Renovascular disease induces mitochondrial damage in swine scattered tubular cells.

Scattered tubular-like cells (STCs) contribute to repair neighboring injured renal tubular cells. Mitochondria mediate STC biology and function but might be injured by the ambient milieu. We hypothesized that the microenviroment induced by the ischemic and metabolic components of renovascular disease impairs STC mitochondrial structure and function in swine, which can be attenuated with mitoprotection. CD24+/CD133+ STCs were quantified in pig kidneys after 16 wk of metabolic syndrome (MetS) or lean diet (Lean) with or without concurrent renal artery stenosis (RAS) (n = 6 each). Pig STCs were isolated and characterized, and mitochondrial structure, membrane potential, and oxidative stress were assessed in cells untreated or incubated with the mitoprotective drug elamipretide (1 nM for 6 h). STC-protective effects were assessed in vitro by their capacity to proliferate and improve viability of injured pig tubular epithelial cells. The percentage of STCs was higher in MetS, Lean + RAS, and MetS + RAS kidneys compared with Lean kidneys. STCs isolated from Lean + RAS and MetS + RAS pigs showed mitochondrial swelling and decreased matrix density, which were both restored by mitoprotection. In addition, mitochondrial membrane potential and ATP production were reduced and production of reactive oxygen species elevated in MetS, Lean + RAS, and MetS + RAS STCs. Importantly, mitoprotection improved mitochondrial structure and function as well as the capacity of MetS + RAS STCs to repair injured tubular cells in vitro. Renovascular disease in swine is associated with a higher prevalence of STCs but induces structural and functional alterations in STC mitochondria, which impair their reparative potency. These observations suggest a key role for mitochondria in the renal reparative capacity of STCs.

Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria.

Metabolic syndrome (MetS) is associated with nutrient surplus and kidney hyperfiltration, accelerating chronic renal failure. The potential involvement of podocyte damage in early MetS remains unclear. Mitochondrial dysfunction is an important determinant of renal damage, but whether it contributes to MetS-related podocyte injury remains unknown. Domestic pigs were studied after 16 wk of diet-induced MetS, MetS treated with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg·kg-1·day-1 sc) for the last month of diet, and lean controls (n = 6 pigs/group). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured using multidetector computed tomography, and podocyte and mitochondrial injury were measured by light and electron microscopy. Urinary levels of podocyte-derived extracellular vesicles (pEVs; nephrin positive/podocalyxin positive) were characterized by flow cytometry. Body weight, blood pressure, RBF, and GFR were elevated in MetS. Glomerular size and glomerular injury score were also elevated in MetS and decreased after ELAM treatment. Evidence of podocyte injury, impaired podocyte mitochondria, and foot process width were all increased in MetS but restored with ELAM. The urinary concentration of pEVs was elevated in MetS pigs and directly correlated with renal dysfunction, glomerular injury, and fibrosis and inversely correlated with glomerular nephrin expression. Additionally, pEV numbers were elevated in the urine of obese compared with lean human patients. Early MetS induces podocyte injury and mitochondrial damage, which can be blunted by mitoprotection. Urinary pEVs reflecting podocyte injury might represent early markers of MetS-related kidney disease and a novel therapeutic target.

Tissue hypoxia, inflammation, and loss of glomerular filtration rate in human atherosclerotic renovascular disease.

