Peritubular Capillaries Research Articles

Risk factors, histopathological features, and graft outcome of transplant glomerulopathy in the absence of donor-specific HLA antibodies

Transplant glomerulopathy is established as a hallmark of chronic antibody-mediated rejection in kidney transplant patients with donor-specific HLA antibodies (HLA-DSA). The clinical importance of transplant glomerulopathy in the absence of HLA-DSA is not well established. To help define this, 954 patients (encompassing 3744 biopsies) who underwent kidney transplantation 2004-2013 were studied with retrospective high-resolution HLA genotyping of both donors and recipients. The risk factors, histopathological appearance and prognosis of cases with transplant glomerulopathy in the absence of HLA-DSA were compared to those cases with HLA-DSA, and the impact of the PIRCHE-II score and eplet mismatches on development of transplant glomerulopathy evaluated. In this cohort, 10.3% developed transplant glomerulopathy, on average 3.2 years post-transplant. At the time of glomerulopathy, 23.5% had persistent pre-transplant or de novo HLA-DSA, while 76.5% were HLA-DSA negative. Only HLA-DSA was identified as a risk factor for glomerulopathy development as eplet mismatches and the PIRCHE-II score did not associate. HLA-DSA negative biopsies with glomerulopathy had less interstitial inflammation, less glomerulitis, and less C4d deposition in the peritubular capillaries compared to the HLA-DSA positive biopsies with glomerulopathy. While graft function was comparable between the two groups, HLA-DSA positive glomerulopathy was associated with a significantly higher risk of graft failure compared to HLA-DSA negative glomerulopathy (Hazard Ratio 3.84; 95% confidence interval 1.94-7.59). Landmark analysis three-years post-transplant showed that HLA-DSA negative patients with glomerulopathy still had a significant increased risk of graft failure compared to patients negative for glomerulopathy (2.62; 1.46-4.72). Thus, transplant glomerulopathy often occurs in the absence of HLA-DSA, independent of HLA molecular mismatches, and represents a different phenotype with less concomitant inflammation and better graft survival compared to that developed in the presence of HLA-DSA.

Role of C5aR1 and C5L2 Receptors in Ischemia-Reperfusion Injury.

The role of C5a receptors (C5aR1 and C5L2) in renal ischemia-reperfusion injury (IRI) is uncertain. We generated an in vitro model of hypoxia/reoxygenation with human proximal tubule epithelial cells to mimic some IRI events. C5aR1, membrane attack complex (MAC) and factor H (FH) deposits were evaluated with immunofluorescence. Quantitative polymerase chain reaction evaluated the expression of C5aR1, C5L2 genes as well as genes related to tubular injury, inflammation, and profibrotic pathways. Additionally, C5aR1 and C5L2 deposits were evaluated in kidney graft biopsies (KB) from transplant patients with delayed graft function (DGF, n = 12) and compared with a control group (n = 8). We observed higher immunofluorescence expression of C5aR1, MAC and FH as higher expression of genes related to tubular injury, inflammatory and profibrotic pathways and of C5aR1 in the hypoxic cells; whereas, C5L2 gene expression was unaffected by the hypoxic stimulus. Regarding KB, C5aR1 was detected in the apical and basal membrane of tubular epithelial cells, whereas C5L2 deposits were observed in endothelial cells of peritubular capillaries (PTC). DGF-KB showed more frequently diffuse C5aR1 staining and C5L2 compared to controls. In conclusion, C5aR1 expression is increased by hypoxia and IRI, both in vitro and in human biopsies with an acute injury. C5L2 expression in PTC could be related to endothelial cell damage during IRI.

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.

Complement Activation in Kidneys of Patients With COVID-19.

