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OBJECTIVE ABO mismatch between donor and recipient occurs in 40% of allogeneic hematopoietic stem cell transplantations (HCT). Different strategies have been described to reduce isohemagglutinins (IHA) before HCT. We describe the effect of selective ABO immunoadsorption (ABO IA) on erythrocyte transfusion rate and the development of post-transplant pure red cell aplasia (ptPRCA). METHODS 63 patients with major ABO incompatibility were retrospectively analyzed. Nine patients with major ABO incompatibility and high-IHA titer were treated by ABO IA before HCT. We analyzed the need for transfusion and the occurrence of ptPRCA. We compared the outcome with patients treated by other methods to reduce IHA. RESULTS In all nine patients treated by ABO IA, IHA decreased in a median four times. PtPRCA occurred in one patient. The median number of transfusions was 8 (range: 0-36) between d0 and d100. In 25 patients with high-IHA titer without treatment or treated by other methods to reduce IHA, the need for transfusions was comparable. No difference in the incidence of ptPRCA was observed. CONCLUSIONS Selective ABO IA is a feasible, safe, and effective method to reduce IHA before HCT in major ABO incompatibility. No effect on transfusion rate or ptPRCA compared to other strategies could be observed.

Medicine 100(10), e24893 (2021). doi:10.1097/MD.0000000000024893, Published by Lippincott Williams & Wilkins, Baltimore, Md.

Mit rund einem Drittel aller Infektionen sind Harnwegsinfekte (HWI) die haufigste Infektionskrankheit von Alten- und Pflegeheimbewohnern. Auch asymptomatische Bakteriurien (ASB) finden sich vermehrt und werden vielfach falsch eingeschatzt. Insbesondere bei Dauerkathetertragern losen sie nicht indizierte Antibiotikatherapien aus. Eine ASB stellt keinen Risikofaktor fur einen symptomatischen HWI dar, Antibiotikagaben fuhren v. a. zu unerwunschten Arzneimittelwirkungen und Resistenzentstehung. Ein Screening fur ASB sollte daher weder innerhalb noch auserhalb von Pflegeheimen erfolgen. Akute Verschlechterungen des mentalen Status sind ebenfalls haufige Trigger einer nicht indizierten Antibiotikagabe, insbesondere bei nicht anamnestizierbaren Patienten. Treten Fieber, Dysurie, neue oder zunehmende Algurie, Strangurie, Pollakisurie oder Inkontinenz auf, sollte ein Urinteststreifen auf Leukozyten und Nitrit, je nach Ergebnis mit anschliesender Kultur, erfolgen. Bei der Wahl des Antibiotikums ist auf eventuelle Komorbiditat, Polypharmazie und die nachlassende Metabolisierungskapazitat im Alter (z. B. abnehmende geschatzte glomerulare Filtrationsrate, eGFR) zu achten. Die Kolonisierung mit multiresistenten Erregern nimmt weiter zu, wunschenswert ist daher die Implementierung von Antibiotic-Stewardship-Programmen fur Pflegeeinrichtungen.

Background Hereditary deficiency of adenine phosphoribosyltransferase causes 2,8-dihydroxyadenine (2,8-DHA) nephropathy, a rare condition characterized by formation of 2,8-DHA crystals within renal tubules. Clinical relevance of rodent models of 2,8-DHA crystal nephropathy induced by excessive adenine intake is unknown. Methods Using animal models and patient kidney biopsies, we assessed the pathogenic sequelae of 2,8-DHA crystal-induced kidney damage. We also used knockout mice to investigate the role of TNF receptors 1 and 2 (TNFR1 and TNFR2), CD44, or alpha2-HS glycoprotein (AHSG), all of which are involved in the pathogenesis of other types of crystal-induced nephropathies. Results Adenine-enriched diet in mice induced 2,8-DHA nephropathy, leading to progressive kidney disease, characterized by crystal deposits, tubular injury, inflammation, and fibrosis. Kidney injury depended on crystal size. The smallest crystals were endocytosed by tubular epithelial cells. Crystals of variable size were excreted in urine. Large crystals obstructed whole tubules. Medium-sized crystals induced a particular reparative process that we term extratubulation. In this process, tubular cells, in coordination with macrophages, overgrew and translocated crystals into the interstitium, restoring the tubular luminal patency; this was followed by degradation of interstitial crystals by granulomatous inflammation. Patients with adenine phosphoribosyltransferase deficiency showed similar histopathological findings regarding crystal morphology, crystal clearance, and renal injury. In mice, deletion of Tnfr1 significantly reduced tubular CD44 and annexin two expression, as well as inflammation, thereby ameliorating the disease course. In contrast, genetic deletion of Tnfr2, Cd44, or Ahsg had no effect on the manifestations of 2,8-DHA nephropathy. Conclusions Rodent models of the cellular and molecular mechanisms of 2,8-DHA nephropathy and crystal clearance have clinical relevance and offer insight into potential future targets for therapeutic interventions.

In Deutschland sind laut Statistischem Bundesamt rund 21 % der Bevolkerung alter als 65 Jahre. Eine stetig zunehmende Zahl dieser Menschen wird zum Nephrologen uberwiesen, beim Vorliegen einer entsprechenden Indikation oft mit konsekutiver Nierenbiopsie. Auch im Alter finden sich bioptisch haufig behandelbare Erkrankungen, welche die Frage nach einer spezifischen Therapie aufwerfen, die nur aufgrund des Alters dem Patienten nicht pauschal versagt werden sollte. Die aktuelle KDIGO-Leitlinie zur Behandlung von Glomerulonephritiden unterscheidet in Empfehlungen fur Kinder und Erwachsene, geht jedoch nur auserst selten direkt auf Altere ein. Der Grund hierfur ist, dass die meisten randomisierten, kontrollierten Studien (moglicherweise wegen der geringeren Komorbiditaten) mit Jungeren durchgefuhrt wurden und die Ergebnisse nur auf die Alteren extrapoliert werden konnen. Zusatzlich sind Glomerulonephritiden seltene Erkrankungen – dementsprechend schwer ist es, ausreichend hohe Patientenzahlen in einem definierten Alter zu rekrutieren. In diesem Artikel wird daher auf allgemeine Aspekte des Alterns eingegangen, die sowohl fur die supportive als auch die spezifische Therapie von Glomerulonephritiden relevant sind. Die Therapieentscheidung sollte bei Alteren individualisiert erfolgen, unter Berucksichtigung des allgemeinen Gesundheitszustandes des Patienten, seiner kognitiven Fahigkeiten, relevanten Begleiterkrankungen, theoretischen Lebenserwartung und seiner personlichen Vorlieben nach grundlicher Aufklarung, Umsetzbarkeit und Symptomkontrolle rucken dabei starker in den Vordergrund.

