Peritubular Capillary Density Research Articles

MiR-486-5p protects against rat ischemic kidney injury and prevents the transition to chronic kidney disease and vascular dysfunction.

Acute kidney injury (AKI) increases the risk for progressive chronic kidney disease (CKD). MicroRNA (miR)-486-5p protects against kidney ischemia-reperfusion (IR) injury in mice, although its long-term effects on the vasculature and development of CKD are unknown. We studied whether miR-486-5p would prevent the AKI to CKD transition in rat, and affect vascular function. Adult male rats were subjected to bilateral kidney IR followed by i.v. injection of liposomal-packaged miR-486-5p (0.5 mg/kg). Kidney function and histologic injury were assessed after 24 h and 10 weeks. Kidney endothelial protein levels were measured by immunoblot and immunofluorescence, and mesenteric artery reactivity was determined by wire myography. In rats with IR, miR-486-5p blocked kidney endothelial cell increases in intercellular adhesion molecule-1 (ICAM-1), reduced neutrophil infiltration and histologic injury, and normalized plasma creatinine (P<0.001). However, miR-486-5p attenuated IR-induced kidney endothelial nitric oxide synthase (eNOS) expression (P<0.05). At 10 weeks, kidneys from rats with IR alone had decreased peritubular capillary density and increased interstitial collagen deposition (P<0.0001), and mesenteric arteries showed impaired endothelium-dependent vasorelaxation (P<0.001). These changes were inhibited by miR-486-5p. Delayed miR-486-5p administration (96 h, 3 weeks after IR) had no impact on kidney fibrosis, capillary density, or endothelial function. In rats, administration of miR-486-5p early after kidney IR prevents injury, and protects against CKD development and systemic endothelial dysfunction. These protective effects are associated with inhibition of endothelial ICAM-1 and occur despite reduction in eNOS. miR-486-5p holds promise for the prevention of ischemic AKI and its complications.

Endothelial Glycocalyx of Peritubular Capillaries in Experimental Diabetic Nephropathy: A Target of ACE Inhibitor-Induced Kidney Microvascular Protection.

Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss.

The relationship of peritubular capillary density with glomerular volume and kidney function in living kidney donors

BackgroundPeritubular capillary rarefaction plays an important role in the progression of chronic kidney disease. Little is known about the relation between peritubular capillary density, glomerular volume and filtration rate in the healthy kidney.MethodsIn this single-center study, we included 69 living kidney donors who donated between 2005 and 2008 and had representative renal biopsies available. In all donors, glomerular filtration rate was measured using 125I-Iothalamate before donation and at five years after donation. Before donation, the increase in glomerular filtration rate after dopamine stimulation was measured. Glomerular volume and peritubular capillary density were determined in biopsies taken at the time of transplantation. Pearson’s correlation coefficient and linear regression were used to assess relations between parameters.ResultsMean donor age was 52 ± 11 years and mean measured glomerular filtration rate was 119 ± 22 mL/min before donation and 82 ± 15 mL/min at five years after donation. While peritubular capillary density (measured by either number of peritubular capillaries/50,000 μm2 or number of peritubular capillaries/tubule) was not associated with measured glomerular filtration rate before or after donation, number of peritubular capillaries/tubule was associated with the increase in measured glomerular filtration rate after dopamine stimulation (St.β = 0.33, p = 0.004), and correlated positively with glomerular volume (R = 0.24, p = 0.047). Glomerular volume was associated with unstimulated measured glomerular filtration rate before donation (St.β = 0.31, p = 0.01) and at five years (St.β = 0.30, p = 0.01) after donation, independent of age.ConclusionsIn summary, peritubular capillary density was not related to unstimulated kidney function before or after kidney donation, in contrast to glomerular volume. However, number of peritubular capillaries/tubule correlated with the increase in glomerular filtration rate after dopamine stimulation in healthy kidneys, and with glomerular volume. These findings suggest that peritubular capillary density and glomerular volume differentially affect kidney function in healthy living kidney donors.Graphical abstract

Evaluation of renal cold ischemia-reperfusion injury with intravoxel incoherent motion diffusion-weighted imaging and blood oxygenation level-dependent MRI in a rat model.

