NAD+ Precursors Reverse Experimental Diabetic Neuropathy in Mice.

Abnormal NAD+ signaling has been implicated in axonal degeneration in diabetic peripheral neuropathy (DPN). We hypothesized that supplementing NAD+ precursors could alleviate DPN symptoms through increasing the NAD+ levels and activating the sirtuin-1 (SIRT1) protein. To test this, we exposed cultured Dorsal Root Ganglion neurons (DRGs) to Nicotinamide Riboside (NR) or Nicotinamide Mononucleotide (NMN), which increased the levels of NAD+, the SIRT1 protein, and the deacetylation activity that is associated with increased neurite growth. A SIRT1 inhibitor blocked the neurite growth induced via NR or NMN. We then induced neuropathy in C57BL6 mice with streptozotocin (STZ) or a high fat diet (HFD) and administered NR or NMN for two months. Both the STZ and HFD mice developed neuropathy, which was reversed through the NR or NMN administration: sensory function improved, nerve conduction velocities normalized, and intraepidermal nerve fibers were restored. The NAD+ levels and SIRT1 activity were reduced in the DRGs from diabetic mice but were preserved with the NR or NMN treatment. We also tested the effect of NR or NMN administration in mice that overexpress the SIRT1 protein in neurons (nSIRT1 OE) and found no additional benefit from the addition of the drug. These findings suggest that supplementing with NAD+ precursors or activating SIRT1 may be a promising treatment for DPN.

NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy.

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.

Renal fibrosis detected by diffusion-weighted magnetic resonance imaging remains unchanged despite treatment in subjects with renovascular disease.

Tissue fibrosis is an important index of renal disease progression. Diffusion-weighted magnetic resonance imaging's (DWI-MRI) apparent diffusion coefficient (ADC) reveals water diffusion is unobstructed by microstructural alterations like fibrosis. We hypothesized that ADC may indicate renal injury and response to therapy in patients with renovascular disease (RVD). RVD patients were treated with medical therapy (MT) and percutaneous transluminal renal angioplasty (MT + PTRA) (n = 11, 3 bilaterally, n = 14 kidneys) or MT (n = 9). ADC and renal hypoxia (R2*) by blood-oxygen-level-dependent MRI were studied before (n = 27) and 3 months after (n = 20) treatment. Twelve patients underwent renal biopsies. Baseline ADC values were correlated with changes in eGFR, serum creatinine (SCr), systolic blood pressure (SBP), renal hypoxia, and renal vein levels of pro-inflammatory marker tumor necrosis-factor (TNF)-α. Renal oxygenation, eGFR, and SCr improved after MT + PTRA. ADC inversely correlated with the histological degree of renal fibrosis, but remained unchanged after MT or MT + PTRA. Basal ADC values correlated modestly with change in SBP, but not in renal hypoxia, TNF-α levels, or renal function. Lower ADC potentially reflects renal injury in RVD patients, but does not change in response to medical or interventional therapy over 3 months. Future studies need to pinpoint indices of kidney recovery potential.

Renal Adiposity Does not Preclude Quantitative Assessment of Renal Function Using Dual-Energy Multidetector CT in Mildly Obese Human Subjects.

RATIONALE AND OBJECTIVES: Multidetector computed tomography (MDCT) is useful for measuring in the research setting single-kidney perfusion and function using iodinated contrast time-attenuation curves. Obesity promotes deposition of intrarenal fat, which might decrease tissue attenuation and thereby interfere with quantification of renal function using MDCT. The purpose of this study was to test the hypothesis that background subtraction adequately accounts for intrarenal fat deposition in mildly obese human subjects during renal contrast enhanced dynamic CT. MATERIALS AND METHODS: We prospectively recruited seventeen human subjects stratified as lean or mildly obese based on body mass index below or over 30 kg/m2, respectively. Renal perfusion was quantified from CT-derived indicator-dilution curves after background subtraction. Dual-energy MDCT images were postprocessed to generate iodine and virtual-noncontrast datasets, and the ratios between kidney/aorta CT numbers and iodine values calculated as surrogates of renal function. RESULTS: Subcutaneous adipose tissue was increased in obese subjects. Virtual-noncontrast maps revealed in obese patients a decrease in basal cortical and medullary attenuation. Overall, basal attenuation inversely correlated with body mass index, in line with renal fat deposition. Contrarily, the kidney/aorta CT attenuation (after background subtraction) and kidney/aorta iodine ratios were similar between lean and obese subjects and correlated directly. These observations show that following background subtraction, the CT number reliably reflects basal tissue attenuation. CONCLUSION: Therefore, our findings support our hypothesis that background subtraction enables reliable assessment of kidney function in mildly obese subjects using MDCT, despite decreased basal attenuation due to renal adiposity.

