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Infection is the second leading cause of death in patients with chronic kidney disease (CKD). Adequate humoral (antibody) and cellular (T cell-driven) immunity are required to minimize pathogen entry and promote pathogen clearance to enable infection control. Vaccination can generate cellular and humoral immunity against specific pathogens and is used to prevent many life-threatening infectious diseases. However, vaccination efficacy is diminished in patients with CKD. Premature ageing of the immune system and chronic systemic low-grade inflammation are the main causes of immune alteration in these patients. In the case of SARS-CoV-2 infection, COVID-19 can have considerable detrimental effects in patients with CKD, especially in those with kidney failure. COVID-19 prevention through successful vaccination is therefore paramount in this vulnerable population. Although patients receiving dialysis have seroconversion rates comparable to those of patients with normal kidney function, most kidney transplant recipients could not generate humoral immunity after two doses of the COVID-19 vaccine. Importantly, some patients who were not able to produce antibodies still had a detectable vaccine-specific T cell response, which might be sufficient to prevent severe COVID-19. Correlates of protection against SARS-CoV-2 have not been established for patients with kidney failure, but they are urgently needed to enable personalized vaccination regimens.

Kidney international 103(2), 391-402 (2022). doi:10.1016/j.kint.2022.09.017, Published by Elsevier, New York, NY

Nach derzeitigem Verstandnis bildet die Erkrankungsgruppe aus Immunkomplex-vermittelter membranoproliferativer Glomerulonephritis (IC-MPGN) und C3-Glomerulopathie (C3G) ein Spektrum ab, welches im Hinblick auf Pathophysiologie und klinischen Verlauf sehr heterogen ist, dessen Vertreter jedoch haufig in der Histologie ein MPGN-Muster aufweisen. Nur wenige Falle der IC-MPGN konnen letztlich einer zugrunde liegenden Ursache zugeordnet werden. Derzeitige Erkenntnisse legen zudem nahe, dass sich IC-MPGN und C3G mehr pathophysiologische Aspekte im Hinblick auf sekundare Ursachen, Autoantikorper und genetische Aspekte teilen, als dies mit Schaffung der neuen Klassifikation zunachst vermutet worden war. Die aktuellen KDIGO(Kidney Disease: Improving Global Outcomes)-Leitlinien bezeichnen die in ihrem entsprechenden Kapitel erfassten Erkrankungen als glomerulare Erkrankungen mit einem „MPGN-Muster“, da sich ein entsprechender Lichtmikroskopiebefund am haufigsten zeigt. Die Kenntnis der zugrunde liegenden Pathophysiologie ist fur die weiterfuhrende Diagnostik zur Abklarung sekundarer Ursachen wegweisend. Eine umfassende Komplementanalytik, begleitet von Antikorperscreening und humangenetischer Analyse, sollte konsequent erfolgen. Die Seltenheit der Erkrankung und die uber die Jahrzehnte unterschiedlich gepragte Klassifikation der Erkrankung haben dazu beigetragen, dass bis heute keine hochwertige Studienevidenz fur das Management dieser Erkrankungen vorliegt. Die derzeit vorliegenden Erkenntnisse bieten aber doch eine gewisse Basis fur die Anwendung verfugbarer Therapieansatze bei den haufig rasch progredienten und nach Nierentransplantation oftmals wiederkehrenden Krankheitsbildern.

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Pathological deposition of collagen is a hallmark of renal fibrosis. We employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis using the collagen-binding agent CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography (CT-FMT) and CNA35 labeled with the near-infrared fluorophore Cy7. Upon i.v. injection, CNA35-Cy7 accumulation was significantly higher in fibrotic kidneys compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of animals and patients with renal fibrosis, CNA35-FITC co-localized with scar collagen type I and III, but not with the basement membrane collagen type IV. Upon i.v. injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in animal models and patients using computer-based morphometry quantification. In conclusion, molecular imaging of collagen using CNA35 enables specific non-invasive quantification of renal fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.

The outcome after living kidney donation was assumed to be comparable to that of the general population. However, recent register studies reveal negative changes in kidney function, quality of life and fatigue. Avoiding methodological issues of previous studies, the Safety of the Living Kidney Donor (SoLKiD) cohort study analyzed the outcome of donors in a multicenter and interdisciplinary fashion. Donor data were collected pre-donation and two-, six- and 12-months post-donation in 20 German transplantation centers. Primary parameters were kidney function, quality of life, and fatigue. Secondary endpoints were blood pressure, hemoglobin, hemoglobin A1c, body mass index, depression and somatization. Parameters were analyzed with non-parametric statistical tests and a mixed model regression for changes in time, their clinical relevance and interaction encompassing 336 donors with mean age of 52 years. Most of the physical secondary parameters, depression, and quality of life showed little or no changes and regained their pre-donation level. Kidney function decreased significantly with a 37% loss of glomerular filtration rate and an increase of donors with chronic kidney disease stage 3 from 1.5% pre-donation to about 50%. Donors consistently showed increased fatigue and somatization. Mental fatigue increased from 10.6% to 28.1%. The main influencing factors for decreased kidney function and increased fatigue were their respective pre-donation levels, and donor age for kidney function and subject stress level in fatigue. Thus, our study showed that a significant number of donors developed clinically relevant changes in physical and mental health and emphasizes the urgent need to inform potential donors about these risks.

