Tissue-Resident Macrophages in Solid Organ Transplantation: Harmful or Protective?

Transplanted organs carry donor immune cells into the recipient, the majority of which are tissue-resident macrophages (TRMs). The role they play in guiding the fate of the transplanted organ toward acceptance or rejection remains elusive. TRMs originate from both embryonic and bone marrow-derived precursors. Embryo-derived TRMs retain the embryonic capability to proliferate, so they are able to self-renew and, theoretically, persist for extended periods of time after transplantation. Bone marrow-derived TRMs do not proliferate and must constantly be replenished by adult circulating monocytes. Recent studies have aimed to clarify the different roles and interactions between donor TRMs, recipient monocytes, and monocyte-derived macrophages (MFs) after organ transplantation. This review aims to shed light on how MFs affect the fate of a transplanted organ by differentiating between the role of donor TRMs and that of MFs derived from graft infiltrating monocytes.

C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

Anti-Phospholipase A2 Receptor 1 and Anti-Cysteine Rich Antibodies, Domain Recognition and Rituximab Efficacy in Membranous Nephropathy: A Prospective Cohort Study.

RATIONALE & OBJECTIVE: Proteinuria and anti-phospholipase A2 receptor 1 (anti-PLA2R1) antibody titers are associated with primary membranous nephropathy (MN) outcomes. We evaluated the association of antibodies against the cysteine-rich (CysR) and C-type lectin 1, 7, and 8 (CTLD1, CTLD7, and CTLD8) domains of PLA2R1 with MN outcomes. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: One-hundred-thirteen consecutive, consenting patients referred to the Nephology Unit of the Azienda-Socio-Sanitaria-Territoriale (ASST) Papa Giovanni XXIII (Bergamo, Italy) with PLA2R1-related, biopsy-proven MN whose persistent nephrotic syndrome (NS) was managed conservatively for>6 months and were monitored with serial evaluations of proteinuria, autoantibodies (by enzyme-linked immunosorbent assay), and clinical outcomes. EXPOSURE: Rituximab. OUTCOME: Complete (proteinuria<0.3g/24h) or partial (proteinuria≥0.3g/24h and<3.0g/24h with>50% reduction vs basal) NS remission. ANALYTICAL APPROACH: Univariable and multivariable Cox regression analyses. RESULTS: All patients had anti-CysR antibodies; 62 (54.9%) were multidomain recognizers. Anti-PLA2R1 and anti-CysR antibody titers were strongly correlated at baseline (P<0.001, r=0.934), 6 months (P<0.001, r=0.964), and 12 months (P<0.001, r=0.944). During a median follow-up of 37.1 (IQR, 20.3-56.9) months, 71 patients (62.8%) achieved either complete or partial remission of their NS. Lower baseline anti-PLA2R1 (HR, 0.997 [95% CI, 0.996-0.999], P=0.002) and anti-CysR [HR, 0.996 [95% CI, 0.993-0.998], P=0.001) titers were associated with a higher probability of remission, along with female sex, lower proteinuria, and lower serum creatinine levels (P<0.05 for all comparisons). Anti-CTLD antibodies were not associated with outcomes. At 6 and 12 months, compared to baseline, anti-PLA2R1 and anti-CysR antibody titers decreased more in patients progressing to partial or complete remission than in those without remission (P<0.05 for all comparisons). LIMITATIONS: Observational design. CONCLUSIONS: In PLA2R1-related MN, anti-PLA2R1 and anti-CysR antibodies similarly predict rituximab efficacy independent of PLA2R1 domain recognition. The choice between these tests should be dictated by feasibility and costs. Evaluating anti-CTLD antibodies appears unnecessary. PLAIN-LANGUAGE SUMMARY: Primary membranous nephropathy (MN), a leading cause of nephrotic syndrome (NS) in adults, is an autoimmune disease caused by autoantibodies binding to the podocyte antigen phospholipase A2 receptor 1 (PLA2R1). We assessed whether the effects of anti-CD20 cytolytic therapy with the monoclonal antibody rituximab are associated with detection rates and levels of anti-PLA2R1 antibodies and antibodies against PLA2R1 domains such as cysteine-rich (CysR), and C-type lectin 1, 7, and 8 (CTLD1, 7, and 8), in patients with PLA2R1-related MN and persistent NS. The probability of rituximab-induced complete or partial NS remission was associated with baseline anti-PLA2R1 and anti-CysR antibody titers, but not with anti-CTLD1, 7 and 8 antibodies or multidomain recognition. Integrated evaluation of anti-PLA2R1 or anti-CysR antibodies with proteinuria and kidney function may play a role in monitoring the effects of rituximab in patients with PLA2R1-related NS and MN.