Most patients who became critically ill following infection with COVID-19 develop severe acute respiratory syndrome (SARS) attributed to a maladaptive or inadequate immune response. The complement system is an important component of the innate immune system that is involved in the opsonization of viruses but also in triggering further immune cell responses. Complement activation was seen in plasma adsorber material that clogged during the treatment of critically ill patients with COVID-19. Apart from the lung, the kidney is the second most common organ affected by COVID-19. Using immunohistochemistry for complement factors C1q, MASP-2, C3c, C3d, C4d, and C5b-9 we investigated the involvement of the complement system in six kidney biopsies with acute kidney failure in different clinical settings and three kidneys from autopsy material of patients with COVID-19. Renal tissue was analyzed for signs of renal injury by detection of thrombus formation using CD61, endothelial cell rarefaction using the marker E-26 transformation specific-related gene (ERG-) and proliferation using proliferating cell nuclear antigen (PCNA)-staining. SARS-CoV-2 was detected by in situ hybridization and immunohistochemistry. Biopsies from patients with hemolytic uremic syndrome (HUS, n = 5), severe acute tubular injury (ATI, n = 7), zero biopsies with disseminated intravascular coagulation (DIC, n = 7) and 1 year protocol biopsies from renal transplants (Ctrl, n = 7) served as controls. In the material clogging plasma adsorbers used for extracorporeal therapy of patients with COVID-19 C3 was the dominant protein but collectin 11 and MASP-2 were also identified. SARS-CoV-2 was sporadically present in varying numbers in some biopsies from patients with COVID-19. The highest frequency of CD61-positive platelets was found in peritubular capillaries and arteries of COVID-19 infected renal specimens as compared to all controls. Apart from COVID-19 specimens, MASP-2 was detected in glomeruli with DIC and ATI. In contrast, the classical pathway (i.e. C1q) was hardly seen in COVID-19 biopsies. Both C3 cleavage products C3c and C3d were strongly detected in renal arteries but also occurs in glomerular capillaries of COVID-19 biopsies, while tubular C3d was stronger than C3c in biopsies from COVID-19 patients. The membrane attack complex C5b-9, demonstrating terminal pathway activation, was predominantly deposited in COVID-19 biopsies in peritubular capillaries, renal arterioles, and tubular basement membrane with similar or even higher frequency compared to controls. In conclusion, various complement pathways were activated in COVID-19 kidneys, the lectin pathway mainly in peritubular capillaries and in part the classical pathway in renal arteries whereas the alternative pathway seem to be crucial for tubular complement activation. Therefore, activation of the complement system might be involved in the worsening of renal injury. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury.

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.

A distinctive distribution of hypoxia-inducible factor-1α in cultured renal tubular cells with hypoperfusion simulated by coverslip placement.

Chronic hypoxia in the renal tubulointerstitium plays a key role in the progression of chronic kidney disease (CKD). It is therefore important to investigate tubular hypoxia and the activity of hypoxia‐inducible factor (HIF)‐1α in response to hypoxia. Rarefaction of the peritubular capillary causes hypoperfusion in CKD; however, the effect of hypoperfusion on HIFs has rarely been investigated. We induced hypoperfusion caused by coverslip placement in human kidney‐2 cells, and observed an oxygen gradient under the coverslip. Immunocytochemistry of HIF‐1α showed a doughnut‐shaped formation on the edge of a pimonidazole‐positive area, which we named the “HIF‐ring”. The oxygen tension of the HIF‐ring was estimated to be between approximately 4 mmHg and 20 mmHg. This result was not compatible with those of past research showing HIF‐1α accumulation in the anoxic range with homogeneous oxygen tension. We further observed the presence of a pH gradient under a coverslip, as well as a shift of the HIF ring due to changes in the pH of the culture medium, suggesting that the HIF ring was formed by suppression of HIF‐1α related to low pH. This research demonstrated that HIF‐1α activation mimics the physiological state in cultured cells with hypoperfusion.

The immunohistochemical expression of von Willebrand factor, T-cadherin, and Caveolin-1 is increased in kidney allograft biopsies with antibody-mediated injury.

