microRNAs (miRNAs) are small, non-coding nucleotides that regulate diverse biological processes. Altered microRNA biosynthesis or regulation contributes to pathological processes including kidney fibrosis. Kidney fibrosis is characterized by deposition of excess extracellular matrix (ECM), which is caused by infiltration of immune cells, inflammatory cells, altered chemokines, and cytokines as well as activation and accumulation of fibroblasts in the kidney. These activated fibroblasts can arise from epithelial cells via epithelial-to-mesenchymal transition (EMT), from bone marrow-derived M2 phenotype macrophages via macrophage-to-mesenchymal transition (MMT), from endothelial cells via endothelial-to-mesenchymal transition (EndMT), from resident fibroblasts, and from bone marrow-derived monocytes and play a crucial role in fibrotic events. Disrupted microRNA biosynthesis and aberrant regulation contribute to the activation of mesenchymal programs in the kidney. miR-29 regulates the interaction between dipeptidyl peptidase-4 (DPP-4) and integrin β1 and the associated active transforming growth factor β (TGFβ) and pro-EndMT signaling; however, miR-let-7 targets transforming growth factor β receptors (TGFβRs) to inhibit TGFβ signaling. N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous anti-fibrotic peptide, which is associated with fibroblast growth factor receptor 1 (FGFR1) phosphorylation and subsequently responsible for the production of miR-let-7. miR-29 and miR-let-7 family clusters participate in crosstalk mechanisms, which are crucial for endothelial cell homeostasis. The physiological level of AcSDKP is vital for the activation of anti-fibrotic mechanisms including restoration of anti-fibrotic microRNA crosstalk and suppression of profibrotic signaling by mitigating DPP-4-associated mesenchymal activation in the epithelial cells, endothelial cells, and M2 phenotype macrophages. The present review highlights recent advancements in the understanding of both the role of microRNAs in the development of kidney disease and their potential as novel therapeutic targets for fibrotic disease states.

Renal artery stenosis (RAS) caused by narrowing of arteries is characterized by microvascular damage. Macrophages are implicated in repair and injury, but the specific populations responsible for these divergent roles have not been identified. Here, we characterized murine kidney F4/80+CD64+ macrophages in three transcriptionally unique populations. Using fate-mapping and parabiosis studies, we demonstrate that CD11b/cint are long-lived kidney-resident (KRM) while CD11chiMϕ, CD11cloMϕ are monocyte-derived macrophages. In a murine model of RAS, KRM self-renewed, while CD11chiMϕ and CD11cloMϕ increased significantly, which was associated with loss of peritubular capillaries. Replacing the native KRM with monocyte-derived KRM using liposomal clodronate and bone marrow transplantation followed by RAS, amplified loss of peritubular capillaries. To further elucidate the nature of interactions between KRM and peritubular endothelial cells, we performed RNA-sequencing on flow-sorted macrophages from Sham and RAS kidneys. KRM showed a prominent activation pattern in RAS with significant enrichment in reparative pathways, like angiogenesis and wound healing. In culture, KRM increased proliferation of renal peritubular endothelial cells implying direct pro-angiogenic properties. Human homologs of KRM identified as CD11bintCD11cintCD68+ increased in post-stenotic kidney biopsies from RAS patients compared to healthy human kidneys, and inversely correlated to kidney function. Thus, KRM may play protective roles in stenotic kidney injury through expansion and upregulation of pro-angiogenic pathways.

