Peritubular Capillaries Research Articles

AVR-RD-01 lentiviral gene therapy reduces Gb3 substrate in endothelial cells of renal peritubular capillaries in a previously untreated classic Fabry disease male patient

AVR-RD-01 lentiviral gene therapy reduces Gb3 substrate in endothelial cells of renal peritubular capillaries in a previously untreated classic Fabry disease male patient

Enhanced thrombospondin-1 causes dysfunction of vascular endothelial cells derived from Fabry disease-induced pluripotent stem cells.

BackgroundFabry disease (FD) is a recessive X-linked lysosomal storage disorder caused by α-galactosidase A (GLA) deficiency. Although the mechanism is unclear, GLA deficiency causes an accumulation of globotriaosylceramide (Gb3), leading to vasculopathy.MethodsTo explore the relationship between the accumulation of Gb3 and vasculopathy, induced pluripotent stem cells generated from four Fabry patients (FD-iPSCs) were differentiated into vascular endothelial cells (VECs). Genome editing using CRISPR-Cas9 system was carried out to correct the GLA mutation or to delete Thrombospondin-1 (TSP-1). Global transcriptomes were compared between wild-type (WT)- and FD-VECs by RNA-sequencing analysis.FindingsHere, we report that overexpression of TSP-1 contributes to the dysfunction of VECs in FD. VECs originating from FD-iPSCs (FD-VECs) showed aberrant angiogenic functionality even upon treatment with recombinant α-galactosidase. Intriguingly, FD-VECs produced more p-SMAD2 and TSP-1 than WT-VECs. We also found elevated TSP-1 in the peritubular capillaries of renal tissues biopsied from FD patients. Inhibition of SMAD2 signaling or knock out of TSP-1 (TSP-1−/−) rescues normal vascular functionality in FD-VECs, like in gene-corrected FD-VECs. In addition, the enhanced oxygen consumption rate is reduced in TSP-1−/− FD-VECs.InterpretationThe overexpression of TSP-1 secondary to Gb3 accumulation is primarily responsible for the observed FD-VEC dysfunction. Our findings implicate dysfunctional VEC angiogenesis in the peritubular capillaries in some of the complications of Fabry disease.FundingThis study was supported by grant 2018M3A9H1078330 from the National Research Foundation of the Republic of Korea.

Inhibition of lysyl oxidase ameliorates renal injury by inhibiting CD44-mediated pericyte detachment and loss of peritubular capillaries

Renal fibrosis is a common pathological manifestation of almost all forms of kidney disease irrespective of the etiological cause. Microvascular rarefaction represents itself as an important phenomenon associated with renal fibrosis and shows strong correlation with decline in renal functions. Lysyl oxidase (LOX) catalyzes crosslinking of extracellular matrix (ECM) proteins including collagens, plays an important role in stabilization of degradation resistant matrix. Since, there seems to be a causal link between deposition of excessive ECM and microvascular rarefaction, we investigated the effects of reduction in renal fibrosis on microvascular rarefaction in acute as well as end stage kidney. We used a well-established unilateral ureteral obstruction (UUO)-induced renal fibrosis model to produce renal fibrosis in animals. We treated animals with a LOX inhibitor, β-aminopropionitrile (BAPN, 100 mg/kg, i.p.) and investigated effects on renal fibrosis and microvascular rarefaction. We observed that LOX inhibition was associated with reduction in collagen deposition in UUO-induced renal fibrosis animal model. Further, ECM normalization by LOX inhibition decreased the loss of peritubular capillaries (PTCs) in fibrotic kidney in acute study while the LOX inhibition failed to inhibit PTCs loss in end stage kidney. The results of present study suggested that inhibition of LOX reduces collagen deposition and renal fibrosis. Further, the reduction in fibrosis fails to protect from PTCs loss in chronic study suggesting the absence of strong link between reduction in fibrosis and improvement in PTCs in an end stage kidney.

Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.