Purpose: Cold ischemia-reperfusion injury (CIRI) is one of the most serious complications following renal transplantation. The current study investigated the feasibility of Intravoxel Incoherent Motion (IVIM) imaging and blood oxygenation level-dependent (BOLD) in the evaluation of different degrees of renal cold ischemia-reperfusion injury in a rat model. Methods: Seventy five rats were randomly divided into three groups (N = 25 for each group): T0: sham-operated group, T2/T4: CIRI groups with different cold ischemia hours (2, 4h, respectively). The rat model of CIRI group was established by left kidney cold ischemia with right nephrectomy. All the rats received a baseline MRI before the surgery. Five rats in each group were randomly selected to undergo an MRI examination at 1h, day 1, day 2 and day 5 after CIRI. The IVIM and BOLD parameters were studied in the renal cortex (CO), the outer stripe of the outer medulla (OSOM), and the inner stripe of the outer medulla (ISOM) followed by histological analysis to examine Paller scores, peritubular capillary (PTC) density, apoptosis rate and biochemical indicators to obtain the contents of serum creatinine (Scr), blood urea nitrogen (BUN), superoxide dismutase (SOD) and malondialdehyde (MDA). Results: The D, D*, PF and T2* values in the CIRI groups were lower than those in the sham-operated group at all timepoints (all p < 0.05). The prolonged cold ischemia times resulted in gradually lower D, D*, PF and T2* values (all p < 0.05). The D and T2* values of cortex and OSOM in Group T0 and T2 returned to the baseline level (all p > 0.05) except Group T4. The D* and PF values of cortex, OSOM and ISOM in Group T2 and T4 still remained below the normal levels (all p < 0.05) except Group T0. D, D*, PF and T2* values were strongly correlated with histopathological (Paller scores, PTC density and apoptosis rate) and the biochemistry indicators (SOD and MDA) (|r|>0.6, p < 0.001). D*, PF and T2* values were moderately to poorly correlated with some biochemistry indicators (Scr and BUN) (|r|<0.5, p < 0.05). Conclusion: IVIM and BOLD can serve as noninvasive radiologic markers for monitoring different degrees of renal impairment and recovery after renal CIRI.

High-throughput image analysis with deep learning captures heterogeneity and spatial relationships after kidney injury

Recovery from acute kidney injury can vary widely in patients and in animal models. Immunofluorescence staining can provide spatial information about heterogeneous injury responses, but often only a fraction of stained tissue is analyzed. Deep learning can expand analysis to larger areas and sample numbers by substituting for time-intensive manual or semi-automated quantification techniques. Here we report one approach to leverage deep learning tools to quantify heterogenous responses to kidney injury that can be deployed without specialized equipment or programming expertise. We first demonstrated that deep learning models generated from small training sets accurately identified a range of stains and structures with performance similar to that of trained human observers. We then showed this approach accurately tracks the evolution of folic acid induced kidney injury in mice and highlights spatially clustered tubules that fail to repair. We then demonstrated that this approach captures the variation in recovery across a robust sample of kidneys after ischemic injury. Finally, we showed markers of failed repair after ischemic injury were correlated both spatially within and between animals and that failed repair was inversely correlated with peritubular capillary density. Combined, we demonstrate the utility and versatility of our approach to capture spatially heterogenous responses to kidney injury.

Clinical Relevance of Computationally Derived Attributes of Peritubular Capillaries from Kidney Biopsies.