Chronic kidney disease affects significant parts of the general population, and novel treatment strategies are warranted. In this issue, Lazzeri et al.1 explore the use of exfoliated cells in the urine of children affected by proteinuric kidney diseases for diagnostic and therapeutic purposes (from patient-to-dish-to-patient). The authors investigate whether the isolated cells represent putative renal progenitor cells (RPCs) and test whether they are similar to a previously isolated putative progenitor population. They find that both cell populations ameliorate doxorubicin-induced nephritis in severe combined immunodeficiency mice. The controversial identity of the isolated cells and their potential use for diagnostic purposes or stem cells therapy are discussed. Chronic kidney disease affects an estimated 5% of the general population. It represents a major risk factor for cardiovascular disease and increased mortality at least as potent as smoking or arterial hypertension. Glomerular diseases are still the most common causes of ESRD. It is time to translate recent advances in our understanding of glomerular diseases into novel improved diagnostic and therapeutic strategies. Several approaches can be used. One is to search for unique intrinsic cells depending on specific signaling pathways, which can be manipulated by specific pharmacologic interventions in situ/in vivo. Alternatively, renal cell populations can be isolated to then be used for diagnostic purposes. Moreover, isolated cells can be expanded in culture and/or manipulated to then be returned back into the diseased organism (i.e., from patient to dish to patient). In this issue of JASN, this latter strategy was explored by Lazzeri et al.1 The authors established cultures of rare exfoliated cells from the urine of children affected by different proteinuric disorders of the kidney. The authors noted that cultured cells expressed a certain combination of markers (CD133+, CD24+, CD106+ [VCAM–1], and uroplakin III negative). In addition, cells coexpressing this combination of markers showed a higher proliferative index and expressed RNA transcripts similar to previously isolated cultures of adult parietal epithelial multipotent progenitor cells (APEMPs). The authors propose that the cultured cells represent the previously postulated fixed intrinsic population of progenitor cells (urinary renal progenitor cells [u-RPCs]).2 To substantiate this, the authors repeated an experiment where doxorubicin-induced renal disease in immunodeficient severe combined immunodeficiency mice was treated by intravenous injection of human APEMPs, u-RPCs, or cultured cells expressing other markers.2 Only the APEMPs and u-RPCs engrafted into the kidney and ameliorated proteinuria. In addition, the u-RPCs engrafted to regenerate podocytes and proximal tubule cells. Finally, the authors explored the tool of cultured u-RPCs for personalized investigations of genetic kidney disorders. They found that u-RPCs from patients with homozygous podocin mutations expressed lower levels of podocin. In a patient with a mutated LMX1B gene, authors found that filamentous actin distribution was altered in cultured u-RPCs. The authors concluded that u-RPCs represent an innovative tool to investigate genetic kidney disorders in more detail.

The current study is based on the hypothesis of mesenchymal stromal cells (MSCs) contributing to soft-tissue calcification and ectopic osteogenesis in chronic kidney disease (CKD). Rat MSCs were transplanted intraperitoneally in an established three-dimensional collagen-based model in healthy control animals and two rat models of CKD and vascular calcification: (1) 5/6 nephrectomy + high phosphorus diet; and (2) adenine nephropathy. As internal controls, collagen gels without MSCs were transplanted in the same animals. After 4 and 8 weeks, MSCs were still detectable and proliferating in the collagen gels (fluorescence-activated cell sorting [FACS] analysis and confocal microscopy after fluorescence labeling of the cells). Aortas and MSC-containing collagen gels in CKD animals showed distinct similarities in calcification (micro-computed tomography [µCT], energy-dispersive X-ray [EDX] analysis, calcium content), induction of osteogenic markers, (ie, bone morphogenic protein 2 [BMP-2], Runt related transcription factor 2 [Runx2], alkaline phosphatase [ALP]), upregulation of the osteocytic marker sclerostin and extracellular matrix remodeling with increased expression of osteopontin, collagen I/III/IV, fibronectin, and laminin. Calcification, osteogenesis, and matrix remodeling were never observed in healthy control animals and non-MSC-containing collagen gels in all groups. Paul Karl Horan 26 (PKH-26)-labeled, 3G5-positive MSCs expressed Runx2 and sclerostin in CKD animals whereas PKH-26-negative migrated cells did not express osteogenic markers. In conclusion, heterotopically implanted MSCs undergo osteogenic differentiation in rat models of CKD-induced vascular calcification, supporting our hypothesis of MSCs as possible players in heterotopic calcification processes of CKD patients.

Mesangioproliferative glomerulonephritis is the most common nephritis worldwide. We examined the effects of low- and high-dose telmisartan, an angiotensin II receptor blocker, in rats with progressive anti-Thy1.1 mesangioproliferative glomerulonephritis in a clinically relevant situation of established renal damage. Uninephrectomized nephritic rats were randomized on day 28 to remain untreated (control treatment; CT), or to receive low- (0.1 mg/kg/day, LT) or high-dose telmisartan (10 mg/kg/day, HT), hydrochlorothiazide + hydralazine (8 + 32 mg/kg/day, HCT + H), or atenolol (100 mg/kg/day, AT). CT and LT rats were hypertensive, whereas HT, HCT + H and AT treatment normalized blood pressures. On day 131, despite similar blood lowering effects, only HT, but not AT or HCT + H, prevented loss of renal function and reduced proteinuria compared to CT. Only HT potently ameliorated glomerulosclerosis, tubulointerstitial damage, cortical matrix deposition, podocyte damage and macrophage infiltration. HT reduced cortical expression of platelet derived growth factor receptor-α and -β as well as transforming growth factor-β1. LT exhibited minor but significant efficacy even in the absence of antihypertensive effects. Transcript array analyses revealed a four-fold down-regulation of renal cortical chemokine (C-C motif) receptor 6 (CCR6) mRNA by HT, which was confirmed at the protein level. Silencing of CCR6 did not alter podocyte function in vitro, thus indicating a predominant role in the tubulo-interstitium. In human kidney biopsies, CCR6 mRNA and mRNA of its ligand chemokine (C-C motif) ligand 20 was up-regulated in patients with progressive IgA nephropathy compared to stable disease. Thus, delayed treatment with high-dose telmisartan exerted a pronounced benefit in progressive mesangioproliferative glomerulonephritis, which extended beyond that of equivalent blood pressure lowering. We identified down-regulation of platelet-derived growth factor receptors and CCR6 as potential mediators of telmisartan-related renoprotection. CCR6 may also regulate the renal outcome in human mesangioprolfierative glomerulonephritis.

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. In vitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis in vivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [ NOV ]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor–β, and fibronectin, and increased PDGF-receptor–α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.

Background/Aims: Intravenous immunoglobulins (IVIG) consist of polyvalent immunoglobulins (Ig), mainly IgG with varying amounts of other Ig depending on preparation. IVIG have renoprotective properties in diseases like lupus nephritis mostly due to their anti-inflammatory effects. The role of polyvalent IgG treatment during the course of experimental progressive mesangioproliferative nephritis is not yet known. Methods: Progressive mesangioproliferative nephritis was induced in male Wistar rats by uninephrectomy and anti-Thy1.1 antibody injection. Rats were treated with either vehicle (phosphate-buffered saline; n = 10) or nonspecific human polyclonal IgG (IgG; n = 10) on days 3, 10 and 17 after disease induction and sacrificed on day 56. Results: During the experiment, IgG treatment prevented weight loss and had a beneficial effect on the rise in serum creatinine and the decline of creatinine clearance. At sacrifice, a significantly lower number of IgG-treated rats had tripled their creatinine or halved their creatinine clearance. Moreover, during the 56 days of follow-up, the IgG-treated group exhibited reduced mortality due to renal failure. At sacrifice, IgG-treated rats had reduced indices of renal fibrosis. Conclusions: IgG treatment is an effective treatment approach in rats with progressive glomerulonephritis. Our data also indicate that studies using specific antibodies need to be controlled for nonspecific IgG effects.