Background Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known. Methods Fifty-seven patients were included in this case–control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane- SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay. Results Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4+ T cells reactive against at least one SARS-CoV-2 protein. CD8+ T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals. Conclusions This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection. Graphic abstract

The randomized, controlled STOP-IgAN trial in patients with IgA nephropathy (IgAN) and substantial proteinuria showed no benefit of immunosuppression added on top of supportive care on renal function over three years. As a follow-up we evaluated renal outcomes in patients over a follow-up of up to ten years in terms of serum creatinine, proteinuria, end-stage kidney disease (ESKD), and death. The adapted primary endpoint was the time to first occurrence of a composite of death, ESKD, or a decline of over 40% in the estimated glomerular filtration rate (eGFR) compared to baseline at randomization into STOP-IgAN. Data were analyzed by Cox-regression models. Follow-up data were available for 149 participants, representing 92% of the patients originally randomized. Median follow-up was 7.4 years (inter quartile range 5.7 to 8.3 years). The primary endpoint was reached in 36 of 72 patients randomized to supportive care and 35 of 77 patients randomized to additional immunosuppression (hazard ratio 1.20; 95% confidence interval 0.75 to 1.92). ESKD occurred in 17 of the patients with supportive care and in 20 of the patients with additional immunosuppression. Additionally, the rates of eGFR loss over 40% and annual eGFR loss did not differ between groups. Two patients died with supportive care and three with additional immunosuppression. Thus, within the limitations of a retrospective study, over a follow-up of up to ten years, and using an adapted primary endpoint, we failed to detect differences in key clinical outcomes in IgAN patients randomized to receive added immunosuppression on top of supportive care versus supportive care alone.

Renin lineage cells (RLCs) serve as a progenitor cell reservoir during nephrogenesis and after renal injury. The maintenance mechanisms of the RLC pool are still poorly understood. Since RLCs were also identified as a progenitor cell population in bone marrow we first considered that these may be their source in the kidney. However, transplantation experiments in adult mice demonstrated that bone marrow–derived cells do not give rise to RLCs in the kidney indicating their non-hematopoietic origin. Therefore we tested whether RLCs develop in the kidney through neogenesis ( de novo differentiation) from cells that have never expressed renin before. We used a murine model to track neogenesis of RLCs by flow cytometry, histochemistry, and intravital kidney imaging. During nephrogenesis RLCs first appear at e14, form a distinct population at e16, and expand to reach a steady state level of 8-10% of all kidney cells in adulthood. De novo differentiated RLCs persist as a clearly detectable population through embryogenesis until at least eight months after birth. Pharmacologic stimulation of renin production with enalapril or glomerular injury induced the rate of RLC neogenesis in the adult mouse kidney by 14% or more than three-fold, respectively. Thus, the renal RLC niche is constantly filled by local de novo differentiation. This process could be stimulated consequently representing a new potential target to beneficially influence repair and regeneration after kidney injury.

Intracellular cAMP, the production of which is catalyzed by the α-subunit of the stimulatory G protein (Gsα), controls renin synthesis and release by juxtaglomerular (JG) cells of the kidney, but may also have relevance for the physiologic integrity of the kidney. To investigate this possibility, we generated mice with inducible knockout of Gsα in JG cells and monitored them for 6 months after induction at 6 weeks of age. The knockout mapped exclusively to the JG cells of the Gsα-deficient animals. Progressive albuminuria occurred in Gsα-deficient mice. Compared with controls expressing wild-type Gsα alleles, the Gsα-deficient mice had enlarged glomeruli with mesangial expansion, injury, and FSGS at study end. Ultrastructurally, the glomerular filtration barrier of the Gsα-deficient animals featured endothelial gaps, thickened basement membrane, and fibrin-like intraluminal deposits, which are classic signs of thrombotic microangiopathy. Additionally, we found endothelial damage in peritubular capillaries and vasa recta. Because deficiency of vascular endothelial growth factor (VEGF) results in thrombotic microangiopathy, we addressed the possibility that Gsα knockout may result in impaired VEGF production. We detected VEGF expression in JG cells of control mice, and cAMP agonists regulated VEGF expression in cultured renin-producing cells. Our data demonstrate that Gsα deficiency in JG cells of adult mice results in kidney injury, and suggest that JG cells are critically involved in the maintenance and protection of the renal microvascular endothelium.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1β cleavage in Aim2(−/−) or WT mice that received WT bone marrow than in WT mice that received Aim2(−/−) bone marrow. Intravital microscopy of the kidney in LysM((gfp/gfp)) mice 5–6 days after UUO demonstrated the significant recruitment of GFP(+) proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX(3)CR1(+) renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1β, as well as the formation of AIM2(+) ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1β levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

BACKGROUND AND OBJECTIVES: The prognostic value of preformed donor-specific HLA antibodies (DSA), which are only detectable by sensitive methods, remains controversial for kidney transplantation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The outcome of 4233 consecutive kidney transplants performed between 2012 and 2015 in 18 German transplant centers was evaluated. Most centers used a stepwise pretransplant antibody screening with bead array tests and differentiation of positive samples by single antigen assays. Using these screening results, DSA against HLA-A, -B, -C, -DRB1 and -DQB1 were determined. Data on clinical outcome and possible covariates were collected retrospectively. RESULTS: Pretransplant DSA were associated with lower overall graft survival, with a hazard ratio of 2.53 for living donation (95% confidence interval [95% CI], 1.49 to 4.29; P

Background Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes.Methods We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18-85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR) 25-75 mL/min per 1.73 m(2) of body surface area, and a urine albumin-to-creatinine ratio (UACR) of 300-5000 mg/g who had received maximum labelled or tolerated renin-angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0.75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders) were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0.75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for >= 30 days) or end-stage kidney disease (eGFR = 90 days, chronic dialysis for >= 90 days, kidney transplantation, or death from kidney failure) in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials. gov, number NCT01858532.Findings Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325) or placebo group (n=1323). Median follow-up was 2.2 years (IQR 1.4-2.9). 79 (6.0%) of 1325 patients in the atrasentan group and 105 (7.9%) of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR] 0.65 [95% CI 0.49-0.88]; p=0.0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3.5%) of 1325 patients in the atrasentan group and 34 (2.6%) of 1323 patients in the placebo group (HR 1.33 [95% CI 0.85-2.07]; p=0.208). 58 (4.4%) patients in the atrasentan group and 52 (3.9%) in the placebo group died (HR 1.09 [95% CI 0.75-1.59]; p=0.65).Interpretation Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Copyright (C) 2019 Elsevier Ltd. All rights reserved.

