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.

TGFb signaling plays a central role in the development of acute and chronic kidney diseases. Previous in vivo studies involved systemic alteration of TGFb signaling, however, limiting conclusions about the direct role of TGFb in tubular cell injury. Here, we generated a double transgenic mouse that inducibly expresses a ligand-independent constitutively active TGFb receptor type 1 (TbR1) kinase specifically in tubular epithelial cells, with expression restricted by the Pax8 promoter. In this model, activation of TGFb 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.

Transforming growth factors beta (TGF-β) are multi-functional cytokines capable of inducing apoptosis in epithelial cells, including glomerular podocytes. We and others have previously shown that podocyte-selective genetic deletion of the microRNA (miR)-processing enzyme, Dicer, caused glomerulosclerosis that was associated with podocyte apoptosis, and the miR-30 family was implicated in the process. Here, we report that apoptosis-associated genes were highly enriched among the predicted targets of miR-30 when compared with randomly selected miRs (26% vs. 4.5 ± 2.1%) or with the known TGF-β-regulated miR-192 (6%), miR-216a (5.1%), and miR-217 (0%). miR-30 family members were abundantly expressed in podocytes in normal mice but were downregulated in albumin/TGF-β transgenic mice with podocyte apoptosis and glomerulosclerosis. In vitro, TGF-β downregulated miR-30s in wildtype and Smad3-deficient, but not Smad2- or Smad2/Smad3-deficient, podocytes. The TGF-β-induced activation of caspase 3 and an increase in TUNEL-positive nuclei were significantly inhibited by the lentivirus-mediated overexpression of miR-30d, but not by a scrambled control miR, in podocytes. TGF-β stimulated the phosphorylation of pro-apoptotic p53 in podocytes with lentiviral expression of a scrambled miR, but not in podocytes expressing miR-30d. In contrast, miR-30d had no effect on the phosphorylation of pro-apoptotic p38 MAP kinase induced by TGF-β. Thus, we report that Smad2-dependent inhibition of miR-30s in podocytes is required for the activation of p53 and the induction of apoptosis by TGF-β. These results demonstrate a novel functional role for miR-30 in podocyte survival and indicate that the loss of miR-30 survival signaling is a novel and specific mechanism of TGF-β-induced podocyte apoptosis during glomerulosclerosis. We propose the therapeutic replacement of miR-30 as a novel strategy to prevent the podocyte apoptosis that is characteristic of progressive glomerular diseases.

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