Mesenchymal stem cells (MSCs) have emerged as ideal cell-based therapeutic candidates for the structural and functional restoration of the diseased kidney. Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of MSCs on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules via paracrine factors to salvage injured cells, but these factors remain unknown.Methods: GDNF was transfected into human adipose mesenchymal stem cells via a lentiviral transfection system, and exosomes were isolated (GDNF-AMSC-exos). Using the unilateral ureteral obstruction (UUO) mouse model and human umbilical vein endothelial cells (HUVECs) against hypoxia/serum deprivation (H/SD) injury models, we investigated whether GDNF-AMSC-exos ameliorate peritubular capillary (PTC) loss in tubulointerstitial fibrosis and whether this effect is mediated by the Sirtuin 1 (SIRT1) signaling pathway. Additionally, by using SIRT1 activators or siRNAs, the roles of the candidate mRNA and its downstream gene in GDNF-AMSC-exo-induced regulation of endothelial cell function were assessed. PTC characteristics were detected by fluorescent microangiography (FMA) and analyzed by the MATLAB software.Results: The green fluorescent PKH67-labeled exosomes were visualized in the UUO kidneys and colocalized with CD81. GDNF-AMSC-exos significantly decreased PTC rarefaction and renal fibrosis scores in mice with UUO. In vitro studies revealed that GDNF-AMSC-exos exerted cytoprotective effects on HUVECs against H/SD injury by stimulating migration and angiogenesis as well as conferring apoptosis resistance. Mechanistically, GDNF-AMSC-exos enhanced SIRT1 signaling, which was accompanied by increased levels of phosphorylated endothelial nitric oxide synthase (p-eNOS). We also confirmed the SIRT1-eNOS interaction in HUVECs by immunoprecipitation. Furthermore, we observed a correlation of the PTC number with the SIRT1 expression level in the kidney in vivo.Conclusion: Our study unveiled a mechanism by which exosomes ameliorate renal fibrosis: GDNF-AMSC-exos may activate an angiogenesis program in surviving PTCs after injury by activating the SIRT1/eNOS signaling pathway.

Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis.

Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to transdifferentiate toward α-SMA+ myofibroblasts leading to interstitial fibrosis. Even if precise pericytes identification requires transmission electron microscopy and the co-immunostaining of several markers (i.e., Gli, NG2 chondroitin sulphate proteoglycan, CD146, desmin or CD73) and emerging new markers (CD248 or TEM1, endosialin), previous studies suggested that PDGFR-β could be used as marker for renal pericytes characterization. Recently, double immunofluorescence staining of PDGFR-β and α-SMA was performed to identify the damage activated pericytes (PDGFR-β+/α-SMA+ cells) in the early phase of fibrosis development. Our data highlighted the crucial role of renal pericytes in the physiopathology of sepsis and I/R associated AKI. In this protocol, we describe the procedure for double immunofluorescence staining of PDGFR-β and α-SMA in swine Formalin-Fixed Paraffin-Embedded (FFPE) kidney biopsies and the method for image analysis and quantification.

In situ multiplex immunofluorescence analysis of the inflammatory burden in kidney allograft rejection: A new tool to characterize the alloimmune response.

The exact composition of leukocyte infiltration during kidney allograft rejection is difficult to comprehend and visualize on the same biopsy slide. Using an innovative technology of multiplex immunofluorescence (mIF), we were able to detect simultaneously NK cells, macrophages, and T cells and to determine their intra- or extravascular localization using an endothelial marker. Twenty antibody-mediated rejection (ABMR), 20 T cell-mediated rejection (TCMR), and five normal biopsies were labeled, with automatic leukocyte quantification and localization. This method was compared to a classic NKp46 immunohistochemistry (IHC) with manual quantification and to mRNA quantification. mIF automatic quantification was strongly correlated to IHC (r=.91, P<.001) and to mRNA expression levels (r>.46, P<.021). T cells and macrophages were the 2 predominant populations involved in rejection (48.0±4.4% and 49.3±4.4%, respectively, in ABMR; 51.8±6.0% and 45.3±5.8% in TCMR). NK cells constituted a rare population in both ABMR (2.7±0.7%) and TCMR (2.9±0.6%). The intravascular compartment was mainly composed of T cells, including during ABMR, in peritubular and glomerular capillaries. However, NK cell and macrophage densities were significantly higher during ABMR in glomerular and peritubular capillaries. To conclude, this study demonstrates the feasibility and utility of mIF imaging to study and better understand the kidney allograft rejection process.