The thorough investigation of peritubular capillary changes during renal fibrosis presented in this issue provides novel insights, not only into the value of elaborate methodological approaches, but also the necessity to carefully dissect pathophysiology using current techniques. This study strengthens the existing view of a uniform progression of renal fibrosis and is consistent with other recent studies pointing toward the relevance of early alterations of peritubular capillaries as initiators of renal fibrosis.

Hypoxia-inducible factor (HIF)-2–triggered erythropoietin production in renal interstitial fibroblast–like cells is the physiologically relevant source of erythropoietin for regulating erythropoiesis. During renal fibrosis, these cells transform into myofibroblasts and lose their ability to produce sufficient erythropoietin leading to anemia. To find if other cells for erythropoietin production might exist in the kidney we tested for the capability of nonepithelial glomerular cells to elaborate erythropoietin. Therefore, HIF transcription factors were stabilized by cell-specific deletion of the von Hippel–Lindau ( VHL ) gene. Inducible deletion of VHL in glomerular connexin40-expressing cells (endothelial, renin-expressing, and mesangial cells) markedly increased glomerular erythropoietin mRNA expression levels, plasma erythropoietin concentrations, and hematocrit values. These changes were mimicked by inducible cell-specific VHL deletion in renin-expressing and in mesangial cells but not in endothelial cells. The increases of erythropoietin production were absent, when VHL was co-deleted with HIF-2. The induction of glomerular erythropoietin expression was associated with the downregulation of juxtaglomerular renin expression, again in a HIF-2-dependent manner. Thus, VHL deletion in renin-expressing and in mesangial cells induces the capability to produce relevant amounts of erythropoietin and to suppress renin expression in the adult kidney if HIF-2 is stabilized.

Basierend auf neuen pathophysiologischen Erkenntnissen der vorausgehenden Jahre, erfolgte im Jahr 2010 die Einfuhrung einer neuen Klassifikation einer Glomerulonephritis mit dominanten oder kodominanten C3-Ablagerungen, deren wesentliche Untergruppe die C3-Glomerulopathie (C3G) darstellt. Wahrend sekundare Ursachen zu einer immunkomplexvermittelten membranoproliferativen Glomerulonephritis (MPGN) fuhren, liegen die Hauptursachen der C3-Glomerulopathie primar in Storungen des Komplementsystems, die dann haufig in Form einer MPGN, aber auch anderer lichtmikroskopischer Erscheinungsbilder apparent werden. Die Kenntnis der zugrunde liegenden Pathophysiologie ist fur die weiterfuhrende Diagnostik zur Abklarung sekundarer Ursachen wegweisend. Eine umfassende Komplementanalytik, begleitet von Antikorperscreening und humangenetischer Analyse, sollte konsequent erfolgen. Wenn auch bis heute durch Studien nicht systematisch validiert, bieten die vorliegenden Erkenntnisse doch eine robuste Grundlage fur die Anwendung verfugbarer Therapieansatze fur diese haufig rasch progredienten und nach Nierentransplantation oftmals wiederkehrenden Krankheitsbilder. In der vorliegenden Ubersicht werden die derzeitigen Erkenntnisse systematisch dargestellt und in Form eines (nationalen) Expertenkonsensus zur Diagnostik der C3-dominanten Glomerulonephritis und der MPGN zusammengefasst.

Nierentransplantierte Patienten haben im Vergleich zur Gesamtbevolkerung ein 2- bis 4-fach erhohtes Tumorrisiko, wobei das Risiko besonders fur Hauttumoren, Lymphome und Nierenzellkarzinome erhoht ist. Ursachlich fur die erhohte Tumorinzidenz ist die durch die immunsuppressive Therapie bedingte eingeschrankte Uberwachungskapazitat des Immunsystems in Bezug auf maligne Veranderungen sowie die erhohte Anfalligkeit fur sog. Onkoviren. Hervorzuheben ist hier die Bedeutung der Primarinfektion mit Epstein-Barr-Virus (EBV) bei der Entstehung von Posttransplantationslymphomen. Allerdings ist auch eine chronische Nierenerkrankung selbst mit einem erhohten Risiko, insbesondere fur Nieren- und Blasentumoren, vergesellschaftet. Bezuglich der Art der immunsuppressiven Medikation bestehen Unterschiede fur das Tumorrisiko. Das gehaufte Auftreten bestimmter Tumoren und ihre Risikofaktoren haben Implikationen fur die Pravention, Fruherkennung und Behandlung von Tumoren nach Nierentransplantation.

Over the past decade, researchers have made substantial progress in characterizing a network of mononuclear phagocytes in the kidney, which variously have been referred to as resident macrophages or dendritic cells. Two recent studies, published in Cell and Kidney International, have identified these resident macrophages/dendritic cells as local immune monitors of peritubular capillaries for circulating antigens and immune complexes. These cells appear to represent an early line of defense against circulating infectious particles and immune complexes, but the resulting inflammatory response may also contribute to interstitial inflammation and kidney disease progression.

DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

Background. To date, no effective immunosuppressive standard for the prevention or treatment of alloantibody production in acute and chronic rejection of renal transplants has been established. Alloantibody formation has been recognized in the well-established rat model of Fischer to Lewis renal transplantation. We used this renal allotransplantation model to test the effectiveness of sirolimus (SRL), bortezomib (BZ) or their combination in an already established humoral rejection situation. Methods. After 3 weeks, transplanted rats were treated either with placebo (P), SRL, BZ or combination of SRL and BZ. Rats were monitored for donor-specific alloantibodies as well as humoral responses in the spleen and bone marrow, renal function and histological changes in the graft. Results. In all three treatment arms, glomerular, tubulointerstitial and vascular changes of chronic antibody-mediated rejection were ameliorated compared to the P group. Production of alloantibodies against components of glomerular basement membrane was reduced in all three treatment arms. The humoral response was strongly reduced, as shown by decreased numbers of IgG-secreting cells, plasma cells and partially B cells in all treatment groups with a trend of SRL/BZ combination being most effective. Infiltration of the graft with inflammatory cells like cytotoxic T cells, T helper cells, B cells and macrophages was efficiently blocked by BZ and the SRL/BZ combination and, except for B cells, by SRL. Conclusions. BZ and SRL represent promising drugs with anti-humoral activity in the situation of an already established chronic humoral or mixed alloimmune response after renal transplantation.