The relationships between renal blood flow (RBF), tissue oxygenation, and inflammatory injury in atherosclerotic renovascular disease (ARVD) are poorly understood. We sought to correlate RBF and tissue hypoxia with glomerular filtration rate (GFR) in 48 kidneys from patients with ARVD stratified by single kidney iothalamate GFR (sGFR). Oxygenation was assessed by blood oxygenation level dependent magnetic resonance imaging (BOLD MRI), which provides an index for the levels of deoxyhemoglobin within a defined volume of tissue (R2*). sGFR correlated with RBF and with the severity of vascular stenosis as estimated by duplex velocities. Higher cortical R2* and fractional hypoxia and higher levels of renal vein neutrophil-gelatinase-associated-lipocalin (NGAL) and monocyte-chemoattractant protein-1 (MCP-1) were observed at lower GFR, with an abrupt inflection below 20 ml/min. Renal vein MCP-1 levels correlated with cortical R2* and with fractional hypoxia. Correlations between cortical R2* and RBF in the highest sGFR stratum (mean sGFR 51 ± 12 ml/min; R = -0.8) were degraded in the lowest sGFR stratum (mean sGFR 8 ± 3 ml/min; R = -0.1). Changes in fractional hypoxia after furosemide were also absent in the lowest sGFR stratum. These data demonstrate relative stability of renal oxygenation with moderate reductions in RBF and GFR but identify a transition to overt hypoxia and inflammatory cytokine release with severely reduced GFR. Tissue oxygenation and RBF were less correlated in the setting of reduced sGFR, consistent with variable oxygen consumption or a shift to alternative mechanisms of tissue injury. Identifying transitions in tissue oxygenation may facilitate targeted therapy in ARVD.

Elevated urinary podocyte-derived extracellular microvesicles in renovascular hypertensive patients.

BACKGROUND: An increased number of podocyte-derived extracellular vesicles (pEVs) may reflect podocyte injury in renal disease. Elevated glomerular pressure and other insults may injure podocytes, yet it remains unclear whether the numbers of pEVs are altered in hypertensive patients. We tested the hypothesis that urinary pEV levels would be elevated in patients with renovascular hypertension (RVH) compared with essential hypertension (EH) or healthy volunteers (HVs). METHODS: We prospectively enrolled patients with EH ( n = 30) or RVH ( n = 31) to study renal blood flow (RBF) and cortical perfusion using multidetector computed tomography under controlled condition (regulated sodium intake and renin-angiotensin blockade). After isolation from urine samples, pEVs (nephrin and podocalyxin positive) were characterized by flow cytometry. Fourteen RVH patients were studied again 3 months after stenting or continued medical therapy. HVs ( n = 15) served as controls. RESULTS: The fraction of pEV among urinary EVs was elevated in RVH compared with HVs and EH (11.4 ± 6.4, 6.8 ± 3.4 and 6.3 ± 3.7%, respectively; P < 0.001) and remained unchanged after 3 additional months of therapy and after controlling for clinical parameters. However, eGFR- and age-adjusted pEV levels did not correlate with any clinical or renal parameters. CONCLUSIONS: In hypertensive patients under controlled conditions, urinary pEV levels are elevated in patients with RVH and low eGFR compared with patients with EH and relatively preserved renal function. These pEVs may reflect podocyte injury secondary to kidney damage, and their levels might represent a novel therapeutic target.

Perirenal Fat Promotes Renal Arterial Endothelial Dysfunction in Obese Swine through Tumor Necrosis Factor-α.

PURPOSE: Perirenal fat is associated with poor blood pressure control and chronic kidney disease but the underlying mechanisms remain elusive. We tested the hypothesis that perirenal fat impairs renal arterial endothelial function in pigs with obesity-metabolic derangements. MATERIALS AND METHODS: We studied 14 domestic pigs after 16 weeks of a high fat/high fructose diet (obesity-metabolic derangement group) or standard chow (lean group). Renal blood flow, glomerular filtration rate and visceral fat volumes were studied in vivo by computerized tomography. Renal arterial endothelial function was also studied ex vivo in organ baths. RESULTS: Pigs with obesity-metabolic derangements demonstrated increased body weight, blood pressure, cholesterol and intra-abdominal fat compared to lean pigs and perirenal fat volume was significantly larger. Renal blood flow and glomerular filtration rate were markedly elevated while urinary protein level was preserved. Ex vivo acetylcholine induced, endothelium dependent vasodilation of renal artery rings was substantially impaired in pigs with obesity-metabolic derangements compared to lean pigs. Endothelial function was further blunted in obesity-metabolic derangement and lean arterial rings by incubation with perirenal fat harvested from pigs with obesity-metabolic derangements but not from lean pigs. It was restored by inhibiting tumor necrosis factor-α. Perirenal fat from pigs with obesity-metabolic derangements also showed increased pro-inflammatory macrophage infiltration and tumor necrosis factor-α expression. CONCLUSIONS: In pigs with obesity-metabolic derangements perirenal fat directly causes renal artery endothelial dysfunction, which is partly mediated by tumor necrosis factor-α.