There are only a few reports evaluating the applicability of endothelial-damage markers analysis by immunohistochemistry (IHC) in kidney allograft samples. This study analyzed the expression of Caveolin-1 (Cav), von Willebrand factor (Vwf), and T-cadherin (Cad) in kidney biopsies and their association with antibody-mediated injury. In this retrospective study, 114 cases with antibody-mediated changes (Banff, 2020) and 72 with interstitial fibrosis/tubular atrophy were selected. IHC for Cav, Vwf and Cad was performed and evaluated according to their qualitative expression in peritubular capillaries. The cases were grouped according to the presence of microvascular inflammation (MVI), donor-specific antibodies (DSA), C4d positivity and antibody-mediated rejection (AMR). A level of significance < 0.05 was adopted. Vwf expression was associated with MVI (p < 0.001), DSA (p = 0.016), C4d (p < 0.001) and AMR (p < 0.001), and was higher in DSA+/C4d+ cases despite MVI (p < 0.001). The expression of Cad correlated with MVI (p = 0.015), C4d (p = 0.005) and AMR (p = < 0.001). Cad was more expressed in chronic AMR compared with acute/active cases (p = 0.001). Cav expression was associated with MVI (p = 0.029) and AMR (p = 0.016) and was also higher in chronic AMR (p = 0.049). A combined score of Vwf and Cad was higher in AMR when compared with C4d without rejection and IF/TA cases (p < 0.001). Vwf, Cad and Cav expression shows association with antibody-mediated injury and may be helpful to support AMR diagnosis.

Preceding T-Cell-Mediated Rejection Is Associated with the Development of Chronic Active Antibody-Mediated Rejection by de Novo Donor-Specific Antibody

Aim: Chronic active antibody-mediated rejection (CAABMR) is an important cause of late-stage renal allograft loss. Early inflammatory events such as acute rejection and infection after transplantation are considered to be the risk factors of de novo donor-specific antibody (dnDSA) production. In this study, we investigated the relationship between pre­disposing T-cell-mediated rejection and dnDSA-positive CAABMR. Methods: We recruited 365 patients who underwent ABO-compatible renal transplantation at our hospital. Among them, 16 patients diagnosed as having dnDSA-positive CAABMR were designated as a CAABMR group, and 38 randomly selected patients were designated as a control group. All biopsies from 1 month after transplantation were included in the study. The presence or absence of borderline changes (BLCs), acute T-cell-mediated rejection (ATMR), microvascular inflammation (MVI), and C4d positive on peritubular capillaries (C4d-P) was examined. Results: In the CAABMR group, BLC/ATMR was found in 12 cases (75%), and the mean duration until appearance of BLC/ATMR was 282.7 ± 328.7 days. C4d-P was found in 11 cases (68.8%), and the mean duration until its appearance was 1,432 ± 1,307 days. MVI was found in all cases, and the mean duration until its appearance was 1,333 ± 1,126 days. The mean duration until diagnosis of CAABMR was 2,268 ± 1,191 days. In the control group, BLC/ATMR was found in 13 cases (34.2%), and the mean duration until the appearance of BLC/ATMR was 173.1 ± 170.4 days. C4d-P was found in 2 cases (5.3%), and the durations until its appearance were 748 and 1,881 days. No cases of MVI were found in the control group. The frequency of BLC/ATMR was significantly higher in the CAABMR group (p < 0.01). Conclusion: Preceding BLC/ATMR is associated with the development of CAABMR with dnDSA.

Constitutive Atg5 overexpression in mouse bone marrow endothelial progenitor cells improves experimental acute kidney injury

BackgroundEndothelial Progenitor Cells have been shown as effective tool in experimental AKI. Several pharmacological strategies for improving EPC-mediated AKI protection were identified in recent years. Aim of the current study was to analyze consequences of constitutive Atg5 activation in murine EPCs, utilized for AKI therapy.MethodsIschemic AKI was induced in male C57/Bl6N mice. Cultured murine EPCs were systemically injected post-ischemia, either natively or after Atg5 transfection (Adenovirus-based approach). Mice were analyzed 48 h and 6 weeks later.ResultsBoth, native and transfected EPCs (EPCsAtg5) improved persisting kidney dysfunction at week 6, such effects were more pronounced after injecting EPCsAtg5. While matrix deposition and mesenchymal transdifferentiation of endothelial cells remained unaffected by cell therapy, EPCs, particularly EPCsAtg5 completely prevented the post-ischemic loss of peritubular capillaries. The cells finally augmented the augophagocytic flux in endothelial cells.ConclusionsConstitutive Atg5 activation augments AKI-protective effects of murine EPCs. The exact clinical consequences need to be determined.