Therapeutic Small Interfering RNA Targeting Complement C3 in a Mouse Model of C3 Glomerulopathy.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.

Empagliflozin protects glomerular endothelial cell architecture in experimental diabetes through the VEGF-A/caveolin-1/PV-1 signaling pathway.

In addition to having blood glucose-lowering effects, inhibitors of sodium glucose cotransporter 2 (SGLT2) afford renoprotection in diabetes. We sought to investigate which components of the glomerular filtration barrier could be involved in the antiproteinuric and renoprotective effects of SGLT2 inhibition in diabetes. BTBR (black and tan, brachyuric) ob/ob mice that develop a type 2 diabetic nephropathy received a standard diet with or without empagliflozin for 10 weeks, starting at 8 weeks of age, when animals had developed albuminuria. Empagliflozin caused marked decreases in blood glucose levels and albuminuria but did not correct glomerular hyperfiltration. The protective effect of empagliflozin against albuminuria was not due to a reduction in podocyte damage as empagliflozin did not affect the larger podocyte filtration slit pore size nor the defective expression of nephrin and nestin. Empagliflozin did not reduce the thickening of the glomerular basement membrane. In BTBR ob/ob mice, the most profound abnormality seen using electron microscopy was in the endothelial aspect of the glomerular capillary, with significant loss of endothelial fenestrations. Remarkably, empagliflozin ameliorated the subverted microvascular endothelial ultrastructure. Caveolae and bridging diaphragms between adjacent endothelial fenestrae were seen in diabetic mice and associated with increased expression of caveolin-1 and the appearance of PV-1. These endothelial abnormalities were limited by the SGLT2 inhibitor. Although no expression of SGLT2 was found in glomerular endothelial cells, SGLT2 was expressed in the podocytes of diabetic mice. VEGF-A, which is a known stimulus for endothelial caveolin-1 and PV-1, was increased in podocytes of BTBR ob/ob mice and normalized by SGLT2 inhibitor treatment. Thus, empagliflozin's protective effect on the glomerular endothelium of diabetic mice could be due to a limitation of the paracrine signaling of podocyte-derived VEGF-A that resulted in a reduction of the abnormal endothelial caveolin-1 and PV-1, with the consequent preservation of glomerular endothelial function and permeability. © 2022 The Pathological Society of Great Britain and Ireland.

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.

Immunophenotypic Alterations in Adult Patients with Steroid-Dependent and Frequently Relapsing Nephrotic Syndrome.

Immune dysregulation plays a key role in the pathogenesis of steroid-dependent/frequently relapsing nephrotic syndrome (SDNS/FRNS). However, in contrast with evidence from the pediatric series, no major B- or T-cell alterations have been described for adults. In these patients, treatment with rituximab allows safe discontinuation of steroids, but long-term efficacy is variable, and some patients experience NS relapses after B cell reconstitution. In this study, we aimed to determine disease-associated changes in the B and T cell phenotype of adult patients with SDND/FRNS after steroid-induced remission. We also investigated whether any of these changes in immune cell subsets could discriminate between patients who developed NS relapses after steroid-sparing treatment with rituximab from those who did not. Lymphocyte subsets in SDNS/FRNS patients (n = 18) were compared to those from patients with steroid-resistant NS (SRNS, n = 7) and healthy volunteers (HV, n = 15). Before rituximab, SDND/FRNS patients showed increased frequencies of total and memory B cells, mainly with a CD38-negative phenotype. Within the T-cell compartment, significantly lower levels of FOXP3+ regulatory T cells (Tregs) were found, mostly due to a reduction in CD45RO+ memory Tregs compared to both SRNS and HV. The levels of CD45RO+ Tregs were significantly lower at baseline in patients who relapsed after rituximab (n = 9) compared to patients who did not (n = 9). In conclusion, patients with SDND/FRNS displayed expansion of memory B cells and reduced memory Tregs. Treg levels at baseline may help identify patients who will achieve sustained remission following rituximab infusion from those who will experience NS relapses.

Hypoimmunogenic Human Pluripotent Stem Cells as a Powerful Tool for Liver Regenerative Medicine.