PDGF-C is a potent mitogen for fibroblasts in vitro. Transgenic PDGF-C overexpression in the heart or liver induces organ fibrosis, and PDGF-C expression is upregulated at sites of interstitial fibrosis in human and rat kidneys; however, the effect of inhibiting PDGF-C on the development of renal fibrosis in vivo is unknown. Renal fibrosis was induced in C57BL/6 mice by unilateral ureteral obstruction (UUO), and then mice were treated with neutralizing anti–PDGF-C antiserum or nonspecific IgG. An increase in PDGF-C expression was observed in fibrotic areas after UUO, contributed in large part by infiltrating macrophages. Treatment with anti–PDGF-C reduced renal fibrosis by 30% at day 5 and reduced interstitial myofibroblast accumulation by 57%. In vitro, PDGF-C was a potent mitogen for renal fibroblasts and induced chemokine expression. In vivo, anti–PDGF-C treatment produced a decrease in the expression of the renal chemokines CCL2 and CCL5 (85 and 67% reductions, respectively), accompanied by a significant decrease in leukocyte infiltration and CCR2 mRNA expression. Further supporting a role of PDGF-C in renal fibrosis, PDGF-C−/− mice demonstrated a reduction in fibrosis and leukocyte infiltration in response to UUO compared with wild-type littermates. In conclusion, specific neutralization or lack of PDGF-C reduces the development of renal inflammation and fibrosis in obstructed mouse kidneys. Leukocyte-derived PDGF-C induces chemokine expression, which may lead to the recruitment of additional leukocytes, creating an amplification loop for renal inflammation and fibrosis.

Bone marrow-derived cells contribute to glomerular cell turnover and repair, but the cell types involved are unknown. Whether rat mesenchymal stem cells (MSC) can accelerate recovery from damage in rat mesangioproliferative anti-Thy1.1 glomerulonephritis was studied. After injection into the left renal artery on day 2 after disease induction, fluorescently labeled MSC were detected in 20 to 50% of glomeruli and rare intrarenal vessels but not in the tubulointerstitium, in contralateral kidneys, or in medium controls. In control experiments, injected mesangial cells were detected less frequently in glomeruli in comparison with injected MSC. In nephritic outbred Wistar rats, MSC injection led to an approximately 50% reduction of mesangiolysis on days 4 and 6 after disease induction, accompanied by three- to four-fold higher intraglomerular cell proliferation on day 4 and more rapid mesangial reconstitution as detected by alpha-smooth muscle actin expression. Injection of MSC into tail veins or intra-arterial injection of mesangial cells instead of MSC failed to reproduce any of these findings. In inbred Lewis rats, anti-Thy1.1 nephritis followed an aggravated course with transient acute renal failure. Acute renal failure was ameliorated by MSC injection into the left renal artery on day 2 after disease induction. Again, MSC led to more rapid recovery from mesangiolysis, increased glomerular cell proliferation, and reduction of proteinuria by 28%. Double immunostaining of 5-bromo-2'-deoxyuridine-labeled MSC for endothelial, mesangial, or monocyte/macrophage antigens showed that 85 to 95% of MSC that localized in glomeruli on day 6 failed to express these markers. In vitro, MSC secreted high amounts of vascular endothelial growth factor and TGF-beta1 but not PDGF-BB. In conclusion, even low numbers of MSC can markedly accelerate glomerular recovery from mesangiolytic damage possibly related to paracrine growth factor release and not to differentiation into resident glomerular cell types or monocytes/macrophages.

Glomerular mesangial cell proliferation and/or matrix accumulation characterizes many progressive renal diseases. PDGF-D was identified recently as a novel mediator of mesangial cell proliferation in vitro and in vivo. This study investigated the long-term consequences of PDGF-D inhibition in vivo. Rats with progressive mesangioproliferative glomerulonephritis (uninephrectomy plus anti-Thy-1.1 antibody) received the PDGF-D-neutralizing, fully human mAb CR002 on days 3, 10, and 17 after disease induction. Glomerular mesangioproliferative changes on day 10 were significantly reduced by anti-PDGF-D treatment as compared with control antibody. Eight weeks after disease induction, anti-PDGF-D therapy significantly ameliorated focal segmental glomerulosclerosis, podocyte damage (de novo desmin expression), tubulointerstitial damage, and fibrosis as well as the accumulation of renal interstitial matrix including type III collagen and fibronectin. Treatment with anti-PDGF-D also reduced the cortical infiltration of monocytes/macrophages on day 56, possibly related to lower renal cortical complement activation (C5b-9 deposition) and/or reduced epithelial-to-mesenchymal transition (preserved cortical expression of E-cadherin and reduced expression of vimentin and alpha-smooth muscle actin). In conclusion, these data provide evidence for a causal role of PDGF-D in the pathogenesis of renal scarring and point to a new therapeutic approach to progressive mesangioproliferative renal disease.

Combined expression of A1 and A20 achieves optimal protection of renal proximal tubular epithelial cells. Background Apoptotic death of renal proximal tubular epithelial cells (RPTECs) is a feature of acute and chronic renal failure. RPTECs are directly damaged by ischemia, inflammatory, and cytotoxic mediators but also contribute to their own demise by up-regulating proinflammatory nuclear factor-kappaB (NF-κB)-dependent proteins. In endothelial cells, the Bcl family member A1 and the zinc finger protein A20 have redundant and dual antiapoptotic and anti-inflammatory effects. We studied the function(s) of A1 and A20 in human RPTECs in vitro. Methods Expression of A1 [reverse transcription-polymerase chain reaction (RT-PCR) and A20 (Northern and Western blot analysis)] in RPTECs was evaluated. A1 and A20 were overexpressed in RPTECs by recombinant adenoviral-mediated gene transfer. Their effect upon inhibitor of NFκB alpha (IκBα) degradation (Western blot), NF-κB nuclear translocation [electrophoretic mobility shift assay (EMSA)], up-regulation of intercellular adhesion molecule-1 (ICAM-1) [fluorescence-activated cell sorter (FACS)] and monocyte chemoattractant protein-1 (MCP-1) (Northern blot) and apoptosis [terminal deoxynucleotiddyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)] and FACS analysis of DNA content) was determined. Results A1 and A20 were induced in RPTECs as part of the physiologic response to tumor necrosis factor (TNF). A20, but not A1, inhibited TNF-induced NF-κB activation by preventing IκBα degradation, hence subsequent up-regulation of the proinflammatory molecules ICAM-1 and MCP-1. Unexpectedly, A20 did not protect RPTECs from TNF and Fas-mediated apoptosis while A1 protected against both stimuli. Coexpression of A1 and A20 in RPTECs achieved additive anti-inflammatory and antiapoptotic cytoprotection. Conclusion A1 and A20 exert differential cytoprotective effects in RPTECs. A1 is antiapoptotic. A20 is anti-inflammatory via blockade of NF-κB. We propose that A1 and A20 are both required for optimal protection of RPTECs from apoptosis (A1) and inflammation (A20) in conditions leading to renal damage.