Background Vascular endothelial growth factor (VEGF) is the most important and tightly regulated angiogenic cytokine in the kidney. Its activity is critical for capillary/glomerular preservation and repair, and recent studies have also demonstrated its relevance for the preservation of podocytes. Methods The present study investigated a large number (n = 153) of renal biopsies from patients with glomerulonephritis (GN) and evaluated the expression and activity of the glomerular VEGF system [VEGF, VEGF-R1, VEGF-R2 and biologically active VEGF as identified by VEGF-VEGF receptor complexes (VEGF-VEGF-R)] in parallel with markers of renal function, injury and repair. Results Whereas glomerular VEGF expression was clearly elevated, VEGF-R expression levels were widely unchanged. In parallel to the overall VEGF expression, the biological activity of VEGF on its receptors was uniformly significantly enhanced. Interestingly, the expression pattern of VEGF-R1 and VEGF-R2 significantly changed during GN where a very prominent podocytic pattern appeared, which was also detected for receptor-bound VEGF. VEGF expression and activity could be linked with indicators of renal injury such as glomerular proliferation and creatinine, respectively. Conclusions This study shows, for the first time, increased podocytic VEGF-VEGF-R binding during human GN, suggesting not only the existence of a glomerular paracrine proangiogenic, but also an autocrine role of the VEGF-VEGF-R system in diseased podocytes.

Background. Activation of the thrombospondin-1 (TSP-1)-TGF-β pathway by glucose and the relevance of TSP-1-dependent activation of TGF-β for renal matrix expansion, renal fibrosis and sclerosis have previously been demonstrated by our group in in vivo and in vitro studies. Design and methods. We investigated renal biopsies (n = 40) and clinical data (n = 30) of patients with diabetic nephropathy. Ten kidneys without evidence of renal disease served as controls. Glomerular and cortical expression of TSP-1, p-smad2/3, fibrosis and glomerular sclerosis (PAS) were assessed by immunhistochemical staining and related with clinical data. Results. Glomerular (g) and cortical (c) TSP-1 were increased during diabetic nephropathy (g: 2.62 ± 2.65; c: 4.5 ± 4.2) compared to controls (g: 0.67 ± 0.7; c: 1.5 ± 1.2). P-smad2/3 was significantly increased (g: 16.7 ± 12.9; c: 148.7 ± 92.8) compared to controls (g: 7.1 ± 3.6; c: 55 ± 25; P < 0.05). TSP-1 was coexpressed with p-smad2/3 as an indicator of TGF-β activation. TSP-1 correlated with enhanced tubulointerstitial p-smad2/3 positivity (r = 0.39 and r = 0.4, P < 0.05) and glomerular p-smad2/3 correlated with proteinuria (r = 0.35, P < 0.05). Conclusions. In summary, the present study suggests a functional activity of the TSP-1/TGF-β axis, especially in the tubulointerstitium of patients with diabetic nephropathy. The positive correlation of glomerular p-smad2/3 positivity with proteinuria further supports the importance of the TSP-1/TGF-β system as a relevant mechanism for progression of human type-2 diabetic nephropathy.

The role of thrombospondin 2 (TSP2) was investigated in an anti-glomerular basement membrane (GBM) nephritis model that compared TSP2-null mice with wild-type (WT) controls. TSP2-null mice were analyzed for kidney function, renal cortical matrix expansion, influx of inflammatory cells, proliferation, and apoptosis, as well as for capillary rarefaction after induction of anti-GBM disease. Whereas the renal cortex of normal control WT mice did not show any detectable TSP2 staining above background, TSP2 protein expression was clearly upregulated in anti-GBM disease. TSP2 deficiency led to an accelerated and enhanced inflammatory response, as indicated by the influx of CD4(+) and CD8a(+) cells and monocytes/macrophages. Glomerular fibrin deposition and a matrix-remodeling response were also observed, as indicated by collagens I and IV staining and a proliferative response within the renal interstitium. These changes were accompanied by increased matrix metalloproteinase 2 activity and enhanced alpha-smooth muscle actin staining in the TSP2-null mice. Neither a compensatory increase in TSP1 nor increased phosphorylation of Smad 2/3, an indicator for TGF-beta activity, was observed. The proliferative response of the peritubular endothelium was accelerated and enhanced, leading to a reversal of capillary rarefaction in TSP2-null mice, whereas interstitial cell death was equivalent to that in WT mice. In conclusion, the lack of the matricellular protein TSP2 in mice accelerates and enhances several responses to renal injury and reveals an important role for TSP2 as a major endogenous antiangiogenic and matrix metalloproteinase 2-regulating factor in renal disease.

The metabotropic receptor P2Y1 is necessary for full ADP-induced platelet activation and is localized on various intrinsic renal cells, including mesangial cells, podocytes, and endothelial cells. To date, nothing is known about the role of the P2Y1 receptor during inflammatory renal disease. The role of the P2Y1 receptor was investigated using 22 P2Y1 gene–deficient (−/−) and 27 wild-type (wt) mice during the time course of passive crescentic nephrotoxic glomerulonephritis. Six P2Y1 −/− and six wt mice served as undiseased controls. Renal tissues were harvested on days 1, 10, and 28 after disease induction. No renal phenotype was found in P2Y1 −/− versus wt mice. In contrast, during crescentic glomerulonephritis, approximately 50% of all wt mice died, whereas all P2Y1 −/− mice survived. Renal function as assessed by creatinine clearance measurements, glomerulosclerosis, and tubulointerstitial injury indices as well as glomerular and interstitial matrix expansion were improved significantly in P2Y1 −/− compared with wt mice. These changes were preceded by reduced glomerular and peritubular capillary rarefaction indices in P2Y1 −/− compared with wt mice. The alteration of the rates of both peritubular apoptosis and endothelial cell proliferation suggests improved capillary preservation in P2Y1 −/− mice early in disease (day 10) and an additional enhanced repair reaction in P2Y1 −/− mice at the late time point (day 28), whereas injury on day 1 seemed to be equivalent in both groups. It is concluded that loss of P2Y1 receptor function safeguards against capillary loss, fibrosis, and death by renal failure during experimental crescentic glomerulonephritis.