The association between peritubular capillary (PTC) density and disease progression has been studied in a variety of kidney diseases using immunohistochemistry. However, other PTC attributes such as PTC shape have not been explored yet. The recent development of computer vision techniques provides the opportunity for the quantification of PTC attributes using conventional stains and whole slide images. To explore the relationship between PTC characteristics and clinical outcome, n=280 periodic acid Schiff-stained kidney biopsies (88 minimal change disease, 109 focal segmental glomerulosclerosis, 46 membranous nephropathy, and 37 IgA nephropathy) from the NEPTUNE digital pathology repository were computationally analyzed. A previously validated deep learning model was applied to segment cortical PTCs. Average PTC aspect ratio (the PTC major to minor axis ratio), size (PTC pixels per PTC segmentation), and density (PTC pixels per unit cortical area) were computed for each biopsy. Cox proportional hazards models were used to assess associations between these PTC parameters and outcome (40% eGFR decline or kidney failure). Cortical PTC characteristics, and interstitial fractional space PTC density were compared between areas of interstitial fibrosis and tubular atrophy (IFTA) and areas without IFTA. When normalized PTC aspect ratio was below 0.6, a 0.1 increase in normalized PTC aspect ratio was significantly associated with disease progression, with a hazard ratio (95% confidence interval) of 1.28 (1.04-1.59) (p=0.019), while PTC density and size were not significantly associated with outcome. Interstitial fractional space PTC density was lower in areas of IFTA compared to non-IFTA areas. Computational image analysis enables quantification of the status of the kidney microvasculature and the discovery of a previously unrecognized PTC biomarker (aspect ratio) of clinical outcome.

3D autofluorescence imaging of hydronephrosis and renal anatomical structure using cryo-micro-optical sectioning tomography.

Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion. Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries. Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm3 and 13.01 ± 3.74 mm3 in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation. Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Potential Involvement of Complement Activation in Kidney Vascular Lesions of Arterionephrosclerosis.

BackgroundComplement dysregulation has been implicated in the pathogenesis of malignant nephrosclerosis with typical pathological manifestation as thrombotic microangiopathy (TMA) in recent studies. The aim of the present study was to evaluate the potential role of complement activation in arterionephrosclerosis, the major pathological change in benign hypertensive nephrosclerosis.MethodsPatients with biopsy-proven arterionephrosclerosis from 2010 to 2018 in our center were retrospectively enrolled in the present study. The clinical data were retrieved from the medical chart record. The pathological changes of renal biopsy were semiquantitatively evaluated. The ratio of inner-/outer-luminal diameter of the arterioles was calculated to evaluate the degree of arteriosclerosis. Immunohistochemical staining of CD34 and CD68 was adopted to evaluate peritubular capillary (PTC) density and macrophage infiltration, respectively. Complement components, including C3d, C4d, C1q, and C5b-9, were detected by immunohistochemical staining in paraffin-embedded sections. IgM and albumin were detected by immunofluorescence staining in frozen renal tissues.ResultsFifty-two patients were enrolled. The mean age was 45.0 ± 12.7 years, with 39 (75%) males. The median duration of hypertension was 66 months (IQR: 24–138 months). A total of 950 arterioles were evaluated, with a mean ratio of the inner/outer luminal diameter of 0.43 ± 0.05. The ratio of the inner-/outer-luminal diameter correlated with eGFR (r = 0.341, p = 0.013), sclerotic/ischemic glomerular lesions (r = –0.364, p = 0.008) and PTC density (r = 0.426, p = 0.002). Seventy-four percent (703/950) of the evaluated arterioles had C3d deposition with various patterns and intensities. The percentage of C3d-positive arterioles ranged from 63.6 to 100.0% in each specimen. The ratio of the inner/outer luminal diameter of arterioles correlated with the intensity of C3d deposition (r = –0.174, p = 0.001). Infiltration of macrophages was observed around C3d-positive arterioles. The percentage of C3d-positive arterioles was correlated with macrophage infiltration in each specimen (r = 0.330, p = 0.018). Occasional C4d-positive staining on arterioles was observed with no deposition of C1q or C5b-9 in arterionephrosclerosis specimens.ConclusionOur findings provide evidence for potential complement activation in the pathogenesis of vascular lesions in arterionephrosclerosis.

Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Elicit Better Preservation of the Intra-Renal Microvasculature Than Renal Revascularization in Pigs with Renovascular Disease.