Podocyte loss contributes to the development of glomerulosclerosis. Although podocyte detachment has been recognized as a new mechanism of podocyte loss in glomerular diseases, its time course and relationship to disease activity are not known. Urinary excretion of viable podocytes was quantified in two models of transient glomerular injury, i.e., rats with puromycin aminonucleoside-induced nephrosis (PAN) and mesangioproliferative nephropathy (anti-Thy 1.1 nephritis model), as well as in a model of continuous glomerular injury, i.e., hypertensive nephropathy (5/6-nephrectomy model), and in aging rats. The number of glomerular Wilm's tumor (WT)-1-positive podocytes and the glomerular expression of cell-cycle proteins in vivo were assessed. Urinary podocyte loss occurred in both primary (PAN) and secondary (anti-Thy 1.1 nephritis) in parallel to the onset of proteinuria. However, subsequently proteinuria persisted despite remission of podocyturia. In continuous glomerular injury, i.e., after 5/6-nephrectomy, podocyturia paralleled the course of proteinuria and of systemic hypertension, whereas no podocyturia became detectable during normal aging (up to 12 mo). Despite podocyte detachment of varying degrees, no decrease in glomerular podocyte counts (i.e., WT-1 positive nuclei) was noted in either disease model. Podocyturia in the PAN and anti-Thy 1.1 nephritis model was preceded by entry of glomerular podocytes into the cell cycle, i.e., cyclin D1, cdc2, and/or proliferating cell nuclear antigen (PCNA) expression. Podocyturia is a widespread phenomenon in glomerular disease and not simply a reflection of proteinuria because it is limited to phases of ongoing glomerular injury. The data suggest that podocyturia may become a more sensitive means to assess the activity of glomerular damage than proteinuria.

R-roscovitine (CYC202) alleviates renal cell proliferation in nephritis without aggravating podocyte injury. Background Cyclin-dependent kinase (CDK) inhibition is a new therapeutic approach to proliferative glomerulonephritides. CDK2 is required for G 1 /S transition and DNA synthesis and is inhibited by CYC202 (R-roscovitine). Since podocytes express CDK2 in nephritis and since loss of podocytes contributes to glomerulosclerosis, the rationale of the present study was to test whether CDK2 inhibition is safe in instances of podocyte injury. Methods Rats with passive Heymann nephritis, a model of membranous glomerulonephritis, were treated (day 3 to 30) with vehicle, low (25 mg/kg/day), or high (50 mg/kg/day) doses of CYC202. Results On day 27, blood pressure was normal in nephritic controls and was dose-dependently reduced by CYC202. Urinary albumin excretion did not differ between the groups on days 9, 16, 23, and 30. To investigate podocyte injury, we assessed the glomerular de novo expression of desmin, which was markedly up-regulated in almost all passive Heymann nephritis glomeruli but was not significantly different between the three groups. No tubulointerstitial de novo expression of desmin or alpha-smooth muscle actin (α-SMA), or tubulointerstitial monocyte/macrophage infiltration was noted in any group. Biologic activity of CYC202 was evident in the form of a dose-dependent decrease in the number of glomerular and tubulointerstitial mitotic figures as compared to vehicle alone. Glomerular immunostaining for cyclin D1, a marker for G 0 to G 1 transition, was significantly decreased in CYC202 treated groups at day 9. Conclusion Whereas inhibition of CDKs by CYC202 reduced intrarenal cell proliferation in passive Heymann nephritis it did not aggravate podocyte damage, suggesting that this novel therapeutic approach is safe in renal diseases characterized by podocyte injury.

PDGF-B is of central importance in mesangioproliferative diseases. PDGF-D, a new PDGF isoform, like PDGF-B, signals through the PDGF betabeta-receptor. The present study first determined that PDGF-D is mitogenic for rat mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. After induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 mAb, glomerular PDGF-D mRNA and protein expression increased significantly from days 4 to 9 in comparison with nonnephritic rats. Peak expression of PDGF-D mRNA occurred 2 d later than peak PDGF-B mRNA expression. In addition, PDGF-D serum levels increased significantly in the nephritic animals on day 7. For investigating the functional role of PDGF-D, neutralizing fully human mAb were generated using the XenoMouse technology. Rats with anti-Thy 1.1-induced nephritis were treated on days 3 and 5 with different amounts of a fully human PDGF-DD-specific neutralizing mAb (CR002), equal amounts of irrelevant control mAb, or PBS by intraperitoneal injection. Specific antagonism of PDGF-D led to a dose-dependent (up to 67%) reduction of glomerular cell proliferation. As judged by double immunostaining for 5-bromo-2'-deoxyuridine and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. Reduction of glomerular cell proliferation in the rats that received CR002 was not associated with reduced glomerular expression of PDGF-B mRNA. PDGF-D antagonism also led to reduced glomerular infiltration of monocytes/macrophages (day 5) and reduced accumulation of fibronectin (day 8). In contrast, no effect was noted in normal rats that received an injection of CR002. These data show that PDGF-D is overexpressed in mesangioproliferative states and can act as an auto-, para-, or even endocrine glomerular cell mitogen, indicating that antagonism of PDGF-D may represent a novel therapeutic approach to mesangioproliferative glomerulonephritides.

BACKGROUND: Chronic rejection with development of transplant arteriosclerosis is the major culprit involved in loss of kidney allografts. The allografts' fate was thought to depend on the intensity of the host immune responses and the potency of immunosuppressive regimens. Recent data suggests that grafts contribute to their own survival by way of up-regulation of "cytoprotective" genes. METHODS: We analyzed the expression of four cytoprotective genes, A20, Bcl-2, Bcl-x(L) and heme oxygenase (HO)-1, in three rat renal allograft models of chronic rejection: Fisher 344-Lewis (F344/Lew), Dark Agouti-Brown Norway (DA/BN), and DA-Wistar-Furth (WF). We chose these genes for their known anti-inflammatory and anti-apoptotic function in endothelial cells (EC) and the atheroprotective function of A20 in smooth muscle cells (SMC). RESULTS: Twenty-eight and 9 weeks following transplantation, F344/Lew and DA/BN transplants had stable graft function. Histopathologic analysis showed moderate tissue damage, minimal cellular infiltrates, and preserved vascular integrity correlating with high expression of A20 in SMC. Conversely, impaired allograft function in the DA/WF combination with substantial transplant arteriosclerosis was noted in 60% of the grafts correlating with absent or decreased A20 expression in EC and SMC. In all combinations, expression of HO-1, Bcl-2, and Bcl-x(L) colocalized with infiltrating cells and was not informative on the graft status. CONCLUSIONS: We demonstrate for the first time a strict correlation between A20 expression in the vessel and the absence of transplant arteriosclerosis in rat kidney-allograft models. This data is similar to data obtained in human kidney allografts and suggests that A20 may represent a novel therapeutic target for the prevention of chronic allograft rejection.