A Computer Model of Oxygen Dynamics in the Cortex of the Rat Kidney at the Cell-Tissue Level.

The renal cortex drives renal function. Hypoxia/reoxygenation are primary factors in ischemia-reperfusion (IR) injuries, but renal oxygenation per se is complex and awaits full elucidation. Few mathematical models address this issue: none captures cortical tissue heterogeneity. Using agent-based modeling, we develop the first model of cortical oxygenation at the cell-tissue level (RCM), based on first principles and careful bibliographical analysis. Entirely parameterized with Rat data, RCM is a morphometrically equivalent 2D-slice of cortical tissue, featuring peritubular capillaries (PTC), tubules and interstitium. It implements hemoglobin/O2 binding-release, oxygen diffusion, and consumption, as well as capillary and tubular flows. Inputs are renal blood flow RBF and PO2 feeds; output is average tissue PO2 (tPO2). After verification and sensitivity analysis, RCM was validated at steady-state (tPO2 37.7 ± 2.2 vs. 36.9 ± 6 mmHg) and under transients (ischemic oxygen half-time: 4.5 ± 2.5 vs. 2.3 ± 0.5 s in situ). Simulations confirm that PO2 is largely independent of RBF, except at low values. They suggest that, at least in the proximal tubule, the luminal flow dominantly contributes to oxygen delivery, while the contribution of capillaries increases under partial ischemia. Before addressing IR-induced injuries, upcoming developments include ATP production, adaptation to minutes–hours scale, and segmental and regional specification.

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.

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

Exosomes Derived from GDNF-Modified Human Adipose Mesenchymal Stem Cells Ameliorate Peritubular Capillary Loss in Tubulointerstitial Fibrosis by Activating the SIRT1/eNOS Signaling Pathway

Background: Glial-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of mesenchymal stem cells on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules, but these factors remain unknown. Methods: GDNF was transfected into human adipose mesenchymal stem cells via a lentiviral transfection system and exosomes were isolated (GDNF-AMSC-exos). Using unilateral ureteral obstruction (UUO) mouse models and human umbilical vein endothelial cells (HUVECs) against hypoxia/serum deprivation (H/SD) injury models we investigated whether GDNF-AMSC-exos ameliorate peritubular capillary (PTC) loss in tubulointerstitial fibrosis, and whether this effect is meditated by SIRT1 signaling pathway. Additionally, by using SIRT1 activators or siRNA, the roles of the candidate mRNA and its downstream gene in GDNF-AMSC-exo-induced regulation of endothelial cell function were assessed. Findings: GDNF-AMSC-exos resulted in significantly decreased PTC rarefaction and renal fibrosis scores in mice with UUO. In vitro studies revealed that GDNF-AMSC-exos exert cytoprotective effects on HUVECs against H/SD injury by stimulating migration, angiogenesis and conferring apoptosis resistance. Mechanistically, GDNF-AMSC-exos enhanced SIRT1 signaling, which accompanied by increased protein levels of phosphorylated eNOS (p-eNOS). In addition, we have confirmed the SIRT1-eNOS interaction in HUVECs by immunoprecipitation. Interpretation: Our study unveils a mechanism by which exosomes ameliorate renal fibrosis: GDNF-AMSC-exos may activate an angiogenesis program in surviving PTCs after injury by activating the SIRT1/eNOS signaling pathway. This finding suggests that inhibition of chronic kidney disease (CKD) progression requires long-term suppression of fibrosis via renal microvascular repair and angiogenesis and that GDNF-AMSC-exos therapy is a promising strategy for achieving this goal. Funding Statement: This work were supported by projects of the National Natural Science Foundation of China (81270769 and 81803473); a project of the Jiangsu Provincial Natural Science Foundation (BK20161172); a project of the Jiangsu Provincial Commission of Health and Family Planning (H201628); projects of the Jiangsu Provincial Post Graduate Innovation Plan (KYCX18-2178, KYCX17-1708, SJCX17-0560, SJCX18-0715); the Municipal Key Research and Development Project of Xuzhou (KC18212); the “Liu ge yi Gong Cheng” project of Jiangsu High-Level Personnel (LGY2016043); a school class project of Xuzhou Medical University (2017KJ13); and the Xuzhou Science and Technology Bureau Applied Basic Research Program( KC18041 and KC 19069). Declaration of Interests: All the authors declared no competing interests. Ethics Approval Statement: Approval of all research involving human participants was obtained from the Institutional Review Board of the Affiliated Hospital of Xuzhou Medical University (permit number: xyfylw2013032). All experiments involving animals followed the animal use protocol enacted by the Institutional Animal Care and Use Committee of Xuzhou Medical University.