Background: Percutaneous transluminal renal angioplasty (PTRA) confers clinical and mortality benefits in select ‘high-risk’ patients with renovascular disease (RVD). Intra-renal-delivered extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) protect the kidney in experimental RVD, but have not been compared side-by-side to clinically applied interventions, such as PTRA. We hypothesized that MSC-derived EVs can comparably protect the post-stenotic kidney via direct tissue effects. Methods: Five groups of pigs (n = 6 each) were studied after 16 weeks of RVD, RVD treated 4 weeks earlier with either PTRA or MSC-derived EVs, and normal controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multi-detector CT, and renal microvascular architecture (3D micro CT) and injury pathways ex vivo. Results: Despite sustained hypertension, EVs conferred greater improvement of intra-renal microvascular and peritubular capillary density compared to PTRA, associated with attenuation of renal inflammation, oxidative stress, and tubulo-interstitial fibrosis. Nevertheless, stenotic kidney RBF and GFR similarly rose in both PTRA- and EV-treated pigs compared RVD + Sham. mRNA sequencing reveled that EVs were enriched with pro-angiogenic, anti-inflammatory, and antioxidants genes. Conclusion: MSC-derived EVs elicit a better preservation of the stenotic kidney microvasculature and greater attenuation of renal injury and fibrosis compared to PTRA, possibly partly attributed to their cargo of vasculo-protective genes. Yet, both strategies similarly improve renal hemodynamics and function. These observations shed light on diverse mechanisms implicated in improvement of post-stenotic kidney function and position EVs as a promising therapeutic intervention in RVD.

Acute Kidney Injury, Microvascular Rarefaction, and Estimated Glomerular Filtration Rate in Kidney Transplant Recipients.

Animal studies suggest that microvascular rarefaction is a key factor in the acute kidney disease to CKD transition. Hence, delayed graft function appears as a unique human model of AKI to further explore the role of microvascular rarefaction in kidney transplant recipients. Here, we assessed whether delayed graft function is associated with peritubular capillary loss and evaluated the association between this loss and long-term kidney graft function. This observational, retrospective cohort study included 61 participants who experienced delayed graft function and 130 who had immediate graft function. We used linear regression models to evaluate associations between delayed graft function and peritubular capillary density expressed as the percentage of efficient cortical area occupied by peritubular capillaries in pre- and post-transplant graft biopsies. eGFRs 1 and 3 years post-transplant were secondary outcomes. Post-transplant biopsies were performed at a median of 113 days (interquartile range, 101-128) after transplantation. Peritubular capillary density went from 15.4% to 11.5% in patients with delayed graft function (median change, -3.7%; interquartile range, -6.6% to -0.8%) and from 19.7% to 15.1% in those with immediate graft function (median change, -4.5%; interquartile range, -8.0% to -0.8%). Although the unadjusted change in peritubular capillary density was similar between patients with and without delayed graft function, delayed graft function was associated with more peritubular capillary loss in the multivariable analysis (adjusted difference in change, -2.9%; 95% confidence interval, -4.0 to -1.8). Pretransplant peritubular capillary density and change in peritubular capillary density were associated with eGFR 1 and 3 years post-transplantation. Perioperative AKI is associated with lower density in peritubular capillaries before transplantation and with loss of peritubular capillaries following transplantation. Lower peritubular capillary density is linked to lower long-term eGFR.