Induced pluripotent stem cells (iPSC) have huge potential as cell therapy for various diseases, given their potential for unlimited self-renewal and capability to differentiate into a wide range of cell types. Although autologous iPSCs represents the ideal source for patient-tailored regenerative medicine, the high costs of the extensive and time-consuming production process and the impracticability for treating acute conditions hinder their use for broad applications. An allogeneic iPSC-based strategy may overcome these issues, but it carries the risk of triggering an immune response. So far, several approaches based on genome-editing techniques to silence human leukocyte antigen class I (HLA-I) or II (HLA-II) expression have been explored to overcome the immune rejection of allogeneic iPSCs. In this study, we employed the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to delete the ß2-Microglobulin (B2M) and the Class II Major Histocompatibility Complex Transactivator (CIITA) genes, essential for the correct surface expression of HLA-I and HLA-II proteins. The resulting hypoimmunogenic iPSC line has a normal karyotype, expresses the pluripotency stem cell markers, and is capable of differentiating into the three embryonic germ layers. Furthermore, we showed that it specifically retains the ability to differentiate towards different liver cells, such as endothelial-like cells, hepatocyte-like cells, and hepatic stellate-like cells. Our results indicate that hypoimmunogenic iPSCs could give a new cost-effective and off-the-shelf opportunity for cell therapy in liver diseases.

Imaging the Kidney with an Unconventional Scanning Electron Microscopy Technique: Analysis of the Subpodocyte Space in Diabetic Mice.

Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE-SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE-SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function.

Sirtuin 3 Deficiency Aggravates Kidney Disease in Response to High-Fat Diet through Lipotoxicity-Induced Mitochondrial Damage.

Sirtuin 3 (SIRT3) is the primary mitochondrial deacetylase that controls the antioxidant pathway and energy metabolism. We previously found that renal Sirt3 expression and activity were reduced in mice with type 2 diabetic nephropathy associated with oxidative stress and mitochondrial abnormalities and that a specific SIRT3 activator improved renal damage. SIRT3 is modulated by diet, and to assess whether Sirt3 deficiency aggravates mitochondrial damage and accelerates kidney disease in response to nutrient overloads, wild-type (WT) and Sirt3-/- mice were fed a high-fat-diet (HFD) or standard diet for 8 months. Sirt3-/- mice on HFD exhibited earlier and more severe albuminuria compared to WT mice, accompanied by podocyte dysfunction and glomerular capillary rarefaction. Mesangial matrix expansion, tubular vacuolization and inflammation, associated with enhanced lipid accumulation, were more evident in Sirt3-/- mice. After HFD, kidneys from Sirt3-/- mice showed more oxidative stress than WT mice, mitochondria ultrastructural damage in tubular cells, and a reduction in mitochondrial mass and energy production. Our data demonstrate that Sirt3 deficiency renders mice more prone to developing oxidative stress and mitochondrial abnormalities in response to HFD, resulting in more severe kidney diseases, and this suggests that mitochondria protection may be a method to prevent HFD-induced renal injury.

Angiotensin-converting enzyme 2: from a vasoactive peptide to the gatekeeper of a global pandemic.

PURPOSE OF REVIEW: We provide a comprehensive overview of angiotensin-converting enzyme 2 (ACE2) as a possible candidate for pharmacological approaches to halt inflammatory processes in different pathogenic conditions. RECENT FINDINGS: ACE2 has quickly gained prominence in basic research as it has been identified as the main entry receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This novel pathogen causes Coronavirus Disease 2019 (COVID-19), a pathogenic condition that reached pandemic proportion and is associated with unprecedented morbidity and mortality. SUMMARY: The renin-angiotensin system is a complex, coordinated hormonal cascade that plays a pivotal role in controlling individual cell behaviour and multiple organ functions. ACE2 acts as an endogenous counter-regulator to the pro-inflammatory and pro-fibrotic pathways triggered by ACE through the conversion of Ang II into the vasodilatory peptide Ang 1-7. We discuss the structure, function and expression of ACE2 in different tissues. We also briefly describe the role of ACE2 as a pivotal driver across a wide spectrum of pathogenic conditions, such as cardiac and renal diseases. Furthermore, we provide the most recent data concerning the possible role of ACE2 in mediating SARS-CoV-2 infection and dictating COVID-19 severity.

Endothelin-targeted new treatments for proteinuric and inflammatory glomerular diseases: focus on the added value to anti-renin-angiotensin system inhibition.