Background. Changes of renal nitric oxide (NO) production have been associated with glomerular hyperfiltration, vascular permeability, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. Several studies demonstrated an up- as well as downregulated expression of NO-synthases (NOS) in experimental diabetic nephropathy. It is still not yet specified whether the regulation and activity of NOS is changed in human diabetic nephropathy. Methods. Renal biopsies and clinical data of 45 patients with diabetic nephropathy and of 10 control subjects were investigated. Glomerular and cortical endothelial NOS (eNOS) and inducible NOS (iNOS) expression were assessed by immunohistochemical staining and related to clinical data such as the duration of diabetes, insulin therapy and arterial hypertension, albuminuria/proteinuria, eGFR according to the formula modification of diet in renal disease (MDRD), presence of vascular complications or diabetic retinopathy. Results. The mean age of patients at biopsy was 60.3 years and the mean duration of diabetes 12.9 years. Expression of cortical and glomerular eNOS was increased in type 2 diabetes (P < 0.05). Increased expression of glomerular and cortical eNOS correlated with more severe vascular complications (r = 0.44; P < 0.05). Glomerular eNOS was strongly increased among different degrees of proteinuria (P < 0.01). In contrast to expression levels of eNOS, the glomerular expression pattern of iNOS changed from an endothelial pattern in glomeruli with preserved morphology towards expression predominantly by inflammatory cells. Conclusions. Thus, increased eNOS expression by the renal endothelium could be demonstrated in type 2 diabetic nephropathy, whereas iNOS was unchanged but spatially differentially expressed. The eNOS expression was related to vascular lesions and the degree of proteinuria.

Several studies have implicated the angiogenic cytokine vascular endothelial growth factor (VEGF) in the development of diabetic nephropathy, but no data are available about its local activity during human disease. Glomeruli from 52 archival biopsies from type II diabetics were evaluated and compared to 10 renal biopsies without kidney disease (controls). Glomerulosclerosis, capillary rarefaction, glomerular and endothelial cell proliferation, apoptosis, VEGF expression, as well as receptor-bound VEGF indicating local VEGF activity, and phosphorylation of the signal transduction molecule Akt were investigated. Owing to substantial heterogeneity of glomerular lesions in individual biopsies, these parameters were correlated with the degree of injury in individual glomeruli rather than biopsies. Severe glomerular capillary rarefaction was linked to the degree of glomerulosclerosis. While cellular apoptosis was detected independent of the stage of injury, endothelial cell proliferation indicating capillary repair was markedly increased only in mildly/moderately injured glomeruli. In controls, VEGF was predominantly expressed in podocytes, whereas receptor-bound VEGF was confined to the glomerular endothelium. VEGF expression was increased in all diabetic glomeruli by many different cell types. In contrast, VEGF receptor activation was increased predominantly in the endothelium of only mildly injured glomeruli, but significantly decreased in more severely injured glomeruli. Diabetic nephropathy is associated with glomerular capillary rarefaction. Despite overall increased glomerular VEGF, the decreased receptor-bound VEGF on the endothelium may be an indicator of an insufficient capillary repair reaction.

Vasodilator-stimulated phosphoprotein (VASP), an actin cytoskeletal protein, is expressed in various cell types including renal cells. In vitro studies provide evidence for a role of VASP regarding platelet activation, cell adhesion, migration, and capillary formation. The in vivo role of VASP was investigated in experimental inflammatory renal disease. Kidneys of healthy VASP deficient (-/-) and wild-type (wt) mice were compared regarding morphology and functional parameters. Passive nephrotoxic nephritis was induced in 28 VASP -/- and 28 wt mice; kidneys were harvested; and tissues were analyzed by morphometric, immunohistochemical, and electron microscopic techniques on days 3, 7, 14, and 28. The time course of disease in VASP -/- mice differed substantially and biphasically from that in wt controls. Early on, VASP -/- mice demonstrated increased platelet influx associated with augmented glomerular and tubulointerstitial inflammation and sclerosis. Whereas renal disease continuously worsened up to day 28 in wt controls, renal disease in VASP -/- mice hardly progressed after day 3 as assessed by various injury indices. This long-term improvement of renal histology in VASP -/- compared with wt mice was associated with remarkable capillary preservation/regeneration up to day 28 mediated via an increased proliferative and a reduced apoptotic activity of VASP-negative peritubular endothelial cells. Despite an enhanced injury response early on, VASP -/- mice are protected from long-term progression of nephrotoxic nephritis, which is associated with improved renal endothelial cell preservation and regeneration.

Thrombospondin-1 is a major activator of TGF-β in fibrotic renal disease in the rat in vivo. Background Transforming growth factor-β (TGF-β), a profibrotic cytokine involved in many scarring processes, has to be activated extracellularly before it can bind to its receptors. Thrombospondin 1 (TSP1), a multifunctional matricellular glycoprotein, has been identified as an activator of TGF-β in in vitro systems and during mouse postnatal development in vivo. TSP1 is expressed de novo in many inflammatory disease processes, including glomerular disease. Methods In this study we investigated whether peptides specifically interfering with the activation process of TGF-β by TSP1 may be able to block activation of TGF-β in an in vivo model of mesangial proliferative glomerulonephritis. Results Continuous intravenous infusion of blocking peptide by minipumps significantly reduced expression of active TGF-β in glomeruli on day 7 of disease as indicated by immunohistochemistry, bioassay, and activation of the TGF-β signal transduction pathway, while total TGF-β expression was unchanged. Inhibition of glomerular TGF-β activation was accompanied by a decrease of glomerular extracellular matrix accumulation and proteinuria, but was without effect on mesangial cell proliferation or influx of monocytes/macrophages. Conclusion TSP1 is a major endogenous activator of TGF-β in experimental inflammatory glomerular disease. Drugs interfering with the activation of TGF-β by locally produced TSP1 may be considered as a future specific treatment of scarring kidney disease.