Evaluation of Renal Fibrosis by Mapping Histology and Magnetic Resonance Imaging

Background: Renal fibrosis is a key driver of progression in chronic kidney disease (CKD). Recent advances in diagnostic imaging techniques have shown promising results for the noninvasive assessment of renal fibrosis. However, the specificity and accuracy of these techniques are controversial because they indirectly assess renal fibrosis. This limits fibrosis assessment by imaging in CKD for clinical practice. To validate magnetic resonance imaging (MRI) assessment for fibrosis, we derived representative models by mapping histology-proven renal fibrosis and imaging in CKD. Methods: Ninety-seven adult Chinese CKD participants with histology were studied. The kidney cortex interstitial extracellular matrix volume was calculated by the Aperio ScanScope system using Masson’s trichrome slices. The kidney cortex microcirculation was quantitatively assessed by peritubular capillary density using CD34 staining. The imaging techniques included intravoxel incoherent motion diffusion-weighted imaging and magnetic resonance elastography (MRE) imaging. Relevant analyses were performed to evaluate the correlations between MRI parameters and histology variables. Multiple linear regression models were used to describe the relationships between a response variable and other variables. The best-fit lines, which minimize the sum of squared residuals of the multiple linear regression models, were generated. Results: MRE values were negatively associated with the interstitial extracellular matrix volume (Rho = −0.397, p < 0.001). The best mapping model of extracellular matrix volume with the MRE value and estimated glomerular filtration rate (eGFR) we obtained was as follows: Interstitial extracellular matrix volume = 218.504 – 14.651 × In(MRE) – 18.499 × In(eGFR). DWI-fraction values were positively associated with peritubular capillary density (Rho = 0.472, p < 0.001). The best mapping model of peritubular capillary density with DWI-fraction value and eGFR was as follows: Peritubular capillaries density = 17.914 + 9.403 × (DWI – fraction) + 0.112 × (eGFR). Conclusions: The study provides histological evidence to support that MRI can effectively evaluate fibrosis in the kidney. These findings picture the graphs of the mapping model from imaging and eGFR into fibrosis, which has significant value for clinical implementation.

Percutaneous transluminal renal angioplasty attenuates poststenotic kidney mitochondrial damage in pigs with renal artery stenosis and metabolic syndrome.

Percutaneous transluminal renal angioplasty (PTRA) has been used to treat renovascular disease (RVD), a chronic condition characterized by renal ischemia and metabolic abnormalities. Mitochondrial injury has been implicated as a central pathogenic mechanism in RVD, but whether it can be reversed by PTRA remains uncertain. We hypothesized that PTRA attenuates mitochondrial damage, renal injury, and dysfunction in pigs with coexisting renal artery stenosis (RAS) and metabolic syndrome (MetS). Four groups of pigs (n = 6 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS + RAS), MetS + RAS treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector computed tomography, and renal tubular mitochondrial structure and function and renal injury ex vivo. PTRA successfully restored renal artery patency, but mean arterial pressure remained unchanged. Stenotic kidney RBF and GFR, which fell in MetS + RAS compared to MetS, rose after PTRA. PTRA attenuated MetS + RAS-induced mitochondrial structural abnormalities in tubular cells and peritubular capillary endothelial cells, decreased mitochondrial H2 02 production, and increased renal cytochrome-c oxidase-IV activity and ATP production. PTRA also improved cortical microvascular and peritubular capillary density and ameliorated tubular injury and tubulointerstitial fibrosis in the poststenotic kidney. Importantly, renal mitochondrial damage correlated with poststenotic injury and dysfunction. Renal revascularization attenuated mitochondrial injury and improved renal hemodynamics and function in swine poststenotic kidneys. This study suggests a novel mechanism by which PTRA might be relatively effective in ameliorating mitochondrial damage and improving renal function in coexisting MetS and RAS.

Characterization of cytotoxic effects of aristolochic acids on the vascular endothelium

Aristolochic acid nephropathy (AAN) is characterized by interstitial fibrosis, proximal tubular atrophy, and hypoxia. A correlation between a reduced peritubular capillary density and the severity of fibrosis has been demonstrated. As calcium, redox and energetic homeostasis are crucial in maintaining endothelial cell function and survival, we aimed to investigate AA-induced disturbances involved in endothelial cell injury. Our results showed a cytotoxic effect of AA on EAhy926 endothelial cells. Exposure of aortic rings to AA impaired vascular relaxation to Acetylcholine (ACh). Increased levels of intracellular reactive oxygen species (ROS) were observed in cells exposed to AA. Pre-treatment with antioxidant N-acetyl cysteine inhibited AA-induced cell death. Superoxide dismutase resulted in restoring ACh-induced relaxation. An increase in intracellular calcium level ([Ca2+]i) was observed on endothelial cells. Calcium chelators BAPTA-AM or APB, a specific inhibitor of IP3R, improved cell viability. Moreover, AA exposure led to reduced AMP-activated protein kinase (AMPK) expression. AICAR, an activator of AMPK, improved the viability of AA-intoxicated cells and inhibited the rise of cytosolic [Ca2+]i levels.This study provides evidence that AA exposure increases ROS generation, disrupts calcium homeostasis and decreases AMPK activity. It also suggests that significant damage observed in endothelial cells may enhance microcirculation defects, worsening hypoxia and tubulointerstitial lesions.