Chronic kidney disease (CKD) is the main cause of end-stage renal disease worldwide arising as a frequent complication of diabetes, obesity, and hypertension. Current therapeutic options, mainly based of inhibition of the renin-angiotensin system (RAS), provide imperfect renoprotection if started at an advanced phase of the disease, and treatments that show or even reverse the progression of CKD are needed. The endothelin (ET) system contributes to the normal renal physiology; however, robust evidence suggests a key role of ET-1 and its cognate receptors, in the progression of CKD. The effectiveness of ET receptor antagonists in ameliorating renal hemodynamics and fibrosis has been largely demonstrated in different experimental models. A significant antiproteinuric effect of ET receptor antagonists has been found in diabetic and non-diabetic CKD patients even on top of RAS blockade, and emerging evidence from ongoing clinical trials highlights their beneficial effects on a wide range of kidney disorders.

Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c+F4/80+ Macrophages through IL-1R8 Regulation.

BACKGROUND: In donor kidneys subjected to ischemia-reperfusion injury during kidney transplant, phagocytes coexpressing the F4/80 and CD11c molecules mediate proinflammatory responses and trigger adaptive immunity in transplantation through antigen presentation. After injury, however, resident renal macrophages coexpressing these surface markers acquire a proreparative phenotype, which is pivotal in controlling inflammation and fibrosis. No data are currently available regarding the effects of transplant-induced ischemia-reperfusion injury on the ability of donor-derived resident renal macrophages to act as professional antigen-presenting cells. METHODS: We evaluated the phenotype and function of intragraft CD11c+F4/80+ renal macrophages after cold ischemia. We also assessed the modifications of donor renal macrophages after reversible ischemia-reperfusion injury in a mouse model of congeneic renal transplantation. To investigate the role played by IL-1R8, we conducted in vitro and in vivo studies comparing cells and grafts from wild-type and IL-R8-deficient donors. RESULTS: Cold ischemia and reversible ischemia-reperfusion injury dampened antigen presentation by renal macrophages, skewed their polarization toward the M2 phenotype, and increased surface expression of IL-1R8, diminishing activation mediated by toll-like receptor 4. Ischemic IL-1R8-deficient donor renal macrophages acquired an M1 phenotype, effectively induced IFNγ and IL-17 responses, and failed to orchestrate tissue repair, resulting in severe graft fibrosis and aberrant humoral immune responses. CONCLUSIONS: IL-1R8 is a key regulator of donor renal macrophage functions after ischemia-reperfusion injury, crucial to guiding the phenotype and antigen-presenting role of these cells. It may therefore represent an intriguing pathway to explore with respect to modulating responses against autoantigens and alloantigens after kidney transplant.

The case of complement activation in COVID-19 multiorgan impact.

The novel coronavirus disease COVID-19 originates in the lungs, but it may extend to other organs, causing, in severe cases, multiorgan damage, including cardiac injury and acute kidney injury. In severe cases, the presence of kidney injury is associated with increased risk of death, highlighting the relevance of this organ as a target of SARS-CoV-2 infection. COVID-19-associated tissue injury is not primarily mediated by viral infection, but rather is a result of the inflammatory host immune response, which drives hypercytokinemia and aggressive inflammation that affect lung parenchymal cells, diminishing oxygen uptake, but also endothelial cells, resulting in endotheliitis and thrombotic events and intravascular coagulation. The complement system represents the first response of the host immune system to SARS-CoV-2 infection, but there is growing evidence that unrestrained activation of complement induced by the virus in the lungs and other organs plays a major role in acute and chronic inflammation, endothelial cell dysfunction, thrombus formation, and intravascular coagulation, and ultimately contributes to multiple organ failure and death. In this review, we discuss the relative role of the different complement activation products in the pathogenesis of COVID-19-associated tissue inflammation and thrombosis and propose the hypothesis that blockade of the terminal complement pathway may represent a potential therapeutic option for the prevention and treatment of lung and multiorgan damage.

Addition of cyclic angiotensin-(1-7) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy.

The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.

The incessant search for renal biomarkers: is it really justified?