Renal Vein Levels of MicroRNA-26a Are Lower in the Poststenotic Kidney.

MicroRNA-26a (miR-26a) is a post-transcriptional regulator that inhibits cellular differentiation and apoptosis. Renal vascular disease (RVD) induces ischemic injury characterized by tubular cell apoptosis and interstitial fibrosis. We hypothesized that miR-26a levels are reduced in the poststenotic kidney and that kidney repair achieved by adipose tissue-derived mesenchymal stem cells (ad-MSCs) is associated with restored miR-26a levels. Renal function and renal miR-26a levels were assessed in pigs with RVD not treated (n=7) or 4 weeks after intrarenal infusion of ad-MSC (2.5×10(5) cells/kg; n=6), patients with RVD (n=12) or essential hypertension (n=12), and healthy volunteers (n=12). In addition, the direct effect of miR-26a on apoptosis was evaluated in a renal tubular cell culture. Compared with healthy control kidneys, swine and human poststenotic kidneys had 45.5±4.3% and 90.0±3.5% lower levels of miR-26a, respectively, which in pigs, localized to the proximal tubules. In pigs, ad-MSC delivery restored tubular miR-26a expression, attenuated tubular apoptosis and interstitial fibrosis, and improved renal function and tubular oxygen-dependent function. In vitro, miR-26a inhibition induced proximal tubular cell apoptosis and upregulated proapoptotic protein expression, which were both rescued by ad-MSC. In conclusion, decreased tubular miR-26a expression in the poststenotic kidney may be responsible for tubular cell apoptosis and renal dysfunction but can be restored using ad-MSC. Therefore, miR-26a might be a novel therapeutic target in renovascular disease.

Experimental coronary artery stenosis accelerates kidney damage in renovascular hypertensive swine.

The impact of coronary artery stenosis (CAS) on renal injury is unknown. Here we tested whether the existence of CAS, regardless of concurrent atherosclerosis, would induce kidney injury and magnify its susceptibility to damage from coexisting hypertension (HT). Pigs (seven each) were assigned to sham, left-circumflex CAS, renovascular HT, and CAS plus HT groups. Cardiac and nonstenotic kidney functions, circulating and renal inflammatory and oxidative markers, and renal and microvascular remodeling were assessed 10 weeks later. Myocardial perfusion declined distal to CAS. Systemic levels of PGF2-α isoprostane, a marker of oxidative stress, increased in CAS and CAS plus HT, whereas single-kidney blood flow responses to acetylcholine were significantly blunted only in CAS plus HT compared with sham, HT, and CAS, indicating renovascular endothelial dysfunction. Tissue expression of inflammatory and oxidative markers were elevated in the CAS pig kidney, and further magnified in CAS plus HT, whereas angiogenic factor expression was decreased. Bendavia, a mitochondria-targeted peptide, decreased oxidative stress and improved renal function and structure in CAS. Furthermore, CAS and HT synergistically amplified glomerulosclerosis and renal fibrosis. Thus, mild myocardial ischemia, independent of systemic atherosclerosis, induced renal injury, possibly mediated by increased oxidative stress. Superimposed HT aggravates renal inflammation and endothelial dysfunction caused by CAS, and synergistically promotes kidney fibrosis, providing impetus to preserve cardiac integrity in order to protect the kidney.

Obesity-metabolic derangement exacerbates cardiomyocyte loss distal to moderate coronary artery stenosis in pigs without affecting global cardiac function.