P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis. Background Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS. Methods The effects of PPADS on nucleotide- or fetal calf serum (FCS)-stimulated proliferation of cultured MC were measured by cell counting and [ 3 H]thymidine incorporation assay. After induction of the anti-Thy1 model, rats received injections of the P2-receptor antagonist PPADS at different doses (15, 30, 60 mg/kg BW). Proliferating mesangial and non-mesangial cells, mesangial cell activation, matrix accumulation, influx of inflammatory cells, mesangiolysis, microaneurysm formation, and renal functional parameters were assessed during anti-Thy1 disease. P2Y-mRNA and protein expression was assessed using RT-PCR and real time PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry. Results In cultured mesangial cells, PPADS inhibited nucleotide, but not FCS-stimulated proliferation in a dose-dependent manner. In the anti-Thy1 model, PPADS specifically and dose-dependently reduced early (day 3), but not late (day 8), glomerular mesangial cell proliferation as well as phenotypic activation of the mesangium and slightly matrix expansion. While no consistent effect was obtained in regard to the degree of mesangiolysis, influx of inflammatory cells, proteinuria or blood pressure, PPADS treatment increased serum creatinine and urea in anti-Thy1 rats. P2Y receptor expression (P2Y 2 and P2Y 6 ) was detected in cultured MC and isolated glomeruli, and demonstrated a transient marked increase during anti-Thy1 disease. Conclusion These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury.

Survival biopsies are frequently applied in rat kidney disease models, but several drawbacks such as surgical kidney trauma, bleeding risk and variable loss of kidney tissue are still unsolved. Therefore, we developed an easy-to-use core biopsy instrument and evaluated whether two consecutive kidney biopsies within the same kidney can be carried out in a standardized manner. On day 0, 18 Lewis rats underwent a right nephrectomy and 9 of these rats a subsequent first biopsy of the left kidney (Bx group). 9 control rats had a sham biopsy of the left kidney (Ctrl group). On day 7, a second kidney biopsy/sham biopsy was performed. On day 42, all animals were sacrificed and their kidneys were removed for histology. Biopsy cylinders contained 57±28 glomeruli per transversal section, representing an adequate sample size. PAS staining showed that the biopsy depth was limited to the renal cortex whereas surgical tissue damage was limited to the area immediately adjacent to the taken biopsy cylinder. On day 42, the reduction of functional renal mass after two biopsies was only 5.2% and no differences of body weight, blood pressure, proteinuria, serum creatinine, glomerulosclerosis, interstitial fibrosis or number of ED-1 positive macrophages were found between both groups. In summary, our apparatus offers a safe method to perform repetitive kidney biopsies with minimal trauma and sufficient sample size and quality even in experimental disease models restricted to one single kidney.

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.

Mesangial cell injury has a major role in many CKDs. Because renin-positive precursor cells give rise to mesangial cells during nephrogenesis, this study tested the hypothesis that the same phenomenon contributes to glomerular regeneration after murine experimental mesangial injury. Mesangiolysis was induced by administration of an anti-mesangial cell serum in combination with LPS. In enhanced green fluorescent protein-reporter mice with constitutively labeled renin lineage cells, the size of the enhanced green fluorescent protein-positive area in the glomerular tufts increased after mesangial injury. Furthermore, we generated a novel Tet-on inducible triple-transgenic LacZ reporter line that allowed selective labeling of renin cells along renal afferent arterioles of adult mice. Although no intraglomerular LacZ expression was detected in healthy mice, about two-thirds of the glomerular tufts became LacZ positive during the regenerative phase after severe mesangial injury. Intraglomerular renin descendant LacZ-expressing cells colocalized with mesangial cell markers α8-integrin and PDGF receptor-β but not with endothelial, podocyte, or parietal epithelial cell markers. In contrast with LacZ-positive cells in the afferent arterioles, LacZ-positive cells in the glomerular tuft did not express renin. These data demonstrate that extraglomerular renin lineage cells represent a major source of repopulating cells for reconstitution of the intraglomerular mesangium after injury.

We investigated the relationship of changes in the microvasculature to age-related structural and functional changes in the kidney to determine whether there was evidence of impaired angiogenesis and whether the loss of microvasculature could be accounted for by changes in the local production of angiogenic or antiangiogenic factors. Glomerular and peritubular capillary number, density, and endothelial cell proliferation were determined in aging (24 months; n = 9) and young (3 months; n = 8) rat kidneys and correlated with renal functional and structural changes and alterations in renal expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aging rats showed a focal decrease in both peritubular capillary (peritubular capillary staining, 5.4% +/- 1.8% versus 11.3% +/- 2.0% per 100 tubules; rarefaction index, 10.6% +/- 4.6% versus 0.6% +/- 0.1%, aging versus young rats; P < 0.05 and P: < 0.001, respectively) and glomerular capillary loops (27.3 +/- 6.9 versus 50.7 +/- 7.4/glomerulus, aging versus young rats; P < 0.001). The number of proliferating endothelial cells was decreased in aging rats compared with young rats (glomerular, 0.04 +/- 0.01 versus 0.15 +/- 0.03 positive cells/glomerular cross-section; peritubular, 0.7 +/- 0.2 versus 4.3 +/- 2.6 positive cells/mm(2); P < 0.05). In the aging kidney, VEGF expression was focally increased in the cortex compared with young rats, whereas a profound decrease was observed in the outer and inner medulla (total area of VEGF expression, 19.2% +/- 11.4% versus 39.3% +/- 7.6%; P < 0.05). Tubular VEGF expression correlated with peritubular capillary density (r(2) = 0.57; P < 0.01) and inversely correlated with tubular osteopontin (r(2) = -0.55; P < 0.05) and macrophage infiltration (r(2) = -0.64; P < 0.01). TSP-1 staining was increased in the glomeruli and tubulointerstitium of the aging rats. Glomerular TSP-1 score correlated inversely with glomerular capillary number (r(2) = -0.89; P < 0.001). Tubulointerstitial TSP-1 also correlated with percentage of positive staining of peritubular capillary (r(2) = -0.59; P < 0.001). Glomerular capillary number showed significant correlation with glomerulosclerosis score, as well as with 24-hour urinary protein excretion. Peritubular capillary density also inversely correlated with interstitial fibrosis score and urinary protein excretion. In conclusion, glomerular and peritubular capillary loss in the aging kidney correlate with alterations in VEGF and TSP-1 expression and also with the development of glomerulosclerosis and tubulointerstitial fibrosis. These findings suggest that impaired angiogenesis associated with progressive loss in renal microvasculature may have a pivotal role in age-related nephropathy.

Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo . Background Thrombospondin 1 (TSP1), a multifunctional, matricellular glycoprotein, is expressed de novo in many inflammatory disease processes, including glomerular disease. Short peptide fragments derived from the type I properdin repeats of the TSP1molecule mimic anti-angiogenic and/or transforming growth factor-β (TGF-β)–activating properties of the whole TSP1 glycoprotein. We investigated the effects of d-reverse peptides derived from the type I domain of TSP1 in experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), as well as their effects on cultured mesangial and glomerular endothelial cells. Methods Effects of TSP peptides on proliferation of mesangial or glomerular endothelial cells in culture after growth arrest or growth factor stimulation (fibroblast growth factor-2, platelet-derived growth factor-BB, 10% fetal calf serum) were measured by [ 3 H]thymidine incorporation assay. Adhesion of rat mesangial cells (MCs) to a TSP-peptide matrix was assayed using an attachment-hexosaminidase assay. TSP peptides were intraperitoneally injected daily in rats that had received an intravenous injection of polyclonal anti-Thy1 antibody to induce mesangial proliferative glomerulonephritis. On biopsies from days 2, 5, and 8 of anti-Thy1 disease, mesangial and glomerular endothelial proliferation, matrix expansion, mesangial activation, and microaneurysm formation were assessed. Functional parameters such as blood pressure and proteinuria were also measured. Results An 18-amino acid peptide (type I peptide) with anti-angiogenic and TGF-β–activating sequences decreased mesangial and glomerular endothelial cell proliferation in vitro and in vivo and reduced microaneurysm formation and proteinuria in experimental glomerulonephritis. Analogues lacking the TGF-β–activating sequence mimicked most effects of the type I peptide. The mechanism of action of these peptides may include antagonism of fibroblast growth factor-2 and alteration of MC adhesion. The TGF-β–activating sequence alone did not have significant effects on mesangial or glomerular endothelial cells in vitro or in experimental kidney disease in vivo. Conclusion Peptides from TSP1 may be promising therapeutics in treating glomerular disease with mesangial and endothelial cell injury.

In this paper we provide an overview of the cellular changes that accompany progressive glomerulosclerosis and tubulointerstitial fibrosis. Emphasis is placed on the role of platelet-derived growth factor (PDGF) in the proliferation of the mesangial cell and interstitial fibroblast. PDGF also stimulates the synthesis and expression of a protein called secreted protein acidic and rich in cysteine (SPARC) and of thrombospondin 1 (TSP1). TSP1 may have an important role in the conversion of transforming growth factor (TGF)-β from its latent to active form. In turn, TGF-β appears to have a major role in mediating the extracellular matrix deposition that occurs with progressive glomerular and interstitial scarring.

Thrombospondin 1 precedes and predicts the development of tubulointerstitial fibrosis in glomerular disease in the rat. Tubulointerstitial fibrosis is one of the most important histologic features that predicts progression in kidney disease. Thrombospondin 1 is an extracellular matrix protein that can activate latent TGF- β , a cytokine implicated in the pathogenesis of tubulointerstitial fibrosis. We examined the expression of thrombospondin 1 in several animal models of glomerulonephritis (anti-Thy1 model, aminonucleoside nephrosis, passive Heymann nephritis) that are associated with tubulointerstitial disease. Thrombospondin 1 mRNA and protein were transiently increased in tubular cells, myofibroblasts and some macrophages in areas of tubulointerstitial injury. Thrombospondin 1 expression always preceded the development of tubulointerstitial fibrosis, and correlated quantitatively and spatially with the later development of interstitial fibrosis. Thrombospondin 1 expression predicted the severity of tubulointerstitial fibrosis better than the degree of macrophage or myofibroblast accumulation. Thrombospondin 1 expression was associated with increased expression and activation of TGF- β 1 and decreased expression of LAP-TGF- β in areas of tubulointerstitial injury. We conclude that thrombospondin 1 is an early marker predicting the development of tubulointerstitial kidney disease. De novo expression of thrombospondin 1 is associated and colocalized with increased expression of TGF- β 1 and decreased expression of LAP-TGF- β during the development of tubulointerstitial disease in vivo . These data are consistent with the possibility that thrombospondin 1 may be an endogenous activator of TGF- β .

The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury. Background Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. Methods The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. Results Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. Conclusions Ezrin likely has a role in the cytoskeletal organization, such as reassembling of actin filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.