PURPOSE OF REVIEW: This review summarizes the most recent and relevant findings in the search for novel biomarkers for the most common renal diseases. RECENT FINDINGS: Unprecedented, fast-paced technical advances in biomedical research have offered an opportunity to identify novel and more specific renal biomarkers in several clinical settings. However, despite the huge efforts made, the molecules identified so far have generally failed to provide relevant information beyond what has already been generated by established biomarkers, such as serum creatinine and proteinuria, whereas the complexity and costs of these technology platforms hamper their widespread implementation. SUMMARY: No novel renal biomarkers have added clear-cut additional value in clinical decision-making. The only exception is anti-phospholipase A2 receptor antibodies, which have been implemented successfully as a diagnostic and prognostic biomarker of membranous nephropathy. This achievement, along with the large number of ongoing collaborative projects worldwide, should lead the renal community to be quite confident regarding the successful qualification of novel and effective diagnostic, prognostic and therapeutic response biomarkers for kidney diseases, hopefully in the next few years.

An Ex Vivo Test of Complement Activation on Endothelium for Individualized Eculizumab Therapy in Hemolytic Uremic Syndrome.

RATIONALE & OBJECTIVE: Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. RESULTS: Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate-activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. LIMITATIONS: The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. CONCLUSIONS: The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

Sirtuins in Renal Health and Disease.

Sirtuins belong to an evolutionarily conserved family of NAD+-dependent deacetylases that share multiple cellular functions related to proliferation, DNA repair, mitochondrial energy homeostasis, and antioxidant activity. Mammalians express seven sirtuins (SIRT1-7) that are localized in different subcellular compartments. Changes in sirtuin expression are critical in several diseases, including metabolic syndrome, diabetes, cancer, and aging. In the kidney, the most widely studied sirtuin is SIRT1, which exerts cytoprotective effects by inhibiting cell apoptosis, inflammation, and fibrosis together with SIRT3, a crucial metabolic sensor that regulates ATP generation and mitochondrial adaptive response to stress. Here, we provide an overview of the biologic effects of sirtuins and the molecular targets thereof regulating renal physiology. This review also details progress made in understanding the effect of sirtuins in the pathophysiology of chronic and acute kidney diseases, highlighting the key role of SIRT1, SIRT3, and now SIRT6 as potential therapeutic targets. In this context, the current pharmacologic approaches to enhancing the activity of SIRT1 and SIRT3 will be discussed.

The Role of Angiotensin II in Parietal Epithelial Cell Proliferation and Crescent Formation in Glomerular Diseases.

Crescentic glomerulonephritis (GN) is a devastating disease with rapidly progressive deterioration in kidney function, which, histologically, manifests as crescent formation in most glomeruli. We previously found that crescents derive from the aberrant proliferation and migration of parietal epithelial cells (PECs)/progenitor cells, and that the angiotensin (ang) II/ang II type-1 (AT1) receptor pathway may participate, together with the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor 4 axis, in the development of those lesions. Herein, we elucidated sequential events and cellular and molecular interactions occurring during crescentic lesion onset and evolution. By analyzing kidney biopsy specimens of patients with extracapillary GN, divided according to the grade of glomerular lesions, we found that the accumulation of macrophages expressing matrix metalloproteinase-12 started manifesting in glomeruli affected by early-stage lesions, whereas AT1 receptor expression could not be detected. In glomeruli with advanced lesions, AT1 receptor expression increased markedly, and the up-regulation of SDF-1, and its receptor C-X-C chemokine receptor 7, was documented on podocytes and PECs, respectively. In vitro studies were instrumental to demonstrating the role of ang II in inducing podocyte SDF-1 production, which ultimately activates PECs. The present findings support the possibility that angiotensin-converting enzyme inhibitor treatment might limit PEC activation and reduce the frequency and extension of crescents in extracapillary GN.

MicroRNAs as Master Regulators of Glomerular Function in Health and Disease.

MicroRNAs (miRNAs) are important regulators of gene expression, and the dysregulation of miRNAs is a common feature of several diseases. More miRNAs are identified almost daily, revealing the complexity of these transcripts in eukaryotic cellular networks. The study of renal miRNAs, using genetically modified mice or by perturbing endogenous miRNA levels, has revealed the important biologic roles miRNAs have in the major cell lineages that compose the glomerulus. Here, we provide an overview of miRNA biogenesis and function in regulating key genes and cellular pathways in glomerular cells during development and homeostasis. Moreover, we focus on the emerging mechanisms through which miRNAs contribute to different diseases affecting the glomerulus, such as FSGS, IgA nephropathy, lupus nephritis, and diabetic nephropathy. In-depth knowledge of miRNA-based gene regulation has made it possible to unravel pathomechanisms, enabling the design of new therapeutic strategies for glomerular diseases for which available therapies are not fully efficacious.