Clinical and Pathological Analyses of Borderline Changes Cases after Kidney Transplantation

Aim: We aimed to perform a clinicopathological analysis of cases presenting with borderline changes (BC) after renal transplantation and discuss whether BC might be clinically or pathologically important. Materials and Methods: BC was diagnosed in 22 renal allograft biopsy specimens obtained from 20 renal transplant recipients between April 2010 and March 2019 after follow-up at the Department of Transplant Surgery, Kidney Center, Toda Chuo General Hospital. Results: BC was diagnosed at a median of 500 days following transplantation. Among the 22 renal allograft biopsy specimens showing evidence of BC, tubulitis was observed in all specimens. Interstitial inflammation was present in 18 specimens (82%), peritubular capillaritis in 14 (64%), interstitial fibrosis (ci) and tubular atrophy (ct) in 4 (18%), and C4d deposition in the peritubular capillary was present in 6 specimens (27%). Glomerulitis and intimal arteritis were not observed. There was no renal graft loss during the observation period, but deterioration of renal allograft function after biopsy occurred in 9 patients (45%). Conclusions: In BC, tubulitis and interstitial inflammation were the main constituents. Because glomerulitis was not observed in our study, we suspect that BC contributes to acute T-cell-mediated rejection. Although BC did not lead to renal graft loss, renal graft function deterioration was seen in nearly half of the patients after the renal graft biopsy. We conclude that BC is important clinically and pathologically and needs to be monitored and treated appropriately.

Alternative complement pathway activation in thrombotic microangiopathy associated with lupus nephritis.

Thrombotic microangiopathy (TMA) in systemic lupus erythematosus is a rare manifestation associated with activation of the complement system. This study aimed to compare plasma and urine complement activation products between patients with active lupus nephritis (aLN) and those with acute TMA plus concomitant active LN (aTMA+aLN). Plasma and urine samples were obtained from 20 patients with aTMA+aLN, 20 patients with aLN matched by the histological activity index, 5 patients with chronic TMA, 20 patients with inactive LN, and 10 kidney donors. Complement fragments C3a, C4a, C4d, Ba, C5a, C5bC9, and factor H were determined by ELISA; and kidney C4d deposition was detected by immunohistochemistry. Patients were followed for > 12 months and complement activation products re-measured after treatment in 10 aTMA+aLN patients. Both aTMA+aLN and aLN groups had increased circulating C3a, Ba, and C5bC9; and decreased circulating C3, C4, C4a, C4d, and factor H. Urinary C3a, C5a, Ba, and C5bC9 were higher in patients with aTMA+aLN than in aLN. After treatment, levels of circulating C3, C4, and factor H increased; while levels of urinary C3a, C5a, Ba, and C5bC9 decreased in patients with aTMA+aLN. These changes were observed at each aTMA episode in two patients studied during repeated TMA episodes. There was no difference in C4d deposition in glomerular capillaries, tubular basement membrane, peritubular capillaries, and arterioles between patients with aLN and those aTMA+aLN. Circulating and urine complement activation products suggest that thrombotic microangiopathy associated with LN is mediated through activation of the alternative complement pathway. Key Points • Immune-complex kidney disease in systemic lupus erythematosus (SLE) is associated with activation of the classical, lectin, and alternative complement pathways • Indirect evidence from measurement of circulating and urinary complement pathway activation products suggests that renal acute thrombotic microangiopathy in SLE is mediated by activation of the alternative complement pathway • C4d kidney immunohistochemistry may be positive in both immune complex nephritis and thrombotic microangiopathy. Therefore, it is not a specific marker of renal thrombotic microangiopathy in SLE.

Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

Peritubular Capillary Rarefaction: An Underappreciated Regulator of CKD Progression.

Peritubular capillary (PTC) rarefaction is commonly detected in chronic kidney disease (CKD) such as hypertensive nephrosclerosis and diabetic nephropathy. Moreover, PTC rarefaction prominently correlates with impaired kidney function and predicts the future development of end-stage renal disease in patients with CKD. However, it is still underappreciated that PTC rarefaction is a pivotal regulator of CKD progression, primarily because the molecular mechanisms of PTC rarefaction have not been well-elucidated. In addition to the established mechanisms (reduced proangiogenic factors and increased anti-angiogenic factors), recent studies discovered significant contribution of the following elements to PTC loss: (1) prompt susceptibility of PTC to injury, (2) impaired proliferation of PTC, (3) apoptosis/senescence of PTC, and (4) pericyte detachment from PTC. Mainly based on the recent and novel findings in basic research and clinical study, this review describes the roles of the above-mentioned elements in PTC loss and focuses on the major factors regulating PTC angiogenesis, the assessment of PTC rarefaction and its surrogate markers, and an overview of the possible therapeutic agents to mitigate PTC rarefaction during CKD progression. PTC rarefaction is not only a prominent histological characteristic of CKD but also a central driving force of CKD progression.

Calcium dobesilate mediates renal interstitial fibrosis and delay renal peritubular capillary loss through Sirt1/p53 signaling pathway

Calcium dobesilate (Cad), a protective agent, protects against microvascular damage, and diseases such as diabetic retinopathy and diabetic nephropathy. However, these vascular protective effects have not been demonstrated in chronic kidney disease (CKD). In this study, we aimed to determine the ability of Cad to protect against renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) and identify the underlying therapeutic mechanisms of Cad during hypoxia/serum deprivation (H/SD) in human umbilical vein endothelial cells (HUVECs). A total of 36 male mice were randomly assigned into 3 groups (12 mice in each group): the Sham-operated group (Sham), the saline solution-treated UUO mice group (UUO), and the Cad administration (intragastrically) group (Cad). The mice in Cad group were administered Cad (100 mg/kg) daily by oral gavage and slaughtered on the 7th and 14th days post-surgery. Six mice from each group were sacrificed by sodium pentobarbital injection on the 7th and 14th day after surgery. Tissue hypoxia, cell apoptosis and fibrotic lesions were detected by Immunostaining and Western blot. Peritubular capillaries (PTCs) injury was measured by a novel technique of fluorescent microangiography (FMA). Endothelial cell-to-mesenchymal transition (EndMT) were identified by immunofluorescence and Western blot. HUVECs proliferation was measured via Cell Counting Kit‑8 assays and Edu staining. Sirt1 and its downstream gene in Cad regulation of endothelial were detected. Hematoxylin-eosin (HE), Masson-trichrome stains and Histological findings showed that Cad administration markedly reduced hypoxia and renal interstitial fibrosis at each time point in UUO. Meanwhile, Cad protect against EndMT process of PTCs by increasing CD31 expression and decreasing α-smooth muscle actin and fibronectin expression. in vitro studies showed that there was a proliferative response of the HUVECs incubated with Cad (10 μM) in H/SD. Sirt1 was suppressed after small interfering RNA (siRNA) was transfected in HUVECs. Mechanistically, Cad enhanced Sirt1 signaling, which was accompanied by increased levels of p53 acetylation (ac-p53). Meanwhile, protein expression of Bcl-2, and VE-cadherin were downregulated, Bax, and α-SMA were upregulated. In summary, the therapeutic effect of Cad in obstructive nephropathy were likely through suppressing EndMT progression and promoting anti-apoptotic effects after via activating the Sirt1/p53 signaling pathway.