Platelet-derived growth factor B-chain (PDGF-B)- and transforming growth factor beta (TGF-beta)-mediated accumulation of extracellular matrix proteins contributes to many progressive renal diseases. In vivo, specific antagonism of either PDGF-B or TGF-beta in experimental mesangioproliferative glomerulonephritis resulted in an almost complete inhibition of matrix protein accumulation, which suggests an interaction between signaling pathways of these two growth factors. Because nothing is known on the nature of this possible interaction, PDGF-B was antagonized in the rat anti-Thy 1.1 model of glomerulonephritis by use of specific aptamers and its effects on the TGF-beta system were investigated. Antagonism of PDGF-B led to a significant reduction of glomerular matrix accumulation compared with scrambled aptamer-treated nephritic controls. PDGF-B antagonism had no effect on the overexpression of glomerular TGF-beta mRNA, TGF-beta protein, or the expression of TGF-beta receptor type I and II mRNA. By immunohistology, it was possible to detect overexpression of the cytoplasmic TGF-beta signaling molecules Smad2 (agonistic) and Smad7 (antagonistic) in glomeruli of nephritic control rats which peaked on day 7 after disease induction, i.e., the peak of mesangial cell proliferation in this model. However, immunohistology and Western blot analysis again revealed no difference in the glomerular expression of both Smad proteins between PDGF-B antagonized and nonantagonized nephritic animals. In addition, no difference in the glomerular expression of phosphorylated Smad2 (P-Smad2) was detected between the differently treated nephritic groups. These observations suggest that the effects of PDGF-B antagonism are independent of TGF-beta in mesangioproliferative glomerulonephritides.

Why is it that only human beings continuously perform acts of heroism? Looking back at our evolutionary history can offer us some potentially useful insight. This review highlights some of the major steps in our evolution-more specifically, the evolution of high blood pressure. When we were fish, the first kidney was developed to create a standardized internal 'milieu' preserving the primordial sea within us. When we conquered land as amphibians, the evolution of the lung required a low systemic blood pressure, which explains why early land vertebrates (amphibians, reptiles) are such low performers. Gaining independence from water required the evolution of an impermeable skin and a water-retaining kidney. The latter was accomplished twice with two different solutions in the two major branches of vertebrate evolution: mammals excrete nitrogenous waste products as urea, which can be utilized by the kidney as an osmotic agent to produce more concentrated urine. Dinosaurs and birds have a distinct nitrogen metabolism and excrete nitrogen as water-insoluble uric acid-therefore, their kidneys cannot use urea to concentrate as well. Instead, some birds have developed the capability to reabsorb water from their cloacae. The convergent development of a separate small circulation of the lung in mammals and birds allowed for the evolution of 'high blood-pressure animals' with better capillarization of the peripheral tissues allowing high endurance performance. Finally, we investigate why mankind outperforms any other mammal on earth and why, to this day, we continue to perform acts of heroism on our eternal quest for personal bliss.

VEGF(165), the most abundant isoform in man, is an angiogenic cytokine that also regulates vascular permeability. Its function in the renal glomerulus, where it is expressed in visceral epithelial and mesangial cells, is unknown. To assess the role of VEGF(165) in glomerular disease, we administered a novel antagonist - a high-affinity, nuclease-resistant RNA aptamer coupled to 40-kDa polyethylene glycol (PEG) - to normal rats and to rats with mesangioproliferative nephritis, passive Heymann nephritis (PHN), or puromycin aminonucleoside nephrosis (PAN). In normal rats, antagonism of VEGF(165) for 21 days failed to induce glomerular pathology or proteinuria. In rats with mesangioproliferative nephritis, the VEGF(165) aptamer (but not a sequence-scrambled control RNA or PEG alone) led to a reduction of glomerular endothelial regeneration and an increase in endothelial cell death, provoking an 8-fold increase in the frequency of glomerular microaneurysms by day 6. In contrast, early leukocyte influx and the proliferation, activation, and matrix accumulation of mesangial cells were not affected in these rats. In rats with PHN or PAN, administration of the VEGF(165) aptamer did not influence the course of proteinuria using various dosages and administration routes. These data identify VEGF(165) as a factor of central importance for endothelial cell survival and repair in glomerular disease, and point to a potentially novel way to influence the course of glomerular diseases characterized by endothelial cell damage, such as various glomerulonephritides, thrombotic microangiopathies, or renal transplant rejection.

Background: The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. Purpose: To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. Methods: Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. Results: Tyrosinase mRNA was detected by RTPCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P

The current study is based on the hypothesis of mesenchymal stromal cells (MSCs) contributing to soft-tissue calcification and ectopic osteogenesis in chronic kidney disease (CKD). Rat MSCs were transplanted intraperitoneally in an established three-dimensional collagen-based model in healthy control animals and two rat models of CKD and vascular calcification: (1) 5/6 nephrectomy + high phosphorus diet; and (2) adenine nephropathy. As internal controls, collagen gels without MSCs were transplanted in the same animals. After 4 and 8 weeks, MSCs were still detectable and proliferating in the collagen gels (fluorescence-activated cell sorting [FACS] analysis and confocal microscopy after fluorescence labeling of the cells). Aortas and MSC-containing collagen gels in CKD animals showed distinct similarities in calcification (micro-computed tomography [µCT], energy-dispersive X-ray [EDX] analysis, calcium content), induction of osteogenic markers, (ie, bone morphogenic protein 2 [BMP-2], Runt related transcription factor 2 [Runx2], alkaline phosphatase [ALP]), upregulation of the osteocytic marker sclerostin and extracellular matrix remodeling with increased expression of osteopontin, collagen I/III/IV, fibronectin, and laminin. Calcification, osteogenesis, and matrix remodeling were never observed in healthy control animals and non-MSC-containing collagen gels in all groups. Paul Karl Horan 26 (PKH-26)-labeled, 3G5-positive MSCs expressed Runx2 and sclerostin in CKD animals whereas PKH-26-negative migrated cells did not express osteogenic markers. In conclusion, heterotopically implanted MSCs undergo osteogenic differentiation in rat models of CKD-induced vascular calcification, supporting our hypothesis of MSCs as possible players in heterotopic calcification processes of CKD patients.

Item does not contain fulltext Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact.

