaling in the tubular epithelium alone is sufficient to cause acute tubular injury and inflammation; therefore, TGFß may be a mechanistic link between acute injury and chronic progression of kidney disease.

Accelerated reendothelialization, increased neovascularization and erythrocyte extravasation after arterial injury in BAMBI-/- mice.

BACKGROUND: Intimal injury rapidly activates TGFß and enhances vascular repair by the growth of endothelial (EC) and vascular smooth muscle cells (VSMC). The response to the TGFß family of growth factors can be modified by BAMBI (BMP, Activin, Membrane Bound Inhibitor) acting as a non-signaling, competitive antagonist of TGFß type I receptors such as ALK 1 and 5. In vivo the effect of BAMBI will depend on its cell-specific expression and of that of the ALK type receptors. We recently reported EC restricted BAMBI expression and genetic elimination of BAMBI resulting in an in vitro and in vivo phenotype characterized by endothelial activation and proliferation involving alternative pathway activation by TGFß through ALK 1. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that BAMBI modulates arterial response to injury via its effects on endothelial repair and arterial wall neovascularization we used a model of femoral arterial denudation injury in wild type (WT) and BAMBI(-/-) mice. Arterial response was evaluated at 2 and 4 weeks after luminal endothelial denudation of femoral arteries. The BAMBI(-/-) genotype mice showed accelerated luminal endothelial repair at 2 weeks and a highly unusual increase in arterial wall neovascularization compared to WT mice. The exuberant intimal and medial neovessel formation with BAMBI(-/-) genotype was also associated with significant red blood cell extravasation. The bleeding into the neointima at 2 weeks transiently increased it's area in the BAMBI(-/-)genotype despite the faster luminal endothelial repair in this group. Vascular smooth muscle cells were decreased at 2 weeks in BAMBI(-/-) mice, but comparable to wild type at 4 weeks. CONCLUSIONS/SIGNIFICANCE: The absence of BAMBI results in a highly unusual surge in arterial wall neovascularization that surprisingly mimiks features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This suggests important effects of BAMBI on arterial EC homeostasis that need to be further studied in a model of inflammatory atherosclerosis.