AVR-RD-01, an Investigational Lentiviral Gene Therapy for Fabry Disease, Reduces Gb3 Substrate in Endothelial Cells of Renal Peritubular Capillaries

AVR-RD-01, an Investigational Lentiviral Gene Therapy for Fabry Disease, Reduces Gb3 Substrate in Endothelial Cells of Renal Peritubular Capillaries

The Role of Mitochondria in Drug-Induced Kidney Injury.

The kidneys utilize roughly 10% of the body’s oxygen supply to produce the energy required for accomplishing their primary function: the regulation of body fluid composition through secreting, filtering, and reabsorbing metabolites and nutrients. To ensure an adequate ATP supply, the kidneys are particularly enriched in mitochondria, having the second highest mitochondrial content and thus oxygen consumption of our body. The bulk of the ATP generated in the kidneys is consumed to move solutes toward (reabsorption) or from (secretion) the peritubular capillaries through the concerted action of an array of ATP-binding cassette (ABC) pumps and transporters. ABC pumps function upon direct ATP hydrolysis. Transporters are driven by the ion electrochemical gradients and the membrane potential generated by the asymmetric transport of ions across the plasma membrane mediated by the ATPase pumps. Some of these transporters, namely the polyspecific organic anion transporters (OATs), the organic anion transporting polypeptides (OATPs), and the organic cation transporters (OCTs) are highly expressed on the proximal tubular cell membranes and happen to also transport drugs whose levels in the proximal tubular cells can rapidly rise, thereby damaging the mitochondria and resulting in cell death and kidney injury. Drug-induced kidney injury (DIKI) is a growing public health concern and a major cause of drug attrition in drug development and post-marketing approval. As part of the article collection “Mitochondria in Renal Health and Disease,” here, we provide a critical overview of the main molecular mechanisms underlying the mitochondrial damage caused by drugs inducing nephrotoxicity.

Development and evaluation of deep learning–based segmentation of histologic structures in the kidney cortex with multiple histologic stains

The application of deep learning for automated segmentation (delineation of boundaries) of histologic primitives (structures) from whole slide images can facilitate the establishment of novel protocols for kidney biopsy assessment. Here, we developed and validated deep learning networks for the segmentation of histologic structures on kidney biopsies and nephrectomies. For development, we examined 125 biopsies for Minimal Change Disease collected across 29 NEPTUNE enrolling centers along with 459 whole slide images stained with Hematoxylin & Eosin (125), Periodic Acid Schiff (125), Silver (102), and Trichrome (107) divided into training, validation and testing sets (ratio 6:1:3). Histologic structures were manually segmented (30048 total annotations) by five nephropathologists. Twenty deep learning models were trained with optimal digital magnification across the structures and stains. Periodic Acid Schiff-stained whole slide images yielded the best concordance between pathologists and deep learning segmentation across all structures (F-scores: 0.93 for glomerular tufts, 0.94 for glomerular tuft plus Bowman’s capsule, 0.91 for proximal tubules, 0.93 for distal tubular segments, 0.81 for peritubular capillaries, and 0.85 for arteries and afferent arterioles). Optimal digital magnifications were 5X for glomerular tuft/tuft plus Bowman’s capsule, 10X for proximal/distal tubule, arteries and afferent arterioles, and 40X for peritubular capillaries. Silver stained whole slide images yielded the worst deep learning performance. Thus, this largest study to date adapted deep learning for the segmentation of kidney histologic structures across multiple stains and pathology laboratories. All data used for training and testing and a detailed online tutorial will be publicly available.

High Phosphate Induces and Klotho Attenuates Kidney Epithelial Senescence and Fibrosis