Objective— Medial artery calcification in patients with chronic kidney disease proceeds through intramembranous ossification resulting from osteoblast-induced calcification of the collagen extracellular matrix. The current study is based on the hypothesis that mesenchymal stem cells (MSC) constitute critical cells for procalcific extracellular matrix remodeling in patients with chronic kidney disease. Methods and Results— Human MSC were cultured in media supplemented with pooled sera from either healthy or uremic patients (20%). Exposure to uremic serum enhanced the proliferation of MSC (cell counting, BrdU incorporation) whereas apoptosis and necrosis were not affected (annexin V and 7-amino-actinomycin staining). Uremic serum–exposed MSC recapitulated osteogenesis by matrix calcification and expression of bone-related genes (bone morphogenetic protein [BMP]-2 receptor, alkaline phosphatase, osteopontin, and Runx2) in 35 days. The uremic serum–induced osteogenesis was completely blocked by a BMP-2/4 neutralizing antibody or the natural antagonist NOGGIN. Calcification and matrix remodeling were further analyzed in a collagen-embedded osteogenesis model recapitulating the vascular collagen I/III environment. The uremic serum–induced calcification was shown to occur along collagen fibers as shown by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and von Kossa staining and was accompanied by extensive matrix remodeling. Conclusion— Uremic serum induced in a BMP-2/4-dependent manner an osteoblast-like phenotype in MSC accompanied by matrix remodeling and calcification.

In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor-β, and fibronectin, and increased PDGF-receptor-α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.

BACKGROUND Renoprotective actions of angiotensin receptor blockers are not well established in normotensive, low-grade proteinuric glomerular diseases. We examined the effect of low-dose telmisartan (LT) and high-dose telmisartan (HT) versus conventional antihypertensive therapy in the rat anti-Thy1.1 model of glomerulonephritis. METHODS Rats were randomized on Day 4 after disease induction to no treatment (CT, control), LT or HT or hydrochlorothiazide + hydralazine (HCT + H). RESULTS All rats remained normotensive: HT and HCT + H reduced blood pressure by 15-20%. LT, HT and HCT + H reduced glomerular endothelial cell proliferation and glomerular and interstitial matrix deposition on Day 14. Only HT reduced podocyte damage and tubular cell dedifferentiation on Day 9 and mesangial cell activation on Day 14. By gene expression analysis arrays, we identified discs-large homolog 1 and angiopoietin-like 4 as potential mediators of the HT effects. In addition, we identified several pathways possibly related to the pleiotropic effects of HT, including growth factor signalling, mammalian target of rapamycin signalling, protein ubiquitination, the Wnt-beta catenin pathway and hypoxia signaling. CONCLUSIONS In summary, treatment with HT, initiated after the induction of disease, ameliorates glomerular and tubulointerstitial damage. We provide the first comprehensive insight into the mechanisms underlying the renoprotective effect of high-dose angiotensin II receptor blockers (ARBs). Our study lays the basis for future investigations on novel pathways affected by ARBs in renal disease.

Publisher Summary Malignancies, mostly related to applications of embryonic stem cells (ESCs), have evolved to be a major concern in stem cell therapies as well. Other concerns relate to the validity of experimental results. Stem cell markers are often prone to technical misinterpretation or may be affected by heterogeneity of the cell preparations. Consequently, this chapter addresses both the medical safety aspects of stem cell therapies and the scientific “safety” of stem cell-related data. It also focuses on safety issues regarding MSCs. However, with the rapid developments in the field, including the first Food and Drug Administration (FDA) approval of an ESC study on spinal cord injury, safety aspects concerning stem cells other than MSCs may gain importance in the near future. Although researchers still have to deal with the lack of indisputable criteria defining MSCs, making it necessary to constantly standardize the widely differing protocols for isolation, expansion and characterization, the emerging (and considerable) therapeutic potential of MSCs renders them strong candidates for human therapies.

Glomerular endothelial cell injury is a key component of a variety of diseases. Factors involved in glomerular endothelial cell repair are promising therapeutic agents for such diseases. Platelet-derived growth factor (PDGF)–C has pro-angiogenic properties; however, nothing is known about such functions in the kidney. We therefore investigated the consequences of either PDGF-C infusion or inhibition in rats with mesangioproliferative glomerulonephritis, which is accompanied by widespread glomerular endothelial cell damage. We also assessed the role of PDGF-C in a mouse model of thrombotic microangiopathy as well as in cultured glomerular endothelial cells. PDGF-C infusion in nephritic rats significantly reduced mesangiolysis and microaneurysm formation, whereas glomerular endothelial cell area and proliferation increased. PDGF-C infusion specifically up-regulated glomerular fibroblast growth factor-2 expression. In contrast, antagonism of PDGF-C in glomerulonephritis specifically reduced glomerular endothelial cell area and proliferation and increased mesangiolysis. Similarly, PDGF-C antagonism in murine thrombotic microangiopathy aggravated the disease and reduced glomerular endothelial area. In conditionally immortalized glomerular endothelial cells, PDGF-C was mitogenic and induced a 27-fold up-regulation of fibroblast growth factor-2 mRNA. PDGF-C also exerted indirect pro-angiogenic effects, since it induced endothelial cell mitogens and pro-angiogenic factors in mesangial cells and macrophages. These results identify PDGF-C as a novel, potent pro-angiogenic factor in the kidney that can accelerate capillary healing in experimental glomerulonephritis and thrombotic microangiopathy.

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance--agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

Background In vitro, the extracellular signal-regulated kinase (ERK) is an intracellular convergence point of multiple stimuli, which affect the cell cycle. However, the role of ERK in cell cycle regulation in vivo is unknown. Methods To address this issue, ERK activity was blocked both in vitro in mesangial cells (MC) and in vivo in experimental glomerulonephritis (GN) by a pharmacological inhibitor (U0126) of the ERK-activating kinase. Results In stimulated MC, inhibition of ERK reduced cyclin-dependent kinase 2 (CDK2) phosphorylation, CDK2 activity and cyclin E/A expression, whereas downregulation of CDK inhibitor p27(Kip1) expression was inhibited. In vivo, U0126 was given to rats in the acute phase of anti-Thy 1.1 GN. We previously showed that glomerular cell proliferation was reduced by 67% upon treatment with the inhibitor compared to nephritic controls. Now, we detected a significant increase in renal CDK2-activity/phosphorylation in the nephritic controls, that was significantly and dose-dependently reduced by ERK inhibition. CDK2 activation was accompanied by an increase in renal expression of cyclins E/A and the enhanced binding of these cyclins to CDK2 in the nephritic controls. These changes were blunted by U0126 treatment. Finally, we noted an increased expression and CDK2-binding of p27(KIP1) protein in the nephritic controls which was decreased in U0126 treated rats. Conclusions Our observations provide the first evidence that ERK is an intracellular regulator of renal CDK2 activity in vivo in a glomerulonephritis model.

Glomerulonephritis (GN) is a major cause of renal failure. This study sought to determine whether intrarenal injection of rat mesenchymal stem cells (MSC) can preserve renal function in a progressive rat model of GN. Early in GN (day 10), fluorescently labeled rat MSC localized to more than 70% of glomeruli, ameliorated acute renal failure, and reduced glomerular adhesions. Fifty days later, proteinuria had progressed in controls to 40 +/- 25 mg/d but stayed low in MSC-treated rats (13 +/- 4 mg/d; P < 0.01). Renal function on day 60 in the MSC group was better than in medium controls. Kidneys of the MSC group as compared with controls on day 60 contained 11% more glomeruli per 1-mm(2) section of cortex but also significantly more collagen types I, III, and IV and alpha-smooth muscle actin. Approximately 20% of the glomeruli of MSC-treated rats contained single or clusters of large adipocytes with pronounced surrounding fibrosis. Adipocytes exhibited fluorescence in their cytoplasm and/or intracellular lipid droplets. Lipid composition in these adipocytes in vivo mirrored that of MSC that underwent adipogenic differentiation in vitro. Thus, in this GN model, the early beneficial effect of MSC of preserving damaged glomeruli and maintaining renal function was offset by a long-term partial maldifferentiation of intraglomerular MSC into adipocytes accompanied by glomerular sclerosis. These data suggest that MSC treatment can be a valuable therapeutic approach only if adipogenic maldifferentiation is prevented.