Kidney-resident macrophages promote a proangiogenic environment in the normal and chronically ischemic mouse kidney.

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.

The Metabolic Syndrome Does Not Affect Development of Collateral Circulation in the Poststenotic Swine Kidney.

BACKGROUND: The collateral circulation is important in maintenance of blood supply to the ischemic kidney distal to renal artery stenosis (RAS). Obesity metabolic syndrome (MetS) preserves renal blood flow (RBF) in the stenotic kidney, but whether this is related to an increase of collateral vessel growth is unknown. We hypothesized that MetS increased collateral circulation around the renal artery. METHODS: Twenty-one domestic pigs were randomly divided into unilateral RAS fed an atherogenic (high-fat/high-fructose, MetS-RAS) or standard diet, or controls (n = 7 each). RBF, glomerular filtration rate (GFR), and the peristenotic collateral circulation were assessed after 10 weeks using multidetector computed tomography (CT) and the intrarenal microcirculation by micro-CT. Vascular endothelial growth factor (VEGF) expression was studied in the renal artery wall, kidney, and perirenal fat. Renal fibrosis and stiffness were examined by trichrome and magnetic resonance elastography. RESULTS: Compared with controls, RBF and GFR were decreased in RAS, but not in MetS-RAS. MetS-RAS formed peristenotic collaterals to the same extent as RAS pigs but induced greater intrarenal microvascular loss, fibrosis, stiffness, and inflammation. MetS-RAS also attenuated VEGF expression in the renal tissue compared with RAS, despite increased expression in the perirenal fat. CONCLUSIONS: MetS does not interfere with collateral vessel formation in the stenotic kidney, possibly because decreased renal arterial VEGF expression offsets its upregulation in perirenal fat, arguing against a major contribution of the collateral circulation to preserve renal function in MetS-RAS. Furthermore, preserved renal function does not protect the poststenotic kidney from parenchymal injury.

Mitoprotection preserves the renal vasculature in porcine metabolic syndrome.

NEW FINDINGS: What is the central question of this study? We hypothesized that chronic mitoprotection would decrease renal vascular remodelling and dysfunction in swine metabolic syndrome. What is the main finding and its importance? This study shows that experimental metabolic syndrome exerts renal microvascular and endothelial cell mitochondrial injury, which were attenuated by mitoprotection, underscoring the contribution of mitochondrial injury to the pathogenesis of metabolic syndrome-induced vascular damage. ABSTRACT: 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-1 ), 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.

Intrarenal fat deposition does not interfere with the measurement of single-kidney perfusion in obese swine using multi-detector computed tomography.

BACKGROUND: Altered vascular structure or function in several diseases may impair renal perfusion. Multi-detector computed tomography (MDCT) is a non-invasive tool to assess single-kidney perfusion and function based on dynamic changes in tissue attenuation during contrast media transit. However, changes in basal tissue attenuation might hamper these assessments, despite background subtraction. Evaluation of iodine concentration using the dual-energy (DECT) MDCT mode allows excluding effects of basal values on dynamic changes in tissue attenuation. We tested whether decreased basal kidney attenuation secondary to intrarenal fat deposition in swine obesity interferes with assessment of renal perfusion using MDCT. METHODS: Domestic pigs were fed a standard (lean) or a high-cholesterol/carbohydrate (obese) diet (n = 5 each) for 16 weeks, and both kidneys were then imaged using MDCT/DECT after iodinated contrast injection. DECT images were post-processed to generate iodine and virtual-non-contrast (VNC) datasets, and the MDCT kidney/aorta CT number (following background subtraction) and DECT iodine ratios calculated during the peak vascular phase as surrogates of renal perfusion. Intrarenal fat was subsequently assessed with Oil-Red-O staining. RESULTS: VNC maps in obese pigs revealed decreased basal cortical attenuation, and histology confirmed increased renal tissue fat deposition. Nevertheless, the kidney/aorta attenuation and iodine ratios remained similar, and unchanged compared to lean pigs. CONCLUSIONS: Despite decreased basal attenuation secondary to renal adiposity, background subtraction allows adequate assessment of kidney perfusion in obese pigs using MDCT. These observations support the feasibility of renal perfusion assessment in obese subjects using MDCT.