Obesity associated with metabolic derangements (ObM) worsens the prognosis of patients with coronary artery stenosis (CAS), but the underlying cardiac pathophysiologic mechanisms remain elusive. We tested the hypothesis that ObM exacerbates cardiomyocyte loss distal to moderate CAS. Obesity-prone pigs were randomized to four groups (n = 6 each): lean-sham, ObM-sham, lean-CAS, and ObM-CAS. Lean and ObM pigs were maintained on a 12-wk standard or atherogenic diet, respectively, and left circumflex CAS was then induced by placing local-irritant coils. Cardiac structure, function, and myocardial oxygenation were assessed 4 wk later by computed-tomography and blood oxygenation level dependent (BOLD) MRI, the microcirculation with micro-computed-tomography, and injury mechanisms by immunoblotting and histology. ObM pigs showed obesity, dyslipidemia, and insulin resistance. The degree of CAS (range, 50-70%) was similar in lean and ObM pigs, and resting myocardial perfusion and global cardiac function remained unchanged. Increased angiogenesis distal to the moderate CAS observed in lean was attenuated in ObM pigs, which also showed microvascular dysfunction and increased inflammation (M1-macrophages, TNF-α expression), oxidative stress (gp91), hypoxia (BOLD-MRI), and fibrosis (Sirius-red and trichrome). Furthermore, lean-CAS showed increased myocardial autophagy, which was blunted in ObM pigs (downregulated expression of unc-51-like kinase-1 and autophagy-related gene-12; P < 0.05 vs. lean CAS) and associated with marked apoptosis. The interaction diet xstenosis synergistically inhibited angiogenic, autophagic, and fibrogenic activities. ObM exacerbates structural and functional myocardial injury distal to moderate CAS with preserved myocardial perfusion, possibly due to impaired cardiomyocyte turnover.

Inflammatory and injury signals released from the post-stenotic human kidney.

AIMS: The mechanisms mediating kidney injury and repair in humans with atherosclerotic renal artery stenosis (ARAS) remain poorly understood. We hypothesized that the stenotic kidney releases inflammatory mediators and recruits progenitor cells to promote regeneration. METHODS AND RESULTS: Essential hypertensive (EH) and ARAS patients (n=24 each) were studied during controlled sodium intake and antihypertensive treatment. Inferior vena cava (IVC) and renal vein (RV) levels of CD34+/KDR+ progenitor cells, cell adhesion molecules, inflammatory biomarkers, progenitor cell homing signals, and pro-angiogenic factors were measured in EH and ARAS, and their gradient and net release compared with systemic levels in matched normotensive controls (n= 24). Blood pressure in ARAS was similar to EH, but the glomerular filtration rate was lower. Renal vein levels of soluble E-Selectin, vascular cell adhesion molecule-1, and several inflammatory markers were higher in the stenotic kidney RV vs. normal and EH RV (P < 0.05), and their net release increased. Similarly, stem-cell homing factor levels increased in the stenotic kidney RV. Systemic CD34+/KDR+ progenitor cell levels were lower in both EH and ARAS and correlated with cytokine levels. Moreover, CD34+/KDR+ progenitor cells developed a negative gradient across the ARAS kidney, suggesting progenitor cell retention. The non-stenotic kidney also showed signs of inflammatory processes, which were more subtle than in the stenotic kidney. CONCLUSION: Renal vein blood from post-stenotic human kidneys has multiple markers reflecting active inflammation that portends kidney injury and reduced function. CD34+/KDR+ progenitor cells sequestered within these kidneys may participate in reparative processes. These inflammation-related pathways and limited circulating progenitor cells may serve as novel therapeutic targets to repair the stenotic kidney.

Obesity-metabolic derangement preserves hemodynamics but promotes intrarenal adiposity and macrophage infiltration in swine renovascular disease.