Microvascular rarefaction distal to renal artery stenosis is linked to renal dysfunction and poor outcomes. Low-energy shockwave therapy stimulates angiogenesis, but the effect on the kidney microvasculature is unknown. We hypothesized that low-energy shockwave therapy would restore the microcirculation and alleviate renal dysfunction in renovascular disease. Normal pigs and pigs subjected to 3 weeks of renal artery stenosis were treated with six sessions of low-energy shockwave (biweekly for 3 consecutive weeks) or left untreated. We assessed BP, urinary protein, stenotic renal blood flow, GFR, microvascular structure, and oxygenation in vivo 4 weeks after completion of treatment, and then, we assessed expression of angiogenic factors and mechanotransducers (focal adhesion kinase and β1-integrin) ex vivo A 3-week low-energy shockwave regimen attenuated renovascular hypertension, normalized stenotic kidney microvascular density and oxygenation, stabilized function, and alleviated fibrosis in pigs subjected to renal artery stenosis. These effects associated with elevated renal expression of angiogenic factors and mechanotransducers, particularly in proximal tubular cells. In additional pigs with prolonged (6 weeks) renal artery stenosis, shockwave therapy also decreased BP and improved GFR, microvascular density, and oxygenation in the stenotic kidney. This shockwave regimen did not cause detectable kidney injury in normal pigs. In conclusion, low-energy shockwave therapy improves stenotic kidney function, likely in part by mechanotransduction-mediated expression of angiogenic factors in proximal tubular cells, and it may ameliorate renovascular hypertension. Low-energy shockwave therapy may serve as a novel noninvasive intervention in the management of renovascular disease.

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The metabolic syndrome (MetS) induces intrarenal microvascular disease, which may involve mitochondrial injury. The mitochondrial cardiolipin-targeting peptide elamipretide (ELAM) improves the microcirculation in post-stenotic kidneys, but its ability to attenuate MetS-induced renal vascular damage is unknown. We hypothesized that chronic treatment with ELAM would decrease renal vascular remodelling and function in swine MetS. Pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with daily injections of ELAM (0.1 mg kg ), and lean control (Lean) animals (n = 6 each). Single-kidney regional perfusion, blood flow and glomerular filtration rate were measured with multi-detector computed tomography (CT). Peritubular capillary (PTC) endothelial cell (EC) mitochondrial density and cardiolipin content were assessed in situ, as were PTC-EC apoptosis and oxidative stress. The spatial density of PTCs (Haematoxylin and Eosin staining) and renal microvessels (micro-CT), and renal artery endothelial function (organ bath) were characterized. Regional perfusion and serum creatinine were preserved in MetS pigs, but renal blood flow and glomerular filtration rate were higher compared with Lean. Mitochondrial density and cardiolipin content were diminished in MetS PTC-ECs, but improved in ELAM-treated pigs, as did PTC density. Elamipretide also attenuated PTC-EC oxidative stress and apoptosis. Furthermore, ELAM improved renal microvascular density, decreased microvascular remodelling and restored endothelial nitric oxide expression and endothelium-dependent relaxation of renal artery segments. In conclusion, MetS-induced mitochondrial alterations might contribute to renal PTC and microvascular loss and might impair renal artery endothelial function in pigs. Mitoprotection with ELAM preserved a hierarchy of renal vessels, underscoring its potential to ameliorate renal vascular injury in MetS.

Hypercholesterolemia is a major risk factor for atherosclerosis. Remaining challenges in the management of atherosclerosis necessitate development of animal models that mimic human pathophysiology. We characterized a novel mutant pig model with DNA transposition of D374Y gain-of-function (GOF) cDNA of chimp proprotein convertase subtilisin/kexin type-9 (PCSK9), and tested the hypothesis that it would develop peripheral vascular remodeling and target organ injury in the kidney. Wild-type or PCSK9-GOF Ossabaw miniature pigs fed a standard or atherogenic diet (AD) (n = 7 each) were studied in vivo after 3 and 6 months of diet. Single-kidney hemodynamics and function were studied using multidetector computed tomography and kidney oxygenation by blood oxygen level-dependent magnetic resonance imaging. The renal artery was evaluated by intravascular ultrasound, aortic stiffness by multidetector computed tomography, and kidney stiffness by magnetic resonance elastography. Subsequent ex vivo studies included the renal artery endothelial function and morphology of abdominal aorta, renal, and femoral arteries by histology. Compared with wild type, PCSK9-GOF pigs had elevated cholesterol, triglyceride, and blood pressure levels at 3 and 6 months. Kidney stiffness increased in GOF groups, but aortic stiffness only in GOF-AD. Hypoxia, intrarenal fat deposition, oxidative stress, and fibrosis were observed in both GOF groups, whereas kidney function remained unchanged. Peripheral arteries in GOF groups showed medial thickening and development of atheromatous plaques. Renal endothelial function was impaired only in GOF-AD. Therefore, the PCSK9-GOF mutation induces rapid development of atherosclerosis in peripheral vessels of Ossabaw pigs, which is exacerbated by a high-cholesterol diet. This model may be useful for preclinical studies of atherosclerosis.