Effect of iodinated contrast media on the oxygen tension in the renal cortico-medullary region of pigs.

Repeated injections of iodinated contrast media (CM) can lead to a deterioration of the renal blood flow, can redistribute blood from the renal cortex to other parts of the kidney and can cause small decreases of the blood flow in cortical capillaries, a significant reduction in blood flow in peritubular capillaries and a significant reduction in blood flow in the vasa recta. Therefore, a study in pigs was designed, to show whether the repeated injection of CM boli, alone, can cause a reduction of oxygenation in the cortico-medullar renal tissue - the region with the highest oxygen demand in the kidney - of pigs.While the mean pO2-value had only decreased by 0.3 mmHg from 29.9±4.3 mmHg to 29.6±4.3 mmHg (p = 0.8799) after the tenth Iodixanol bolus, it decreased by 5.9 mmHg from 34.0±4.3 mmHg to 28.1±4.3 mmHg after the tenth Iopromide bolus (p = 0.044). This revealed a remarkable difference in the influence of these CM on the oxygen partial pressure in the kidney.Repeated applications of CM had a significant influence on the renal oxygen partial pressure. In line with earlier studies showing a redistribution of blood from the cortex to other renal areas, this study revealed that Iodixanol - in contrast to Iopromide - induced no changes in the pO2 in the cortico-medullar region which confirms that Iodixanol did not hinder the flow of blood through the renal micro-vessels. These results are in favor of a hypothesis from Brezis that a microcirculatory disorder might be the basis for the development of CI-AKI.

Significant polar vasculosis in a patient with a 30-year history of type 2 diabetes.

SummaryWe report the renal histology of a 66-year-old man with hypertension, cardiovascular disease, and a 30-year history of type 2 diabetes mellitus with proliferative diabetic retinopathy, diabetic neuropathy, and diabetic foot status post toe amputation. Urinary protein excretion was 1.4 g/gCr, serum creatinine level 0.86 mg/dL, estimated glomerular filtration rate 69 mL/min/1.73 m2, and HbA1c 13–15%, despite using insulin. Light microscopy showed global glomerulosclerosis in 37% of the glomeruli, but the remaining glomeruli were intact. Significant polar vasculosis was present, while arteriolar sclerosis was mild. Electron microscopy revealed a thickened glomerular basement membrane, which is compatible with the early stage of diabetic glomerulopathy. The presented case was unique because glomerular changes seen typically in diabetes were not seen in the patient, despite the long-standing history of diabetes and diabetic comorbidities, while prominent polar vasculosis was found. Polar vascular formation helps preserve the glomeruli by allowing hyperosmotic blood bypass the glomeruli; this decreases intraglomerular pressure and minimizes glomerular endothelial damage.Learning points:A 66-year-old man with a 30-year history of type 2 diabetes mellitus with poor glycemic control underwent renal biopsy, which showed scarce glomerular changes typically seen in diabetic kidney disease and instead revealed significant polar vasculosis.Past studies demonstrated that the increased small vessels around the vascular hilus in diabetic patients originated from the afferent arterioles and drained into the peritubular capillaries.Polar vascular formation may preserve glomerular function by allowing the blood flow to bypass the glomeruli and decreasing the intraglomerular pressure, which minimizes endothelial damage of the glomerular tufts.