Renal fibrosis is the final common pathway of most progressive renal diseases. C5 was recently identified as a risk factor for liver fibrosis. This study investigated the role of C5 in the development of renal tubulointerstitial fibrosis by (1) induction of renal fibrosis in wild-type and C5(-/-) mice by unilateral ureteral ligation (UUO) and (2) investigation of the effects of a C5a receptor antagonist (C5aRA) in UUO. In C5(-/-) mice, when compared with wild-type controls, markers of renal fibrosis (Sirius Red, type I collagen, fibronectin, alpha-smooth muscle actin, vimentin, and infiltrating macrophages) were significantly reduced on day 5 of UUO. On day 10, fibronectin mRNA and protein expression were still reduced in the C5(-/-) mice. Cortical mRNA of all PDGF isoforms and of TGF-beta(1) (i.e., central mediators of renal disease) were significantly reduced in C5(-/-) mice when compared with controls. Renal tubular cell expression of the C5aR was sparse in normal cortex but markedly upregulated after UUO. Treatment of wild-type UUO mice with C5aRA also led to a significant reduction of cortical Sirius Red staining, fibronectin protein expression, and PDGF-B mRNA expression on day 5. Neither genetic C5 deficiency nor C5aRA treatment caused any histologic changes in the nonobstructed kidneys. In cultured murine cortical tubular cells, C5a stimulated production of TGF-beta(1), and this was inhibited by C5aRA. Using a combined genetic and pharmacologic approach, C5, in particular C5a, is identified as a novel profibrotic factor in renal disease and as a potential new therapeutic target.

Background. Arresting or regressing kidney scarring is of major clinical relevance. Platelet-derived growth factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage. Methods. Using this model, we now assessed the effects of CR002 (n ¼ 15) vs irrelevant control IgG (n ¼ 17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided. Results. In vitro, CR002 inhibited the PDGF-D- but not the PDGF-B-induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002-treated group was transiently reduced between days 49 and 77 (� 19 to � 23% in comparison with the controls; P < 0.05). On day 100, CR002 treatment reduced the number of rats that had doubled their serum creatinine (CR002: 40 vs controls: 71%; P < 0.05). Compared with controls, the CR002 animals, on day 100, significantly lowered glomerular expression of vimentin and collagens as well as tubulointerstitial damage scores, interstitial fibrosis, vimentin and cortical PDGF-D mRNA levels. Conclusions. PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e. the final common pathway of most renal diseases.

Keywords: Alport’s syndrome; basement membrane;bone marrow transplantationIn the 9 May 2006 issue of Proc Natl Acad Sci USA,Sugimoto and colleagues [1] described fascinating dataon a potential approach to treat Alport’s syndrome, arare genetic disease leading to renal failure, which so farcould not be cured. The work was highly publicized anddiscussed in both scientific journals and the lay press.Alport’s syndrome derives from a mutation of eitherthe a3, a4ora5 chain of type IV collagen, i.e. collagentypes that constitute basement membranes in the renalglomerulus, the ear and the eye. Mice that aregenetically deficient of the a3(IV)-chain (‘Alportmice’) develop a renal phenotype very similar to thatof Alport patients (Figure 1), i.e. proteinuria, glomer-ulonephritis and subsequent tubulointerstitial fibrosisstarting at 8 weeks of age and leading to death due torenal failure at 20–23 weeks. In the study by Sugimotoet al. [1], 8 week-old Alport mice were lethallyirradiated and then received an allogenic unfraction-ated bone marrow transplant from either LacZ mice,i.e. mice with a normal collagen production plusexpression of the LacZ marker in all cells, or fromanother Alport mouse. Whereas the latter had no effecton the phenotype, the allogenic bone marrow led tomarkedly reduced proteinuria upon follow-up, andimproved renal function as well as renal histology.LacZ-positive cells constituted about 10% of theglomerular cells and were found in podocyte andmesangial cell locations. The glomerular stainingpattern for the a3- and a5(IV) chains was partiallyrestored. If these data can be confirmed and extendedby showing that the treatment delays death from renalfailure in Alport mice (see subsequently), there wouldbe several major implications of this study.

Postischemic acute renal failure (ARF) is common and often fatal. Cellular mechanisms include cell adhesion, cell infiltration and generation of oxygen free radicals, and inflammatory cytokine production. Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ("statins") directly influence inflammatory mechanisms. The hypothesis that ischemia-induced ARF could be ameliorated with statin treatment was investigated and possible molecular mechanisms were analyzed in a uninephrectomized rat model. Male Sprague-Dawley rats were pretreated with cerivastatin (0.5 mg/kg) or vehicle for 3 d. Ischemic ARF was induced by left renal artery clipping for 45 min, while the right kidney was being removed. After 24 h of ARF, serum creatinine levels were increased 7.5-fold in vehicle-treated control animals with ARF, compared with sham-operated animals (P < 0.005). Statin treatment reduced the creatinine level elevation by 40% (P < 0.005). Simultaneously, ischemia-induced severe decreases in GFR were significantly ameliorated by statin treatment (sham operation, 0.95 +/- 0.09 ml/min, n = 13; ischemia without treatment, 0.06 +/- 0.02 ml/min, n = 9; ischemia with statin pretreatment, 0.21 +/- 0.03 ml/min, n = 11; P < 0.001). Furthermore, statin pretreatment prevented the occurrence of tubular necrosis, with marked loss of the brush border, tubular epithelial cell detachment, and tubular obstruction in the S3 segment of the outer medullary stripe. In addition, monocyte and macrophage infiltration was almost completely prevented, intercellular adhesion molecule-1 upregulation was greatly decreased, and inducible nitric oxide synthase expression was reduced. Fibronectin and collagen IV expression was reduced, approaching levels observed in sham-operated animals. In vehicle-treated rats with ARF, mitogen-activated protein kinase extracellular activated kinase-1/2 activity was increased and the transcription factors nuclear factor-kappaB and activator protein-1 were activated. Statin treatment reduced this activation toward levels observed in sham-operated rats. The data suggest that hydroxy-3-methylglutaryl coenzyme A reductase inhibition protects renal tissue from the effects of ischemia-reperfusion injury and thus reduces the severity of ARF. The chain of events may involve anti-inflammatory effects, with inhibition of mitogen-activated protein kinase activation and the redox-sensitive transcription factors nuclear factor-kappaB and activator protein-1.

