Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

CD137-selektierte leukämiereaktive T-Zellen gesunder Spender zur adoptiven Immuntherapie stammzelltransplantierter Leukämiepatienten = CD137-selected leukaemia-reactive T Cells from healthy donors for adoptive immunotherapy in stem-cell-transplanted leukaemia-patients

Bei stammzelltransplantierten Patienten, die ein Rezidiv ihrer Leukämie erleiden, kann eine Donor-Lymphozyten-Infusion (DLI) dauerhafte vollständige Leukämieremissionen induzieren. T-Zellen in der DLI vermitteln sowohl den potentiell kurativen Graft-versus-Leukaemia (GVL) Effekt, als auch die potentiell lebensbedrohliche Graft-versus-Host Disease (GVHD). Hingegen könnte die Infusion von leukämiereaktiven T-Zellen einen selektiven GVL Effekt und einen Langzeitschutz vor Rezidiven durch eine spezifisch gegen die Leukämie gerichtete Immunantwort und Immunität vermitteln. Unsere Arbeitsgruppe hat Protokolle zur in vitro Generierung leukämiereaktiver T-Zellen entwickelt, die hohe zytotoxische Aktivität gegen akute myeloische Leukämie-Blasten (AML) bei minimaler Reaktion auf mögliche GVHD Zielstrukturen zeigen. Für die klinische Anwendung sind diese Protokolle jedoch zu aufwändig, wobei vor allem eine erhebliche Verkürzung der Kulturzeit auf wenige Wochen erforderlich ist. Diese Verkürzung der in vitro Kulturzeit könnte das Wachstum von T-Zellen vom central memory oder frühen effector memory Phänotyp fördern, für die eine bessere in vivo Effektorfunktion und längere Persistenz im Rezipienten verglichen mit T-Zellen aus Langzeitkultur gezeigt werden konnte. Der Aktivierungsmarker und Kostimulations-Rezeptor CD137 kann zur Erkennung und Isolation antigenspezifischer T-Zellen genutzt werden, ohne dass dafür das von den T-Zellen erkannte Peptidepitop bekannt sein muss. Eine CD137-vermittelte Anreicherung mit Hilfe von clinical grade Materialien könnte verwendet werden, um DLI-Produkte mit leukämiespezifischen T-Zellen herzustellen, die sich sowohl durch eine effizientere T-Zell Generierung durch in vitro Selektion und Kostimulation, als auch durch eine verbesserte Spezifität des T-Zell-Produkts auszeichnen. Lymphozyten-Leukämie Cokulturen (mixed lymphocyte leukaemia cultures) wurden mit CD8 T-Zellen gesunder Spender und HLA-identischen oder einzel-HLA-mismatch AML-Blasten angesetzt und wöchentlich restimuliert. Nach zwei Wochen wurden die T-Zellen 12 Stunden nach Restimulation über den Marker CD137 positiv isoliert und anschließend separat weiterkultiviert. Die isolierten Fraktionen und unseparierten Kontrollen wurden im ELISPOT-Assay und im Chrom-Freisetzungstest an Tag 5 nach der Restimulation getestet. Es wurden keine konsistent nachweisbaren Vorteile im Hinblick auf Wachstum und Funktion der isolierten CD137-positiv Fraktion im Vergleich zur unseparierten Kontrolle gefunden. Verschiedene Isolationsmethoden, Patient-Spender-Systeme, Methoden zur Restimulation, Temperaturbedingungen, Zytokinkombinationen und Methoden der Zytokinzugabe sowie zusätzliche Feeder-Zellen oder AML-Blasten konnten Wachstum, funktionelle Daten und die deutlichen Zellverluste während der Isolation nicht entscheidend beeinflussen. Vitalfärbungen zeigten, dass aktivierungsinduzierter Zelltod CD137-positiver Zellen zu diesen Ergebnissen beitragen könnte. Im Gegensatz zur Stimulation mit AML-Blasten wurden erfolgreiche CD137-Anreicherungen für peptidstimulierte T-Zellen publiziert. Unterschiedliche CD137-Expressionskinetiken, aktivierungsinduzierter Zelltod und regulatorische T-Zellen sind mögliche Faktoren aufgrund derer die CD137-Anreicherung in diesem spezifischen Kontext ungeeinet sein könnte. Der stimulatorische Effekt eines CD137-Signals auf AML-reaktive CD8 T-Zellen wurde mit Hilfe von CD3/CD28 und CD3/CD28/CD137 Antikörper-beschichteten magnetischen beads untersucht. Für Nierenzellkarzinom-reaktive T-Zellen war die Stimulation mit CD3/CD28/CD137 beads genauso effektiv wie mit Tumorzellen und effektiver als mit CD3/CD28 beads. Beide Arten von beads waren für eine Stimulation während der ersten Wochen der Zellkultur geeignet, sodass ein zusätzliches CD137-Signal für die länger anhaltende Expansion tumorreaktiver T-Zellen zur klinischen Anwendung nützlich sein könnte. Die bead-Expansion veränderte die IFN-Sekretion im ELISPOT nicht, aber verursachte eine mäßige Verschlechterung der Zytotoxizität im Chrom-Freisetzungstest. Im Gegensatz dazu zeigten bei AML-reaktiven T-Zellen beide Arten von beads einen nicht apoptosevermittelten, dosisabhängigen zellschädigenden Effekt, der zu einer raschen Abnahme der Zellzahl in Kulturen mit beads führte. Unerwünschte Effekte auf die T-Zell-Funktionalität durch bead-Stimulation sind in der Literatur beschrieben, dennoch gibt es aktuell keine Veröffentlichungen, die eine fundierte Erklärung für den Effekt auf AML-reaktive T-Zellen bieten könnten. Abgesehen von Literaturdaten, die darauf hindeuten, dass CD137 ein vielversprechendes Kandidatenmolekül für die Anreicherung und Expansion von AML-reaktiven T-Zellen sein könnte, zeigen die eigenen Daten sowohl zur CD137-Isolation als auch zur bead-Stimulation, dass für diese spezielle Anwendung CD137 ein ungeeigneter Aktivierungsmarker und Kostimulations-Ligand ist.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Sirolimus and everolimus reduce albumin endocytosis in proximal tubule cells via an angiotensin II-dependent pathway

The proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) often induce proteinuria due to glomerular but also tubular dysfunction in transplant patients. The beneficial effect of angiotensin converting enzyme inhibitors (ACE-I) and angiotensin II (Ang II) type 1 receptor blockers (ARB) has been reported.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

Urinary soluble HLA-DR is a potential biomarker for acute renal transplant rejection

BACKGROUND.: Urine is a potentially rich source of biomarkers for monitoring kidney dysfunction. In this study, we have investigated the potential of soluble human leukocyte antigen (sHLA)-DR in the urine for noninvasive monitoring of renal transplant patients. METHODS.: Urinary soluble HLA-DR levels were measured by sandwich enzyme-linked immunosorbent assay in 103 patients with renal diseases or after renal transplantation. sHLA-DR in urine was characterized by Western blotting and mass spectrometry. RESULTS.: Acute graft rejection was associated with a significantly elevated level of urinary sHLA-DR (P<0.0001), compared with recipients with stable graft function or healthy individuals. A receiver operating characteristic curve analysis showed the area under the curve to be 0.88 (P<0.001). At a selected threshold, the sensitivity was 80% and specificity was 98% for detection of acute renal transplant rejection. sHLA-DR was not exosomally associated and was of lower molecular weight compared with the HLA-DR expressed as heterodimer on the plasma membrane of antigen-presenting cells. CONCLUSIONS.: sHLA-DR excreted into urine is a promising indicator of renal transplant rejection.