Pathogenesis of Fabry nephropathy: The pathways leading to fibrosis

BackgroundKidney is one of the main target organs in Fabry disease, a lysosomal X-linked genetic disorder. Renal involvement is characterized by proteinuria and progressive chronic kidney disease leading to end-stage renal disease. Pathogenic mechanisms in the progression of renal damage in Fabry disease are not thoroughly known yet. The lysosomal Gb3 deposition is the first step of complex pathological pathways resulting in renal sclerosis/fibrosis. Our hypothesis is that Fabry disease associated cellular alterations in tubular cells induce the production of TGF-β1, which mediate transdifferentiation of renal cells into myofibroblasts resulting in fibrosis of renal tissue. ObjectivesThe aim of this work is to study the mechanisms leading to fibrosis in kidney from human Fabry patients. MethodsFifteen renal biopsies from naïve Fabry patients were included. Histological and immunohistochemical analysis was carried out. ResultsPositive staining for TGF-β1 was found in tubular epithelial cells in biopsies from Fabry patients. Apoptosis was determined by active caspase 3 staining in tubular and mesangial glomerular cells. Due to TGF-β1 is the main profibrotic cytokine and induces accumulation of myofibroblasts, we performed a study for its marker α-smooth muscle actin (α-SMA). This study revealed expression of α-SMA on pericytes surrounding peritubular capillaries, smooth muscle cells of blood vessels, mesangial cells and periglomerular zone.TGF-β1 is produced mainly by tubular cells in Fabry kidney biopsies probably inducing cellular trans-differentiation of renal cells into myofibroblasts. A positive staining for a marker of myofibroblasts was present, affirming the presence of those profibrotic cells. ConclusionsThese results show for the first time that TGF-β1 is expressed in human renal tissue from Fabry patients, and that this profibrotic cytokine is mainly produced by proximal tubular cells. In addition both, peritubular interstitium and glomeruli, presented cells positive for myofibroblasts markers.

TAFRO syndrome as a cause of glomerular microangiopathy: a case report and literature review

BackgroundTAFRO syndrome is a systemic inflammatory disorder that manifests as thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Renal dysfunction is frequently complicated with TAFRO syndrome, however, it is challenging to perform kidney biopsy in patients with TAFRO syndrome in the presence of thrombocytopenia. Renal histology in TAFRO syndrome mainly shows membranoproliferative glomerulonephritis (MPGN)-like lesions or thrombotic microangiopathy (TMA)-like glomerulopathy. We review our case and previous reports of TAFRO syndrome with kidney biopsy findings and discuss the renal pathophysiology of TAFRO syndrome.Case presentationWe describe a previously healthy 48- year-old woman with TAFRO syndrome. Kidney biopsy performed before the treatment showed diffuse global endocapillary proliferative changes with endothelial cell swelling, double contours of partial capillary walls, and mesangiolysis, consistent with TMA-like glomerulopathy. Glucocorticoid therapy including steroid pulse was ineffective and she developed anasarca, renal dysfunction and oliguria. Hemodialysis was required. However, the anti-Interleukin (IL)-6 receptor antibody (tocilizumab) therapy was very effective. An increase in urinary volume was achieved about 2 weeks after the tocilizumab therapy and hemodialysis was discontinued. To investigate the renal pathophysiology of TAFRO syndrome, we performed immunohistological staining of vascular endothelial growth factor (VEGF)-A, CD34, and D2–40, in our case and a normal control kidney. Glomerular VEGF-A was especially positive in podocytes both, in the control and in the case, with no significant difference and there was a significant increase of VEGF-A staining area in the cortical peritubular capillaries in the case. Both glomerular and renal cortical CD34 expression were significantly decreased in our case. D2–40 expression in cortex was not significantly different.ConclusionsWe reviewed our case and other 10 previous reports about renal biopsy findings in TAFRO syndrome and found that glomerular microangiopathy was a common finding. IL-6-VEGF-axis-induced glomerular microangiopathy may play a crucial role in developing acute kidney injury in TAFRO syndrome and the anti-IL-6 receptor antibody therapy may be useful for TAFRO syndrome refractory to glucocorticoids. About the pathophysiology of VEGF in TAFRO syndrome, VEGF balance in the glomerulus and perhaps in the peritubular capillary system as well may be critical. Further investigation is needed.

Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Recent data suggested a causative role of uric acid (UA) in the development of renal disease, in which endothelial dysfunction is regarded as the key mechanism. Endothelial-to-mesenchymal transition (EndoMT) and shedding of the glycocalyx are early changes of endothelial dysfunction. We investigated whether UA induced EndoMT in HUVECs and an animal model of hyperuricemia fed with 2% oxonic acid for 4 wk. UA induced EndoMT in HUVECs with a generation of reactive oxygen species via the activation of membranous NADPH oxidase (from 15 min) and mitochondria (from 6 h) along with glycocalyx shedding (from 6 h), which were blocked by probenecid. GM6001, an inhibitor of matrix metalloproteinase, alleviated UA-induced glycocalyx shedding and EndoMT. Antioxidants including N-acetyl cysteine, apocynin, and mitotempo ameliorated EndoMT; however, they did not change glycocalyx shedding in HUVECs. In the kidney of hyperuricemic rats, endothelial staining in peritubular capillaries (PTCs) was substantially decreased with a de novo expression of α-smooth muscle actin in PTCs. Plasma level of syndecan-1 was increased in hyperuricemic rats, which was ameliorated by allopurinol. UA caused a phenotypic transition of endothelial cells via induction of oxidative stress with glycocalyx shedding, which could be one of the mechanisms of UA-induced endothelial dysfunction and kidney disease.-Ko, J., Kang, H.-J., Kim, D.-A., Kim, M.-J., Ryu, E.-S., Lee, S., Ryu, J.-H., Roncal, C., Johnson, R. J., Kang, D.-H. Uric acid induced the phenotype transition of vascular endothelial cells via induction of oxidative stress and glycocalyx shedding.

Haemorrhagic kidney syndrome may not be a variation of infectious salmon anaemia

Haemorrhagic kidney syndrome (HKS) of Atlantic salmon is a condition of unknown aetiology that was responsible in the late 1990's for large scale losses in Eastern Canada in sea-caged fish. Lesions included renal interstitial haemorrhage, plus acute renal tubular necrosis with eosinophilic casts. Affected fish were shown to have erythrocyte inclusion body virus (EIBS) but the significance of this infection was unknown at the time. Following the initial report describing this syndrome, data were published showing that infectious salmon anaemia virus (ISAV) was involved with HKS, that lesions typical of HKS could be reproduced using extracts of kidney from fish with HKS, and that these extracts contained ISAV. Even though no liver lesions were ever seen (the hallmark of ISA) the conclusion was reached that HKS was a pathological variation of ISA.Piscine orthoreovirus (PRV) is an emerging viral pathogen of both Atlantic and Pacific salmon. In the former, it is causally linked to heart and skeletal muscle inflammation (HSMI), while in Pacific salmon, a range of jaundice syndromes and distinctive renal tubular necrosis are reported. The similarity of the renal lesions in Pacific salmon to those seen in HKS prompted a re-evaluation of HKS, using in-situ hybridization to identify and localize both PRV and ISAV, using the archived material from which HKS was originally described. We show here the presence of both ISAV and PRV in affected tissues, concentrated in lesions.These findings show that fish with HKS had a dual viral infection, and that HKS was not, therefore, necessarily due to ISAV alone. Given the similarity between the renal lesions in HKS and those in chinook salmon with PRV, these findings also suggest that PRV was a more plausible aetiological candidate. It is just as likely, however, that both viruses were required, and that they acted in a synergistic fashion. The lesions in renal tubules in HKS, partly driven by haemoglobin and partly by one or both viruses, probably led to release of locally high levels of vasoactive factors that were able to target the peritubular capillaries of the renal portal system, leading to necrosis and interstitial haemorrhage.