Axon degeneration in diabetic peripheral neuropathy (DPN) is associated with impaired NAD+ metabolism. We tested whether the administration of NAD+ precursors, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), prevents DPN in models of Type 1 and Type 2 diabetes. NMN was administered to streptozotocin (STZ)-induced diabetic rats and STZ-induced diabetic mice by intraperitoneal injection at 50 or 100 mg/kg on alternate days for 2 months. mice The were fed with a high fat diet (HFD) for 2 months with or without added NR at 150 or 300 mg/kg for 2 months. The administration of NMN to STZ-induced diabetic rats or mice or dietary addition of NR to HFD-fed mice improved sensory function, normalized sciatic and tail nerve conduction velocities, and prevented loss of intraepidermal nerve fibers in skin samples from the hind-paw. In adult dorsal root ganglion (DRG) neurons isolated from HFD-fed mice, there was a decrease in NAD+ levels and mitochondrial maximum reserve capacity. These impairments were normalized in isolated DRG neurons from NR-treated mice. The results indicate that the correction of NAD+ depletion in DRG may be sufficient to prevent DPN but does not significantly affect glucose tolerance, insulin levels, or insulin resistance.

Apoptosis of macrophages and smooth muscle cells is linked to atherosclerotic plaque destabilization. The apoptotic cascade leads to exposure of negatively charged phosphatidylethanolamine (PE) on the outer leaflet of the cell membrane, thereby making apoptosis detectable using probes targeting PE. The objective of this study was to exploit capabilities of a PE-specific imaging probe, 99mTc-duramycin, in localizing atherosclerotic plaque and assessing plaque evolution in apolipoprotein-E knockout (ApoE-/-) mice. Methods Atherosclerosis was induced in ApoE-/- mice by feeding an atherogenic diet. 99mTc-duramycin images were acquired using a small-animal SPECT imager. Six ApoE-/- mice at 20 weeks of age (Group I) were imaged and then sacrificed for ex vivo analyses. Six additional ApoE-/- mice (Group II) were imaged at 20 and 40 weeks of age before sacrifice. Six ApoE wild-type (ApoE+/+) mice (Group III) were imaged at 40 weeks as controls. Five additional ApoE-/- mice (40 weeks of age) (Group IV) were imaged with a 99mTc-labeled inactive peptide, 99mTc-LinDUR, to assess 99mTc-duramycin targeting specificity. Results Focal 99mTc-duramycin uptake in the ascending aorta and aortic arch was detected at 20 and 40 weeks in the ApoE-/- mice but not in ApoE+/+ mice. 99mTc-duramycin uptake in the aortic lesions increased 2.2-fold on quantitative imaging in the ApoE-/- mice between 20 and 40 weeks. Autoradiographic and histological data indicated significantly increased 99mTc-duramycin uptake in the ascending aorta and aortic arch associated with advanced plaques. Quantitative autoradiography showed that the ratio of activity in the aortic arch to descending thoracic aorta, which had no plaques and radioactive uptake, was 2.1 times higher at 40 weeks than at 20 weeks (6.62±0.89 vs. 3.18±0.29, P<0.01). There was barely detectable focal uptake of 99mTc-duramycin in the aortic arch of ApoE+/+ mice. No detectable 99mTc-LinDUR uptake was observed in the aortas of ApoE-/- mice. Conclusions PE-targeting properties of 99mTc-duramycin in the atherosclerotic mouse aortas were noninvasively characterized. 99mTc-duramycin is promising in localizing advanced atherosclerotic plaques.