Dialysis and Renal Transplantation in HIV-Infected Patients: a European Survey

OBJECTIVES:: To determine prevalence and characteristics of end-stage renal diseases (ESRD) [dialysis and renal transplantation (RT)] among European HIV-infected patients. METHODS:: Cross-sectional multicenter survey of EuroSIDA clinics during 2008. RESULTS:: Prevalence of ESRD was 0.5%. Of 122 patients with ESRD 96 were on dialysis and 26 had received a RT. Median age was 47 years, 73% were males and 43% were black. Median duration of HIV infection was 11 years. Thirty-three percent had prior AIDS; 91% were receiving antiretrovirals; and 88% had undetectable viral load. Median CD4T-cell count was 341 cells per cubic millimetre; 20.5% had hepatitis C coinfection. Most frequent causes of ESRD were HIV-associated nephropathy (46%) and other glomerulonephritis (28%). Hemodialysis (93%) was the most common dialysis modality; 34% of patients were on the RT waiting list. A poor HIV control was the reason for exclusion from RT waiting list in 22.4% of cases. All the RT recipients were all alive at the time of the survey. Acute rejection was reported in 8 patients (30%). Functioning graft was present in 21 (80%). CONCLUSIONS:: This is the first multinational cross-sectional study of ESRD among European HIV population. Low prevalence of ESRD was found. Two-thirds of patients were excluded from RT for non-HIV/AIDS-related pathologies. Most patients had a functioning graft despite a high acute rejection rate.

Persistent decline in estimated but not measured glomerular filtration rate on tenofovir may reflect tubular rather than glomerular toxicity

Tenofovir disoproxil fumarate (TDF) has been associated with proximal renal tubulopathy and reduction in estimated glomerular filtration rate (eGFR), without accounting for the tubular secretion of creatinine.

β-Caryophyllene ameliorates cisplatin-induced nephrotoxicity in a cannabinoid 2 receptor-dependent manner

(E)-β-caryophyllene (BCP) is a natural sesquiterpene found in many essential oils of spice (best known for contributing to the spiciness of black pepper) and food plants with recognized anti-inflammatory properties. Recently it was shown that BCP is a natural agonist of endogenous cannabinoid 2 (CB(2)) receptors, which are expressed in immune cells and mediate anti-inflammatory effects. In this study we aimed to test the effects of BCP in a clinically relevant murine model of nephropathy (induced by the widely used antineoplastic drug cisplatin) in which the tubular injury is largely dependent on inflammation and oxidative/nitrative stress. β-caryophyllene dose-dependently ameliorated cisplatin-induced kidney dysfunction, morphological damage, and renal inflammatory response (chemokines MCP-1 and MIP-2, cytokines TNF-α and IL-1β, adhesion molecule ICAM-1, and neutrophil and macrophage infiltration). It also markedly mitigated oxidative/nitrative stress (NOX-2 and NOX-4 expression, 4-HNE and 3-NT content) and cell death. The protective effects of BCP against biochemical and histological markers of nephropathy were absent in CB(2) knockout mice. Thus, BCP may be an excellent therapeutic agent to prevent cisplatin-induced nephrotoxicity through a CB(2) receptor-dependent pathway. Given the excellent safety profile of BCP in humans it has tremendous therapeutic potential in a multitude of diseases associated with inflammation and oxidative stress.

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle's loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl--dependent HCO3- secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1-/- mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1-/- mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO3- secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.

Involvement of drug-specific T cells in acute drug-induced interstitial nephritis

Drug-induced interstitial nephritis can be caused by a plethora of drugs and is characterized by a sudden impairment of renal function, mild proteinuria, and sterile pyuria. For investigation of the possible pathomechanism of this disease, drug-specific T cells were analyzed, their function was characterized, and these in vitro findings were correlated to histopathologic changes that were observed in kidney biopsy specimens. Peripheral blood mononuclear cells from three patients showed a proliferative response to only one of the administered drugs, namely flucloxacillin, penicillin G, and disulfiram, respectively. The in vitro analysis of the flucloxacillin-reactive cells showed an oligoclonal immune response with an outgrowth of T cells bearing the T cell receptor Vbeta9 and Vbeta21.3. Moreover, flucloxacillin-specific T cell clones could be generated from peripheral blood, they expressed CD4 and the alphabeta-T cell receptor, and showed a heterogeneous cytokine secretion pattern with no clear commitment to either a Th1- or Th2-type response. The immunohistochemistry of kidney biopsies of these patients revealed cell infiltrations that consisted mostly of T cells (CD4+ and/or CD8+). An augmented presence of IL-5, eosinophils, neutrophils, CD68+ cells, and IL-12 was observed. In agreement with negative cytotoxicity assays, no cytotoxicity-related molecules such as Fas and perforin were detected by immunohistochemistry. The data indicate that drug-specific T cells are activated locally and orchestrate a local inflammation via secretion of various cytokines, the type of which depends on the cytokine pattern secreted and which probably is responsible for the renal damage.