Glomerular mesangial cell proliferation and/or mesangial matrix accumulation characterizes many progressive renal diseases. Rats with progressive mesangioproliferative glomerulonephritis were treated from day 3 to day 7 after disease induction with a high-affinity oligonucleotide aptamerantagonist against platelet-derived growth factor-B chain (PDGF-B). In comparison with nephritic rats that received vehicle or a scrambled aptamer, treatment with the PDGF-B aptamer led to a significant reduction of mesangioproliferative changes, glomerular hypertrophy, podocyte damage, and glomerular macrophage influx on day 8. Both nephritic control groups subsequently developed progressive proteinuria and decreased renal function. On day 100, glomerulosclerosis, tubulointerstitial damage, glomerular and interstitial accumulation of types III and IV collagen, and overexpression of transforming growth factor-β were widespread. All of these chronic changes were prevented in rats that received the PDGF-B aptamer, and their functional and morphologic parameters on day 100 were largely indistinguishable from non-nephritic rats. These data provide the first evidence for a causal role of PDGF in the pathogenesis of renal scarring and point to a new, highly effective therapeutic approach to progressive, in particular mesangioproliferative, renal disease.

The presence or absence of melanoma cells in human peripheral blood has recently been shown to be associated with disease prognosis, including overall survival. The detection of tyrosinase mRNA-positive circulating melanoma cells by reverse transcription-polymerase chain reaction (RT-PCR) has been limited to disseminated tumours expressing measurable amounts of this melanocyte-specific enzyme. To biologically classify both melanotic and amelanotic melanomas and to evaluate the clinical and prognostic relevance of tumour cell microcontamination, we examined autologous peripheral blood stem cell (PBSC) harvests from patients with advanced malignant melanoma prior to dose-escalated chemotherapy. To assay heterogeneous melanoma cell antigen expression, we developed a highly sensitive RT-PCR using four melanoma- and one tumour-associated antigen as molecular markers. Expression of the melanocyte-associated transcripts of tyrosinase, MART1/Melan-A, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) as well as the tumour-specific transcript of MAGE-3 was analysed by RT-PCR in PBSC harvests from 31 patients. Seven of the 31 PBSC harvests tested positive for one or more molecular markers: two patients for tyrosinase only, and one patient for MAGE-3 only, one patient for tyrosinase and MAGE-3, one for tyrosinase and MART1/Melan-A, and two patients for tyrosinase, MART1/Melan-A, TRP-2 and MAGE-3. mRNA-positive patients exhibited a significantly impaired overall survival (P = 0.0032), with a median survival of 3 months as opposed to 10 months in PBSC mRNA-negative patients. In conclusion, the use of this multiple-marker microcontamination assay allowed for molecular and prognostic classification of advanced malignant melanoma.

Different activation of mitogen-activated protein kinases in ex- perimental proliferative glomerulonephritis. Mitogen-activated protein (MAP) kinases are critical for cell signaling goals such as cellular proliferation and induction of apoptosis. We examined whether MAP kinases, as a point of convergence for multiple extracellular stimuli, are activated in proliferative glomerulone- phritis (GN) in vivo. Accelerated crescentic anti-glomerular base- ment membrane (GBM) GN was induced in rats preimmunized with rabbit IgG by administration of rabbit anti-rat GBM serum. Whole cortical tissue and isolated glomeruli were then subjected to kinase activity assays and Western blot analysis. Cortical activity of the archetypal MAP kinase, extracellular signal-regu- lated kinase (ERK), was increased significantly one, three, and seven days after induction of GN. In contrast, activation of MAP kinases with antiproliferative actions, stress-activated protein kinase, and p38 MAP kinase was detectable only in the early stages of proliferative GN (days one and three), implying that different MAP kinases serve distinct roles in the pathogenesis of GN. A major focus for research into the pathogenesis of glomerulonephritis (GN) has been to define extracellular growth-promoting factors controlling cell growth during proliferative forms of GN. Several cytokines are implicated in the pathogenesis of proliferative GN and may account for hypercellularity (1). Mitogen-activated protein (MAP) kinases are important mediators in the intracellular net- work of interacting proteins transducing extracellular cues to intracellular responses (2). Because in vitro studies have shown MAP kinases to be a key point in the control of cellular growth, these might be crucial mediators of cellular proliferation in renal diseases mediated by extracellular stimuli. Extracellular signal-regulated kinase (ERK), the best described mammalian MAP kinase, regulates the expression of many genes by phosphorylating several tran- scription factors (2). ERK's activity is regulated by MAP kinase/ERK kinase (MEK), which phosphorylates both threonine and tyrosine regulatory sites in ERK (2). In vitro studies have established the pivotal role of the ERK signaling pathway in the control of cellular proliferation (2). In contrast to ERK, the recently described MAP kinases stress-activated protein kinase (SAPK) and p38 MAP ki- nase are thought to inhibit cell growth and to induce apoptosis (2, 3). Although much is known about the func- tion of MAP kinases in vitro, little is known about their role in physiological or pathophysiological conditions or their activation in vivo.

Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Background More than half of the new patients admitted to dialysis therapy in some centers are diagnosed with type IIb diabetes, that is, diabetes associated with obesity. This study searched for a common final pathway of renal damage in this progressive renal disease. Methods The evolution of biochemical and morphological renal changes was examined in 6- to 60-week-old Zucker rats ( fa / fa -rats), a model of obesity associated with type II diabetes. Results fa / fa -rats exhibited pronounced hyperinsulinemia and hyperlipidemia at 6 weeks and became diabetic after 14 weeks of age. Significant focal segmental glomerulosclerosis was first noted in 18-week-old fa / fa -rats and tubulointerstitial damage and proteinuria in 40-week-old fa / fa -rats. A comparison of kidneys of six-week-old fa / fa -and lean control ( Fa /?) rats by immunohistology revealed a 1.8-fold increase in glomerular monocyte/macrophage counts in fa / fa -rats and a significant increase in de novo desmin expression in podocytes. Electron microscopy demonstrated an increase in the number of podocyte mitochondria and intracytoplasmic protein and fat droplets. Podocyte desmin scores markedly increased until week 18 in fa / fa -rats, whereas glomerular monocyte/macrophage counts peaked at 3.2-fold at week 14. Podocyte desmin expression, but not glomerular macrophage infiltration, correlated with damage in adjacent tubular cells, as evidenced by their de novo expression of vimentin. Progressive glomerular hypertrophy was detected in fa / fa -rats after 10 weeks. GBM width was significantly increased in 14-week-old fa / fa -rats as compared with lean controls. Mesangial cell activation ( de novo expression of α-smooth muscle actin) and proliferation was low to absent throughout the observation period in fa / fa -rats. Renal cell death counts (TUNEL) remained unchanged in 6- to 40-week-old fa / fa -rats. Tubulointerstitial myofibroblast formation and matrix accumulation occurred late during the study duration in fa / fa -rats. Conclusion These data suggest that early progressive podocyte damage and macrophage infiltration is associated with hyperlipidemia and type IIb diabetes mellitus, and antedates both the development of glomerulosclerosis and tubulointerstitial damage.