Efficacy of the pharmacologic chaperone migalastat in a subset of male patients with the classic phenotype of Fabry disease and migalastat-amenable variants: data from the phase 3 randomized, multicenter, double-blind clinical trial and extension study

PurposeOutcomes in patients with Fabry disease receiving migalastat during the phase 3 FACETS trial (NCT00925301) were evaluated by phenotype. MethodsData were evaluated in two subgroups of patients with migalastat-amenable GLA variants: “classic phenotype” (n=14; males with residual peripheral blood mononuclear cell α-galactosidase A <3% normal and multiorgan system involvement) and “other patients” (n=36; males not meeting classic phenotype criteria and all females). Endpoints included estimated glomerular filtration rate (eGFR), left ventricular mass index (LVMi), Gastrointestinal Symptoms Rating Scale diarrhea subscale (GSRS-D), renal peritubular capillary (PTC) globotriaosylceramide (GL-3) inclusions, and plasma globotriaosylsphingosine (lyso-Gb3). ResultsBaseline measures in the classic phenotype patients suggested a more severe phenotype. At month 24, mean (SD) annualized change in eGFRCKD-EPI with migalastat was −0.3 (3.76) mL/min/1.73m2 in the classic phenotype subgroup; changes in LVMi, GSRS-D, and lyso-Gb3 were −16.7 (18.64) g/m2, −0.9 (1.66), and −36.8 (35.78) nmol/L, respectively. At month 6, mean PTC GL-3 inclusions decreased with migalastat (−0.8) and increased with placebo (0.3); switching from placebo to migalastat, PTC inclusions decreased by −0.7. Numerically smaller changes in these endpoints were observed in the other patients. ConclusionMigalastat provided clinical benefit to patients with Fabry disease and amenable variants, regardless of disease severity.

Combination of Glomerular C4d and Morphologic Glomerular Lesions as a Possible Indicator in the Diagnosis of Acute or Chronic Active Antibody-Mediated Rejection

BackgroundLinear C4d staining in the peritubular capillaries is considered a sensitive and useful marker of active or chronic active antibody-mediated rejection (ABMR) in transplanted kidneys. However, the diagnostic significance of glomerular C4d deposits (gC4d) is still undetermined. The aim of this study is to evaluate the association of gC4d with clinicopathologic features and to assess its diagnostic value. MethodsFrom 2013 to 2018, a total of 158 cases of allograft kidney biopsy specimens were obtained from the Korea University Anam Hospital. The histologic features were evaluated according to the Banff classification. The gC4d were determined through immunohistochemical analyses and classified based on scores of 0 to 3 according to the extent of gC4d. ResultsA total of 73 cases (46.2%) showed gC4d, and 37 cases (23.4%), 23 cases (14.6%), and 13 cases (8.2%) were classified with a score of 1+, 2+, and 3+, respectively. The gC4d showed a significant correlation with antibody-associated histologic lesions, including peritubular capillaritis, glomerulitis, and transplant glomerulopathy (P < .001). However, gC4d showed no significant association with cell-mediated injuries such as tubulitis, interstitial inflammation, acute tubular necrosis, and thrombotic microangiopathy. Although positive gC4d alone was associated with nonspecific findings without ABMR, most cases of gC4d combined with glomerulitis or transplant glomerulopathy showed typical histologic features of ABMR, clinically with higher antibody titers and severe functional deterioration. ConclusionsGlomerular C4d deposits may be an alternate useful marker in the diagnosis of active or chronic active ABMR when combined with histologic features of glomerular lesions.