Obesity-metabolic disorders (ObM) often accompany renal artery stenosis (RAS). We hypothesized that the coexistence of ObM and RAS magnifies inflammation and microvascular remodeling in the stenotic kidney (STK) and aggravates renal scarring. Twenty-eight obesity-prone Ossabaw pigs were studied after 16 wk of a high-fat/high-fructose diet or standard chow including ObM-sham, ObM-RAS, Lean-RAS, or Lean-sham (normal control) groups. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed by multidetector computed tomography (CT), renal oxygenation and tubular transport capability by blood-oxygen-level-dependent MRI, and microcirculation by micro-CT for vessel density, and Western blotting for protein expressions of angiogenic factors (VEGF/FLK-1). Renal vein and inferior vena cava levels of inflammatory cytokines were measured to evaluate systemic and kidney inflammation. Macrophage (MØ) infiltration and subpopulations, fat deposition in the kidney, and inflammation in perirenal and abdominal fat were also examined. GFR and RBF were decreased in Lean-STK but relatively preserved in ObM-STK. However, ObM-STK showed impaired tubular transport function, suppressed microcirculation, and stimulated glomerulosclerosis. ObM diet interacted with RAS to blunt angiogenesis in the STK, facilitated the release of inflammatory cytokines, and led to greater oxidative stress than Lean-STK. The ObM diet also induced fat deposition in the kidney and infiltration of proinflammatory M1-MØ, as also in perirenal and abdominal fat. Coexistence of ObM and RAS amplifies renal inflammation, aggravates microvascular remodeling, and accelerates glomerulosclerosis. Increased adiposity and MØ-accentuated inflammation induced by an ObM diet may contribute to structural injury in the post-STK kidney.

Transition from obesity to metabolic syndrome is associated with altered myocardial autophagy and apoptosis.

OBJECTIVE: Transition from obesity to metabolic-syndrome (MetS) promotes cardiovascular diseases, but the underlying cardiac pathophysiological mechanisms are incompletely understood. We tested the hypothesis that development of insulin resistance and MetS is associated with impaired myocardial cellular turnover. METHODS AND RESULTS: MetS-prone Ossabaw pigs were randomized to 10 weeks of standard chow (lean) or to 10 (obese) or 14 (MetS) weeks of atherogenic diet (n=6 each). Cardiac structure, function, and myocardial oxygenation were assessed by multidetector computed-tomography and Blood Oxygen Level-Dependent-MRI, the microcirculation with microcomputed-tomography, and injury mechanisms by immunoblotting and histology. Both obese and MetS showed obesity and dyslipidemia, whereas only MetS showed insulin resistance. Cardiac output and myocardial perfusion increased only in MetS, yet Blood Oxygen Level-Dependent-MRI showed hypoxia. Inflammation, oxidative stress, mitochondrial dysfunction, and fibrosis also increased in both obese and MetS, but more pronouncedly in MetS. Furthermore, autophagy in MetS was decreased and accompanied by marked apoptosis. CONCLUSIONS: Development of insulin resistance characterizing a transition from obesity to MetS is associated with progressive changes of myocardial autophagy, apoptosis, inflammation, mitochondrial dysfunction, and fibrosis. Restoring myocardial cellular turnover may represent a novel therapeutic target for preserving myocardial structure and function in obesity and MetS.

Chronic renovascular hypertension is associated with elevated levels of neutrophil gelatinase-associated lipocalin.

BACKGROUND: Renovascular hypertension (RVH) is characterized by chronic inflammation of the stenotic kidney and progressive renal dysfunction. Neutrophil gelatinase-associated lipocalin (NGAL), an acute phase protein induced in inflammatory conditions and ischemia, is a novel biomarker for acute kidney injury. We hypothesized that chronic RVH would be associated with increased renal and circulating NGAL levels. METHODS: We prospectively measured renal vein and inferior vena cava (IVC) levels of NGAL and inflammatory cytokines in essential hypertensive (EH) and RVH patients, during constant sodium intake and anti-hypertensive regimens, and compared them with systemic levels in age-matched normotensive subjects (n = 22 each). In addition, we measured urinary NGAL and kidney injury molecule (KIM)-1 in all patients. RESULTS: Blood pressure, serum creatinine, estimated glomerular filtration rate (eGFR), lipid panels and medications were similar in RVH and EH. Systemic, stenotic and contralateral renal vein levels of NGAL were all similarly elevated in RVH versus normal hypertension and EH (P < 0.05), as were renal vein levels of inflammatory markers like tumor necrosis factor-α. Furthermore, renal vein NGAL levels inversely correlated with eGFR, and directly with renal vein (but not systemic) levels of inflammatory markers. Urinary levels of NGAL and KIM-1 were elevated in both EH and RVH, as were systemic levels of C-reactive protein. CONCLUSIONS: Chronic RVH is associated with elevated NGAL levels, likely due to ongoing kidney and systemic inflammation and ischemia. These findings may also imply the occurrence of the inflammation process in chronic RVH, which might contribute to the poorer outcomes of RVH compared with EH patients.

Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.

Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin.

Increased glomerular filtration rate in early metabolic syndrome is associated with renal adiposity and microvascular proliferation.

Metabolic syndrome (MetS) is associated with glomerular hyperfiltration and is a risk factor for chronic kidney disease, but the underlying mechanisms are poorly defined. This study tested the hypothesis that increased glomerular filtration rate (GFR) in early MetS is associated with renal adiposity and microvascular proliferation. Twelve MetS-prone Ossabaw pigs were randomized to 10 wk of a standard (lean, n = 6) or atherogenic (MetS, n = 6) diet. Kidney hemodynamics and function, perirenal fat volume, and tubular dynamics were assessed in vivo by multidetector computed tomography (CT) and blood oxygen level-dependent (BOLD)-MRI. Microvascular architecture was assessed ex vivo with micro-CT. Candidate injury mechanisms were evaluated in kidney tissue by Western blotting and histology. Basal GFR, renal blood flow, and renal cortical perfusion and volume were elevated in the MetS group. Perirenal and kidney tissue fat, proximal-nephron intratubular fluid concentration, and endothelial nitric oxide synthase expression were increased in MetS. GFR levels correlated with tissue triglyceride levels. Elevated spatial density of 20- to 40-µm cortical microvessels was accompanied by mild oxidative stress, inflammation, and with proximal tubular vacuolization. Medullary size and perfusion were relatively preserved, and BOLD-MRI showed intact medullary tubular response to furosemide. Increased GFR in early MetS is associated with renal adiposity and microvascular proliferation, which involve mainly the renal cortex and precede significant activation of oxidative stress and inflammation. Renal adiposity and proliferative microvessels may represent novel therapeutic targets for preserving renal function in early MetS.

Germline PKHD1 mutations are protective against colorectal cancer.

The autosomal recessive polycystic kidney disease (ARPKD) gene, PKHD1, has been implicated in the genesis or growth of colorectal adenocarcinoma, as a high level of somatic mutations was found in colorectal tumor tissue. To determine whether carriers of a single PKHD1 mutation are at increased risk of colorectal carcinoma, we assessed the prevalence of the commonest European mutation, T36M. First, we assayed a European cohort of ARPKD patients and found T36M was responsible for 13.1% of mutations. We then investigated two European cohorts with colorectal adenocarcinoma versus two control cohorts of similar age and gender. Screening for the most common PKHD1 mutation, T36M, we detected 15:3,603 (0.42%) controls versus 1:3,767 (0.027%) colorectal cancer individuals, indicating that heterozygous PKHD1 mutations are not a risk factor and are protective (p=0.0002). We also show that the carriage rate for PKHD1 mutations in the European population is higher than previous accepted at 3.2% (1:31 genomes).

Magnetization Transfer Imaging Predicts Porcine Kidney Recovery After Revascularization of Renal Artery Stenosis.

MATERIALS AND METHODS: Stenotic kidney (STK) and contralateral kidney magnetization transfer ratios (MTRs; Mt/M0) were measured at 3.0-T magnetic resonance imaging, at offset frequencies of 600 and 1000 Hz, before and 1 month post-PTRA in 7 RVD pigs. Stenotic kidney MTR was correlated to renal perfusion, renal blood flow (RBF), and glomerular filtration rate (GFR), determined using multidetector computed tomography and with ex vivo renal fibrosis (trichrome staining). Untreated RVD (n = 6) and normal pigs (n = 7) served as controls. RESULTS: Renovascular disease induced hypertension and renal dysfunction. Blood pressure and renal perfusion were unchanged post-PTRA, but GFR and RBF increased. Baseline cortical STK-MTR predicted post-PTRA renal perfusion and RBF, and MTR changes associated inversely with changes in perfusion and normalized GFR. Stenotic kidney MTR at 600 Hz showed closer association with renal parameters, but both frequencies predicted post-PTRA cortical fibrosis. CONCLUSIONS: Renal STK-MTR, particularly at 600 Hz offset, is sensitive to hemodynamic changes after PTRA in swine RVD and capable of noninvasively predicting post-PTRA kidney perfusion, RBF, and fibrosis. Therefore, STK-MTR may be a valuable tool to predict renal hemodynamic and functional recovery, as well as residual kidney fibrosis after revascularization in RVD.