Summary: Mesangial injury accompanies many glomerular diseases and diabetic nephropathy. Whereas in some diseases the injury resolves, in other cases it may progress to sclerosis. We have studied mesangial injury in the Thy 1 model in rats. In this model, the injection of anti-Thy 1 antibody results in complement-mediated killing of

Cyclin kinase inhibitors are increased during experimental membranous nephropathy: Potential role in limiting glomerular epithelial cell proliferation in vivo. The inadequate proliferative response of the visceral glomerular epithelial cell (GEC) following injury in vivo may contribute to the development of progressive glomerulosclerosis in many forms of glomerular disease. Cell proliferation is ultimately controlled by cell-cycle regulatory proteins, including cyclins that bind to cyclin dependent kinases (CDK), and the active complex formed is necessary for progression through the cell-cycle. By inhibiting cyclin-CDK complexes, cyclin kinase inhibitors arrest the cell-cycle and prevent proliferation. To determine the mechanisms that may be responsible for the lack of GEC proliferation in vivo, we examined GEC expression of specific cell-cycle proteins in normal rats and in the passive Heymann nephritis (PHN) model of membranous nephropathy, where the GEC are the target of complement-mediated injury. Following antibody deposition and complement activation there was a marked up-regulation in the cyclin kinase inhibitors p21 and p27 in rats with PHN. By associating with cyclin A-CDK2 complexes, p21 and p27 limited the kinase activity of CDK2. Giving bFGF to rats with PHN was associated with an increase in GEC mitosis and ploidy and a decrease in expression of p21, but not CDK2 or p27. Furthermore, apoptosis was not present in PHN, but was increased in rats given bFGF. In conclusion, this study shows that the low proliferative capacity of the GEC in vivo in response to immune injury may be due to an increase in the expression of specific cyclin kinase inhibitors. The increase in mitosis in PHN rats given bFGF may be due to a decrease in p21. Thus, changes in cell cycle regulatory proteins may regulate the response of GEC to injury and underlie the development of progressive glomerulosclerosis in diseases of the GEC.

Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC-antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection.

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during antiglomerular basement membrane (GBM) nephritis. Therefore, p38 alpha conditional knockout mice (MxCre-p38 alpha(Delta/Delta)) were used to examine the role of p38 alpha in anti-GBM induced nephritis. Both wild type and MxCre-p38 alpha(Delta/Delta) mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38 alpha(Delta/Delta) mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro-and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38 alpha(Delta/Delta) mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38 alpha(Delta/Delta) mice. In conclusion, p38 alpha is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs.

SPARC is expressed in renal interstitial fibrosis and in renal vascular injury. Tubulointerstitial inflammation and fibrosis are critical determinants for renal function and prognosis in a variety of human nephropathies. Yet, the pathophysiology of the injury remains obscure. We investigated the expression of SPARC (secreted protein acidic and rich in cysteine) by immunohistochemistry and in situ hybridization in experimental models characterized by tubulointerstitial fibrosis and matrix expansion in rats. SPARC is a secreted glycoprotein that has been demonstrated to affect cellular interaction with matrix proteins, modulate cell proliferation, bind to and/or inhibit growth factors such as PDGF and bFGF, and regulate angiogenesis. Interstitial expression of SPARC was most prominent in passive Heyman nephritis (PHN), chronic cyclosporine A (CsA) nephropathy, and the remnant kidney model and, to a lesser extent, in angiotensin II (Ang II)-infused animals. SPARC protein and mRNA were substantially increased at sites of tubulointerstitial fibrosis/matrix expansion. In the PHN model, SPARC protein was expressed by interstitial fibroblasts that also produced α-smooth muscle actin (“myofibroblasts”) and correlated both temporally (r = 0.97) and spatially with sites of type I collagen deposition. Interstitial cell proliferation preceded the development of interstitial fibrosis, and maximal SPARC expression (d15) coincided with the initial decline in interstitial proliferation. In the Ang II-infusion model, which is characterized by arteriolopathy and tubulointerstitial injury, an increase in SPARC protein and mRNA was also seen in injured blood vessels. SPARC was shown to be expressed by vascular smooth muscle cells and also by cells in the adventitia of hypertrophied arteries. In summary, SPARC was transiently expressed by interstitial fibroblasts at sites of tubulointerstitial injury and fibrosis, and by smooth muscle cells and cells in the adventitia of injured arteries in the Ang II-model. In addition to its proposed role in extracellular matrix deposition, the antiproliferative properties of SPARC might contribute to the resolution of interstitial fibroblast proliferation in the PHN model.

Changes in cell cycle protein expression during experimental mesangial proliferative glomerulonephritis. A characteristic response to mesangial cell injury is proliferation, which is closely linked to mesangial matrix accumulation and the progression of glomerular disease. Cell proliferation in non-renal cells in vitro is regulated at the level of the cell-cycle by specific cyclins and their catalytic partners, cyclin dependent kinases (CDK). Cyclin kinase inhibitors (CKI) prevent proliferation by inhibiting cell-cycle progression. However, the expression of cell-cycle regulatory proteins in the kidney and in renal disease is unknown. To determine this we studied the expression of cell-cycle proteins in vivo in normal rats and rats with experimental mesangial proliferative glomerulonephritis (Thy1 model). Normal quiescent rat glomeruli have a differential expression for CKI's, where p27 Kip1 is highly expressed, and the levels for p21 (Cip1, Waf1, Sdi1, Cap20) (p21) are low. The onset of mesangial cell proliferation in Thy1 glomerulonephritis is associated with a reduction in p27 Kip1 levels when mesangial cell proliferation is maximal. Mesangial cell proliferation in vivo is also associated with an increase in glomerular expression of cyclin A, and an increase in expression and activity for CDK2. The resolution of mesangial cell proliferation was associated with a return to baseline levels for p27 Kip1 , while the expression for p21 increased substantially. Furthermore, mesangial cell p21 expression was maintained following the resolution of proliferation. These results provide evidence for a complex interplay of cell-cycle regulatory proteins during the glomerular response to injury in vivo . The marked increase in CDK2 expression during mesangial cell proliferation and the sustained increase in p21 expression following the resolution of mesangial cell proliferation suggests that the in vivo expression of certain cell-cycle proteins may differ from that described in non-renal cells in vitro .