MiR-218 Expressed in Endothelial Progenitor Cells Contributes to the Development and Repair of the Kidney Microvasculature

Ischemia due to hypoperfusion is one of the most common forms of acute kidney injury. We hypothesized that kidney hypoxia initiates the up-regulation of miR-218 expression in endothelial progenitor cells (EPCs) to guide endocapillary repair. Murine renal artery-derived EPCs (CD34+/CD105-) showed down-regulation of mmu-Mir218-5p/U6 RNA ratio after ischemic injury, while in human renal arteries, MIR218-5p expression was up-regulated after ischemic injury. MIR218 expression was clarified in cell culture experiments in which increases in both SLIT3 and MIR218-2-5p expressions were observed after 5 minutes of hypoxia. ROBO1 transcript, a downstream target of MIR218-2-5p, showed inverse expression to MIR218-2-5p. EPCs transfected with a MIR218-5p inhibitor in three-dimensional normoxic culture showed premature capillary formation. Organized progenitor cell movement was reconstituted when cells were co-transfected with Dicer siRNA and low-dose Mir218-5p mimic. A Mir218-2 knockout was generated to assess the significance of miR-218-2 in a mammalian model. Mir218-2-5p expression was decreased in Mir218-2-/- embryos at E16.5. Mir218-2-/- decreased CD34+ angioblasts in the ureteric bud at E16.5 and were nonviable. Mir218-2+/- decreased peritubular capillary density at postnatal day 14 and increased serum creatinine after ischemia in adult mice. Systemic injection of miR-218-5p decreased serum creatinine after injury. These experiments demonstrate that miR-218 expression can be triggered by hypoxia and modulates EPC migration in the kidney.

The double edge of anti-CD40 siRNA therapy: It increases renal microcapillar density but favours the generation of an inflammatory milieu in the kidneys of ApoE\u2212/\u2212 mice

BackgroundChronic kidney disease (CKD) is associated with endothelial dysfunctions thus prompting links between microcirculation (MC), inflammation and major cardiovascular risk factors.Purpose of the studyWe have previously reported that siRNA-silencing of CD40 (siCD40) reduced atherosclerosis (ATH) progression. Here, we have deepened on the effects of the siCD40 treatment by evaluating retrospectively, in stored kidneys from the siCD40 treated ApoE−/− mice, the renal microcirculation (measured as the density of peritubular capillaries), macrophage infiltration and NF-κB activation.MethodsKidneys were isolated after 16 weeks of treatment with the anti-CD40 siRNA (siCD40), with a scrambled control siRNA (siSC) or with PBS (Veh. group). Renal endothelium, infiltrating macrophages and activated NF-κB in endothelium were identified by immunohistochemistry, while the density of stained peritubular capillaries was quantified by image analysis.ResultsATH was associated with a reduction in renal MC, an effect reversed by the anti-CD40 siRNA treatment (3.8 ± 2.7% in siCD40; vs. 1.8 ± 0.1% in siSC; or 1.9 ± 1.6% in Veh.; p < 0.0001). Furthermore, siCD40 treatment reduced the number of infiltrating macrophages compared to the SC group (14.1 ± 5.9 cells/field in siCD40; vs. 37.1 ± 17.8 cells/field in siSC; and 1.3 ± 1.7 cells/field in Veh.; p = 0.001). NF-κB activation also peaked in the siSC group, showing lower levels in the siCD40 and Veh. groups (63 ± 60 positive cells/section in siCD40; vs. 152 ± 44 positive cells/section in siSC; or 26 ± 29 positive cells/section in veh.; p = 0.014). Lastly, serum creatinine was also increased in the siCD40 (3.4 ± 3.3 mg/dL) and siSC (4.6 ± 3.0 mg/dL) groups when compared with Veh. (1.1 ± 0.9 mg/dL, p = 0.1).ConclusionsAnti-CD40 siRNA therapy significantly increased the density of peritubular capillaries and decreased renal inflammation in the ATH model. These data provide a physiological basis for the development of renal diseases in patients with ATH. Furthermore, our results also highligth renal off-target effects of the siRNA treatment which are discussed.Graphical abstract