Characterization of PKD protein-positive exosome-like vesicles.

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Low-Energy Shockwave Treatment Promotes Endothelial Progenitor Cell Homing to the Stenotic Pig Kidney.

Endothelial progenitor cells (EPCs) patrols the circulation and contributes to endothelial cell regeneration. Atherosclerotic renal artery stenosis (ARAS) induces microvascular loss in the stenotic kidney (STK). Low-energy shockwave therapy (SW) can induce angiogenesis and restore the STK microcirculation, but the underlying mechanism remains unclear. We tested the hypothesis that SW increases EPC homing to the swine STK, associated with capillary regeneration. Normal pigs and pigs after 3 wk of renal artery stenosis were treated with six sessions of low-energy SW (biweekly for three consecutive weeks) or left untreated. Four weeks after completion of treatment, we assessed EPC (CD34+/KDR+) numbers and levels of the homing-factor stromal cell-derived factor (SDF)-1 in the inferior vena cava and the STK vein and artery, as well as urinary levels of vascular endothelial growth factor (VEGF) and integrin-1ß. Subsequently, we assessed STK morphology, capillary count, and expression of the proangiogenic growth factors angiopoietin-1, VEGF, and endothelial nitric oxide synthase ex vivo. A 3-wk low-energy SW regimen improved STK structure, capillary count, and function in ARAS+SW, and EPC numbers and gradients across the STK decreased. Plasma SDF-1 and renal expression of angiogenic factors were increased in ARAS+SW, and urinary levels of VEGF and integrin-1ß tended to rise during the SW regimen. In conclusion, SW improves ischemic kidney capillary density, which is associated with, and may be at least in part mediated by, promoting EPCs mobilization and homing to the stenotic kidney.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Regulatory T Cells to Ameliorate Chronic Kidney Injury.

Metabolic syndrome (MetS) profoundly changes the contents of mesenchymal stem cells and mesenchymal stem cells-derived extracellular vesicles (EVs). The anti-inflammatory TGF-ß (transforming growth factor-ß) is selectively enriched in EVs from Lean but not from MetS pigs, but the functional impact of this endowment remains unknown. We hypothesized that Lean-EVs more effectively induce regulatory T cells in injured kidneys. Five groups of pigs (n=7 each) were studied after 16 weeks of diet-induced MetS and unilateral renal artery stenosis (RAS; MetS+RAS). Two groups of MetS+RAS were treated 4 weeks earlier with an intrarenal injection of either Lean-EVs or MetS-EVs. MetS+RAS had lower renal volume, renal blood flow, and glomerular filtration rate than MetS pigs. Compared with Lean-EVs, MetS-EVs were less effective in improving renal function and decreasing tubular injury and fibrosis in MetS+RAS. Lean-EVs upregulated TGF-ß expression in stenotic kidney and increased regulatory T cells numbers more prominently. Furthermore, markedly upregulated anti-inflammatory M2 macrophages reduced proinflammatory M1 macrophages, and CD8+ T cells were detected in stenotic kidneys treated with Lean-EVs compared with MetS-EVs, and renal vein levels of interleukin-1ß were reduced. In vitro, coculture of Lean-EVs with activated T cells led to greater TGF-ß-dependent regulatory T cells induction than did MetS-EVs. Therefore, the beneficial effects of mesenchymal stem cells-derived EVs on injured kidneys might be partly mediated by their content of TGF-ß signaling components, which permitting increased Treg preponderance. Modulating EV cargo and transforming their functionality might be useful for renal repair.