Effect of donor-specific transfusions on the outcome of renal allografts in the cyclosporine era

Despite the introduction of new immunosuppressive agents, a steady decline of functioning renal allografts after living donation is observed. Thus nonpharmacological strategies to prevent graft loss have to be reconsidered, including donor-specific transfusions (DST). We introduced a cyclosporine-based DST protocol for renal allograft recipients from living-related/unrelated donation. From 1993 to 2003, 200 ml of whole blood, or the respective mononuclear cells from the potential living donor were administered twice to all of our 61 recipient candidates. The transplanted subjects were compared with three groups of patients without DST from the Collaborative Transplant Study (Heidelberg, Germany) during a 6-year period. Six patients were sensitized without delay for a subsequent cadaveric kidney. DST patients had less often treatment for rejection and graft survival was superior compared with subjects from the other Swiss transplant centers (n = 513) or from Western Europe (n = 7024). To diminish the probability that superior results reflect patient selection rather than effects of DST, a 'matched-pair' analysis controlling for relevant factors of transplant outcome was performed. Again, this analysis indicated that recipients with DST had better outcome. Thus, our observation suggests that DST improve the outcome of living kidney transplants even when modern immunosuppressive drugs are prescribed.

[Bleeding complication due to accumulation of low-molecular-weight heparin in a patient with renal insufficiency]

We report of a 71-year-old woman with a history of chronic analgesic nephropathy, who underwent coronary angiography. Because of anterior ventricular aneurysm, anticoagulation with nadroparine was installed. Continued ACE-inhibitor and ASA with additional intravenous contrast substance lead to acute tubular necrosis with rapid decline of the renal function. Due to accumulation of the low molecular weight heparin, the patient developed an extensive retroperitoneal haematoma with circulatory shock and temporary anuric kidney failure. Low molecular weight heparins are commonly used during percutaneous coronary interventions. They are as safe and efficient compared to unfractioned heparin. But due to their renal elimination, they have to be monitored by measuring anti-factor Xa-activity if creatinine-clearance is <30 ml/min.

Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Sirolimus ameliorates the enhanced expression of metalloproteinases in a rat model of autosomal dominant polycystic kidney disease

BACKGROUND: Remodelling of matrix and tubular basement membranes (TBM) is a characteristic of polycystic kidney disease. We hypothesized that matrix and TBM degradation by metalloproteinases (MMPs) could promote cyst formation. We therefore investigated the renal expression of MMPs in the Han:SPRD rat model of autosomal dominant polycystic kidney disease (ADPKD) and examined the effect of sirolimus treatment on MMPs. METHODS: 5-week-old male heterozygous (Cy/+) and wild-type normal (+/+) rats were treated with sirolimus (2 mg/kg/day) through drinking water for 3 months. RESULTS: The mRNA and protein levels of MMP-2 and MMP-14 were markedly increased in the kidneys of heterozygous Cy/+ animals compared to wild-type +/+ as shown by RT-PCR and Western blot analyses for MMP-2 and MMP-14, and by zymography for MMP-2. Strong MMP-2 expression was detected by immunoperoxidase staining in cystic epithelial cells that also displayed an altered, thickened TBM. Tissue inhibitor of metalloproteinases-2 (TIMP-2) expression was not changed in Cy/+ kidneys. Sirolimus treatment leads to decreased protein expression of MMP-2 and MMP-14 in Cy/+, whereas MMP-2 and MMP-14 mRNA levels and TIMP-2 protein levels were not affected by sirolimus. CONCLUSION: In summary, in kidneys of the Han:SPRD rat model of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with a marked improvement of MMP-2 and MMP-14 overexpression, and this correlated also with less matrix and TBM alterations and milder cystic disease.