Losartan protects against intermittent hypoxia-induced peritubular capillary loss by modulating the renal renin-angiotensin system and angiogenesis factors.

Obstructive sleep apnea is characterized by chronic intermittent hypoxia (CIH), which is a risk factor for renal peritubular capillary (PTC) loss, and angiotensin II receptor blockers can alleviate PTC loss. However, the mechanism by which losartan (an angiotensin II receptor blocker) reduces CIH-induced PTC loss and attenuates kidney damage is still unknown. Thus, in this study, we examined the protective effects of losartan against CIH-induced PTC loss and explored the underlying mechanisms in rat CIH model. The immunohistochemical staining of CD34 and morphological examination showed that CIH reduced PTC density and damaged tubular epithelial cells. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR, and western blot analysis results revealed that CIH increased the expression of hypoxia inducible factor-1α (HIF-1α), angiotensin II (Ang II), angiotensin II type 1 receptor (AT1R), pro-angiogenesis factor vascular endothelial growth factor (VEGF), and anti-angiogenesis factor thrombospondin-1 (TSP-1) in the renal cortex of rats. CIH may up-regulate VEGF expression and simultaneously increase TSP-1 production. By histopathological, immunohistochemistry, ELISA, RT-qPCR, and western blot analysis, we found that the expressions of renal renin-angiotensin system (RAS), HIF-1α, VEGF, and TSP-1 were decreased, and PTC loss and tubular epithelial cell injury were attenuated with losartan treatment. Losartan ameliorated CIH-induced PTC loss by modulating renal RAS to improve the crosstalk between endothelial cells and tubular epithelial cells and subsequently regulate the balance of angiogenesis factors. Our study provided novel insights into the mechanisms of CIH-induced kidney damage and indicated that losartan could be a potential therapeutic agent for renal protection by alleviating CIH-induced PTC loss.

Capillary rarefaction is more closely associated with CKD progression after cisplatin, rhabdomyolysis, and ischemia-reperfusion-induced AKI than renal fibrosis.

Acute kidney injury (AKI) is a strong independent predictor of mortality and often results in incomplete recovery of renal function, leading to progressive chronic kidney disease (CKD). Many clinical trials have been conducted on the basis of promising preclinical data, but no therapeutic interventions have been shown to improve long-term outcomes after AKI. This is partly due to the failure of preclinical studies to accurately model clinically relevant injury and long-term outcomes on CKD progression. Here, we evaluated the long-term effects of AKI on CKD progression in three animal models reflecting diverse etiologies of AKI: repeat-dose cisplatin, rhabdomyolysis, and ischemia-reperfusion injury. Using transdermal measurement of glomerular filtration rate as a clinically relevant measure of kidney function and quantification of peritubular capillary density to measure capillary rarefaction, we showed that repeat-dose cisplatin caused capillary rarefaction and decreased renal function in mice without a significant increase in interstitial fibrosis, whereas rhabdomyolysis-induced AKI led to severe interstitial fibrosis, but renal function and peritubular capillary density were preserved. Furthermore, long-term experiments in mice with unilateral ischemia-reperfusion injury showed that restoration of renal function 12 wk after a contralateral nephrectomy was associated with increasing fibrosis, but a reversal of capillary rarefaction was seen at 4 wk. These data demonstrate that clear dissociation between kidney function and fibrosis in these models of AKI to CKD progression and suggest that peritubular capillary rarefaction is more strongly associated with CKD progression than renal fibrosis.