Mitoprotection attenuates myocardial vascular impairment in porcine metabolic syndrome.

Metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPCs). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS. Pigs were studied after 16 wk of diet-induced MetS, MetS treated for the last 4 wk with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg/kg sc once daily), and lean controls ( n = 6 each). Cardiac remodeling and function were assessed in vivo by multidetector-computed tomography (CT), and coronary artery and sinus blood samples were collected. EC mitochondrial density, apoptosis, oxidative stress, endothelial nitric oxide synthase immunoreactivity, myocardial microvascular density (three-dimensional microcomputed tomography), and coronary endothelial function (organ bath) were assessed ex vivo. The number and arteriovenous gradient of CD34+/KDR+ EPCs were calculated by FACS (a negative net gradient indicating EPC retention). MetS and MetS + ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS animals compared with lean animals but normalized in MetS + ELAM animals. ELAM also attenuated EC oxidative stress and apoptosis and improved subendocardial microvascular density. ELAM-induced vasculoprotection was reflected by decreased coronary retention of EPCs. ELAM also partly improved endothelial nitric oxide synthase immunoreactivity, coronary endothelial function, and vessel maturity, whereas myocardial perfusion was unaffected. Chronic mitoprotection improved coronary EC mitochondrial density and decreased vascular remodeling and dysfunction. However, additional mitochondria-independent mechanisms likely contribute to MetS-induced cardiac vascular injury. NEW & NOTEWORTHY The present study shows that chronic mitoprotection preserved coronary endothelial cell mitochondria and decreased vascular injury, subendocardial microvascular loss, coronary retention of endothelial progenitor cells, and release of markers of vascular injury. However, myocardial perfusion remained blunted, suggesting that additional mitochondria-independent mechanisms likely contribute to metabolic syndrome-induced cardiac vascular injury.

Peripheral vascular atherosclerosis in a novel PCSK9 gain-of-function mutant Ossabaw miniature pig model.

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.

Low-Energy Shockwave Therapy Improves Ischemic Kidney Microcirculation.

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 ß1-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.

Detection of atherosclerotic plaques in ApoE-deficient mice using (99m)Tc-duramycin.

UNLABELLED: Apoptosis of macrophages and smooth muscle cells is linked to atherosclerotic plaque destabilization. The apoptotic cascade leads to exposure of 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, (99m)Tc-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. (99m)Tc-duramycin images were acquired using a small-animal SPECT imager. Six ApoE(-/-) mice at 20weeks of age (Group I) were imaged and then sacrificed for ex vivo analyses. Six additional ApoE(-/-) mice (Group II) were imaged at 20 and 40weeks of age before sacrifice. Six ApoE wild-type (ApoE(+/+)) mice (Group III) were imaged at 40weeks as controls. Five additional ApoE(-/-) mice (40weeks of age) (Group IV) were imaged with a (99m)Tc-labeled inactive peptide, (99m)Tc-LinDUR, to assess (99m)Tc-duramycin targeting specificity. RESULTS: Focal (99m)Tc-duramycin uptake in the ascending aorta and aortic arch was detected at 20 and 40weeks in the ApoE(-/-) mice but not in ApoE(+/+) mice. (99m)Tc-duramycin uptake in the aortic lesions increased 2.2-fold on quantitative imaging in the ApoE(-/-) mice between 20 and 40weeks. Autoradiographic and histological data indicated significantly increased (99m)Tc-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 or radioactive uptake, was 2.1 times higher at 40weeks than at 20weeks (6.62±0.89 vs. 3.18±0.29, P<0.01). There was barely detectable focal uptake of (99m)Tc-duramycin in the aortic arch of ApoE(+/+) mice. No detectable (99m)Tc-LinDUR uptake was observed in the aortas of ApoE(-/-) mice. CONCLUSIONS: PE-targeting properties of (99m)Tc-duramycin in the atherosclerotic mouse aortas were noninvasively characterized. (99m)Tc-duramycin is promising in localizing advanced atherosclerotic plaques.