Epidemiology and Pathophysiology of Glomerular C4d Staining in Native Kidney Biopsies

IntroductionRoutine C4d staining in renal transplantation has stimulated its use in kidney biopsies with glomerulonephritis (GN). Methodical description on staining patterns in the native kidney is not available.MethodsWe retrospectively evaluated C4d staining in formalin-fixed paraffin-embedded sections from 519 native kidney biopsies (bx) with and without glomerular disease.ResultsStrong C4d staining was consistently present in immune-complex GN, including lupus nephritis (LN) (n = 68), membranous GN (n = 24), membranoproliferative glomerulonephritis (MPGN) pattern (n = 22), fibrillary GN (n = 3), and proliferative GN with monoclonal IgG (n = 3). C4d stained all cases of postinfectious GN (n = 7) amyloidosis (n = 20) and C1q GN (n = 3). In contrast, IgA nephropathy (IgAN) (n = 34), was negative in 62% of bx, with the rest staining variably. The E1 Oxford classification score correlated with capillary wall C4d staining (P = 0.05). C4d marked the glomerular and arteriolar lesions in thrombotic microangiopathy (TMA; n = 16), the glomerular sclerotic segments in focal segmental glomerulosclerosis (FSGS; n = 77), and marked areas of necrosis in crescentic GN (n = 21). In diabetic glomerulopathy (n = 70), C4d marked advanced insudative lesions but was negative otherwise. C4d weakly stained the mesangium, or was negative in normal biopsies (n = 13), minimal change disease (MCD; n = 21), thin basement membrane disease (n = 20), Alport (n = 3), IgM nephropathy (n = 2), C3 glomerulopathy (n = 5), acute interstitial nephritis (n = 12), acute tubular necrosis (n = 22), ischemic glomerulopathy/nephrosclerosis (n = 23), and other miscellaneous processes (n = 14). Staining in tubular basement membranes and peritubular capillaries was most common in lupus.ConclusionBased on reliable staining in lupus and membranous GN, C4d staining is potentially useful as a screening and diagnostic tool, if only paraffin-embedded tissue is available. Knowledge of C4d staining patterns in normal and pathological tissues enhances its diagnostic value.

SAT-126 PROGRESSION OF RENAL MICROVASCULAR ABNORMALITIES AND THE EFFECT OF SIROLIMUS ON ANGIOGENESIS IN EXPERIMENTAL POLYCYSTIC KIDNEY DISEASE

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure, and is characterized by the development of hundreds of fluid-filled cysts in the kidney. The enlargement of these cysts leads to interstitial fibrosis, loss of normal renal parenchyma, and eventually end-stage renal disease (ESRD). Progressive cyst growth is also accompanied by neoangiogenesis and the loss of normal renal microvasculature. The aim of this study was to determine the progression of renal microvascular abnormalities in PKD, and whether this is altered by sirolimus, a cyst growth inhibitor with anti-angiogenic properties. Kidney sections from Lewis Polycystic Kidney (LPK) rats and juvenile cystic kidney (jck) mice were examined at the early (week 1 LPK; week 4 jck), established (week 3 to 12 LPK; week 8 jck) and end-stage phases of cystic renal disease (week 24 LPK; week 12 jck). Angiogenesis and peritubular capillary loss were assessed by immunohistology using antibodies against endothelial cells (RECA-1 for LPK rats and CD34 for jck mice). In addition, LPK rats and jck mice were treated with sirolimus or vehicle during the established phase of disease (starting at 9 weeks of age in LPK rats and 4 weeks of age in jck mice), to monitor its effect on kidney weight, cyst area, and renal vasculature. Angiogenesis was evident during the established phase of cystic renal disease in both LPK rats and jck mice, and persisted with progression. In LPK rats, peritubular capillary loss occurred rapidly during the early cystic stage, whereas in jck mice this loss was only evident at the end-stage cystic phase. Treatment with sirolimus demonstrated a slight but significant decrease in percentage two kidney weight to total body weight (2KW/TBW; 10.5%) compared to vehicle in LPK rats, but this was not observed in the jck mice. Sirolimus treatment did not alter angiogenesis or the loss of peritubular capillaries in either model. These data indicate that angiogenesis is an early feature of cystic renal disease, persists over time, and is accompanied by the loss of peritubular capillaries. Sirolimus did not alter angiogenesis in LPK rats or jck mice when administered in the established phase of disease. Future studies could test treatment at earlier time points, or inhibitors of alternate pathways of angiogenesis could also be studied as a strategy to reduce neoangiogenesis and cyst progression in PKD.