Chronic exposure to hypoxia attenuates renal injury and innate immunity activation in the remnant kidney model.

Hypoxia is thought to influence the pathogenesis of chronic kidney disease, but direct evidence that prolonged exposure to tissue hypoxia initiates or aggravates chronic kidney disease is lacking. We tested this hypothesis by chronically exposing normal rats and rats with 5/6 nephrectomy (Nx) to hypoxia. In addition, we investigated whether such effect of hypoxia would involve activation of innate immunity. Adult male Munich-Wistar rats underwent Nx (n = 54) or sham surgery (sham; n = 52). Twenty-six sham rats and 26 Nx rats remained in normoxia, whereas 26 sham rats and 28 Nx rats were kept in a normobaric hypoxia chamber (12% O2) for 8 wk. Hypoxia was confirmed by immunohistochemistry for pimonidazole. Hypoxia was confined to the medullary area in sham + normoxia rats and spread to the cortical area in sham + hypoxia rats, without changing the peritubular capillary density. Exposure to hypoxia promoted no renal injury or elevation of the content of IL-1β or Toll-like receptor 4 in sham rats. In Nx, hypoxia also extended to the cortical area without ameliorating the peritubular capillary rarefaction but, unexpectedly, attenuated hypertension, inflammation, innate immunity activation, renal injury, and oxidative stress. The present study, in disagreement with current concepts, shows evidence that hypoxia exerts a renoprotective effect in the Nx model instead of acting as a factor of renal injury. The mechanisms for this unexpected beneficial effect are unclear and may involve NF-κB inhibition, amelioration of oxidative stress, and limitation of angiotensin II production by the renal tissue.

Combination of Functional Magnetic Resonance Imaging and Histopathologic Analysis to Evaluate Interstitial Fibrosis in Kidney Allografts.

Recent developments indicated that functional magnetic resonance imaging (MRI) could potentially provide noninvasive assessment of kidney interstitial fibrosis in patients with kidney diseases, but direct evidence from histopathology is scarce. We aimed to explore the diagnostic utilities of functional MRI for the evaluation of kidney allograft interstitial fibrosis. We prospectively examined 103 kidney transplant recipients who underwent for-cause biopsies and 20 biopsy-proven normal subjects with functional MRI. Histomorphometric analyses of interstitial fibrosis and peritubular capillary densities were performed on digitally scanned Masson's trichrome- and CD34-stained slides, respectively. The performances of functional MRI to discriminate interstitial fibrosis were assessed by calculating the area under the curve using receiver-operating characteristic curve. Main pathologic findings in this single-center cohort were representative of common diagnostic entities in the kidney allografts, with rejection (32%) and glomerulonephritides (31%) accounting for the majority of diagnoses. Apparent diffusion coefficient from diffusion-weighted imaging correlated with interstitial fibrosis (ρ=-0.77; P<0.001). Additionally, decreased arterial spin labelings were accompanied by peritubular capillary density reductions (r=0.77; P<0.001). Blood oxygen level-dependent (BOLD) imaging demonstrated cortical hypoxia with increasing interstitial fibrosis (ρ=0.61; P<0.001). The area under the curve for the discrimination of ≤25% versus >25% interstitial fibrosis and ≤50% versus >50% interstitial fibrosis were 0.87 (95% confidence interval [95% CI], 0.79 to 0.93) and 0.88 (95% CI, 0.80 to 0.93) by apparent diffusion coefficient, 0.92 (95% CI, 0.85 to 0.97) and 0.94 (95% CI, 0.87 to 0.98) by arterial spin labeling, 0.81 (95% CI, 0.72 to 0.88) and 0.86 (95% CI, 0.78 to 0.92) by perfusion fraction, 0.79 (95% CI, 0.69 to 0.87) and 0.85 (95% CI, 0.76 to 0.92) by BOLD imaging, respectively. Functional MRI measurements were strongly correlated with kidney allograft interstitial fibrosis. The performances of functional MRI for discriminating ≤50% versus >50% interstitial fibrosis were good to excellent.