Cellular senescence is an irreversible cell growth arrest and is associated with aging and age-related diseases. High plasma phosphate (Pi) and deficiency of Klotho contribute to aging and kidney fibrosis, a pathological feature in the aging kidney and chronic kidney disease. This study examined the interactive role of Pi and Klotho in kidney senescence and fibrosis. Homozygous Klotho hypomorphic mice had high plasma Pi, undetectable Klotho in plasma and kidney, high senescence with massive collagen accumulation in kidney tubules, and fibrin deposits in peritubular capillaries. To examine the Pi effect on kidney senescence, a high (2%) Pi diet was given to wild-type mice. One week of high dietary Pi mildly increased plasma Pi, and upregulated kidney p16/p21 expression, but did not significantly decrease Klotho. Two weeks of high Pi intake led to increase in plasminogen activator inhibitor (PAI)-1, and decrease in kidney Klotho, but still without detectable increase in kidney fibrosis. More prolonged dietary Pi for 12 weeks exacerbated kidney senescence and fibrosis; more so in heterozygous Klotho hypomorphic mice compared to wild-type mice, and in mice with chronic kidney disease (CKD) on high Pi diet compared to CKD mice fed a normal Pi diet. In cultured kidney tubular cells, high Pi directly induced cellular senescence, injury and epithelial-mesenchymal transition, and enhanced H2O2-induced cellular senescence and injury, which were abrogated by Klotho. Fucoidan, a bioactive molecule with multiple biologic functions including senescence inhibition, blunted Pi-induced cellular senescence, oxidation, injury, epithelial-mesenchymal transition, and senescence-associated secretary phenotype. In conclusion, high Pi activates senescence through distinct but interconnected mechanisms: upregulating p16/p21 (early), and elevating plasminogen activator inhibitor-1 and downregulating Klotho (late). Klotho may be a promising agent to attenuate senescence and ameliorate age-associated, and Pi-induced kidney degeneration such as kidney fibrosis.

Renal intravascular large B cell lymphoma: the first case report in Korea and a review of the literature.

Herein, we describe the first case of renal intravascular large B cell lymphoma in Korea occurring in a 66-year-old female. She presented with mild fever and dyspnea. On physical and laboratory evaluations, hemophagocytic lymphohistiocytosis was suspected, but the bone marrow biopsy results were unremarkable. During the work-up, massive proteinuria developed, which led to a renal biopsy. The renal architecture was relatively well-preserved, but the glomeruli were hypercellular with the infiltration of atypical, large lymphoid cells with increased nucleus-cytoplasm ratio and clumped chromatin. Similar cells were also present in the peritubular capillaries. The tumor cells exhibited membranous staining for CD20 and CD79a. After the diagnosis of intravascular large B cell lymphoma, the patient received rituximab-based chemotherapy under close follow-up.

Nanoparticles exhibit greater accumulation in kidney glomeruli during experimental glomerular kidney disease.

Loss and dysfunction of glomerular podocytes result in increased macromolecule permeability through the glomerular filtration barrier and nephrotic syndrome. Current therapies can induce and maintain disease remission, but cause serious and chronic complications. Nanoparticle drug carriers could mitigate these side effects by delivering drugs to the kidneys more efficiently than free drug through tailoring of carrier properties. An important extrinsic factor of nanoparticle biodistribution is local pathophysiology, which may drive greater nanoparticle deposition in certain tissues. Here, we hypothesized that a “leakier” filtration barrier during glomerular kidney disease would increase nanoparticle distribution into the kidneys. We examined the effect of nanoparticle size and disease state on kidney accumulation in male BALB/c mice. The effect of size was tested using a panel of fluorescent polystyrene nanoparticles of size 20–200 nm, due to the relevance of this size range for drug delivery applications.Experimental focal segmental glomerulosclerosis was induced using an anti‐podocyte antibody that causes abrupt podocyte depletion. Nanoparticles were modified with carboxymethyl‐terminated poly(ethylene glycol) for stability and biocompatibility. After intravenous injection, fluorescence from nanoparticles of size 20 and 100 nm, but not 200 nm, was observed in kidney glomeruli and peritubular capillaries. During conditions of experimental focal segmental glomerulosclerosis, the number of fluorescent nanoparticle punctae in kidney glomeruli increased by 1.9‐fold for 20 and 100 nm nanoparticles compared to normal conditions. These findings underscore the importance of understanding and leveraging kidney pathophysiology in engineering new, targeted drug carriers that accumulate more in diseased glomeruli to treat glomerular kidney disease.