Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Patterns of differentiation of renin lineage cells during nephrogenesis.

Developmentally heterogeneous renin-expressing cells serve as progenitors for mural, glomerular, and tubular cells during nephrogenesis and are collectively termed renin lineage cells (RLCs). In this study, we quantified different renal vascular and tubular cell types based on specific markers and assessed proliferation and de novo differentiation in the RLC population. We used kidney sections of mRenCre-mT/mG mice throughout nephrogenesis. Marker positivity was evaluated in whole digitalized sections. At embryonic day 16, RLCs appeared in the developing kidney, and the expression of all stained markers in RLCs was observed. The proliferation rate of RLCs did not differ from the proliferation rate of non-RLCs. RLCs expanded mainly by de novo differentiation (neogenesis). Fractions of RLCs originating from the stromal progenitors of the metanephric mesenchyme (renin-producing cells, vascular smooth muscle cells, and mesangial cells) decreased during nephrogenesis. In contrast, aquaporin-2-positive RLCs in the collecting duct system, which embryonically emerges almost exclusively from the ureteric bud, expanded postpartum. The cubilin-positive RLC fraction in the proximal tubule, deriving from the cap mesenchyme, remained constant. In summary, RLCs were continuously detectable in the vascular and tubular compartments of the kidney during nephrogenesis. Therein, various patterns of RLC differentiation that depend on the embryonic origin of the cells were identified.NEW & NOTEWORTHY The unifying feature of the renal renin lineage cells (RLCs) is their origin from renin-expressing progenitors. RLCs evolve to an embryologically heterogeneous large population in structures with different ancestry. RLCs are also targets for the widely used renin-angiotensin-system blockers, which modulate their phenotype. Unveiling the different differentiation patterns of RLCs in the developing kidney contributes to understanding changes in their cell fate in response to homeostatic challenges and the use of antihypertensive drugs.

Renin cells with defective Gsα/cAMP signaling contribute to renal endothelial damage.

Synthesis of renin in renal renin-producing cells (RPCs) is controlled via the intracellular messenger cAMP. Interference with cAMP-mediated signaling by inducible knockout of Gs-alpha (Gsα) in RPCs of adult mice resulted in a complex adverse kidney phenotype. Therein, glomerular endothelial damage was most striking. In this study, we investigated whether Gsα knockout leads to a loss of RPCs, which itself may contribute to the endothelial injury. We compared the kidney phenotype of three RPC-specific conditional mouse lines during continuous induction of recombination. Mice expressing red fluorescent reporter protein tdTomato (tdT) in RPCs served as controls. tdT was also expressed in RPCs of the other two strains used, namely with RPC-specific Gsα knockout (Gsα mice) or with RPC-specific diphtheria toxin A expression (DTA mice, in which the RPCs should be diminished). Using immunohistological analysis, we found that RPCs decreased by 82% in the kidneys of Gsα mice as compared with controls. However, the number of tdT-positive cells was similar in the two strains, demonstrating that after Gsα knockout, the RPCs persist as renin-negative descendants. In contrast, both renin-positive and tdT-labeled cells decreased by 80% in DTA mice suggesting effective RPC ablation. Only Gsα mice displayed dysregulated endothelial cell marker expression indicating glomerular endothelial damage. In addition, a robust induction of genes involved in tissue remodelling with microvascular damage was identified in tdT-labeled RPCs isolated from Gsα mice. We concluded that Gsα/renin double-negative RPC progeny essentially contributes for the development of glomerular endothelial damage in our Gsα-deficient mice.

Origin and Consequences of Necroinflammation.

When cells undergo necrotic cell death in either physiological or pathophysiological settings in vivo, they release highly immunogenic intracellular molecules and organelles into the interstitium and thereby represent the strongest known trigger of the immune system. With our increasing understanding of necrosis as a regulated and genetically determined process (RN, regulated necrosis), necrosis and necroinflammation can be pharmacologically prevented. This review discusses our current knowledge about signaling pathways of necrotic cell death as the origin of necroinflammation. Multiple pathways of RN such as necroptosis, ferroptosis, and pyroptosis have been evolutionary conserved most likely because of their differences in immunogenicity. As the consequence of necrosis, however, all necrotic cells release damage associated molecular patterns (DAMPs) that have been extensively investigated over the last two decades. Analysis of necroinflammation allows characterizing specific signatures for each particular pathway of cell death. While all RN-pathways share the release of DAMPs in general, most of them actively regulate the immune system by the additional expression and/or maturation of either pro- or anti-inflammatory cytokines/chemokines. In addition, DAMPs have been demonstrated to modulate the process of regeneration. For the purpose of better understanding of necroinflammation, we introduce a novel classification of DAMPs in this review to help detect the relative contribution of each RN-pathway to certain physiological and pathophysiological conditions.

ADMA reduction does not protect mice with streptozotocin-induced diabetes mellitus from development of diabetic nephropathy.

BACKGROUND AND AIMS: Cardiovascular disease is the major cause of morbidity and mortality in the world. Diabetes and its complications, such as diabetic nephropathy, dramatically increase cardiovascular risk. Association studies suggest that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, plays a role in the pathogenesis of diabetic nephropathy. The major pathway of ADMA catabolism is hydrolysis by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that lowering ADMA by overexpression of DDAH1 protects from development of diabetic nephropathy. METHODS: Diabetes was induced with streptozotocin (STZ) in wild type and DDAH1 transgenic mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. ADMA levels were assessed by isotope-dilution tandem mass spectrometry, creatinine by standard laboratory methods and albumin by ELISA. Kidney tissues were stained for markers of glomerular cells, cell matrix, inflammation and cell proliferation. RESULTS: STZ led to development of diabetes in all injected mice. Transgenic overexpression of DDAH1 led to a decrease in plasma ADMA levels in healthy animals. Diabetic state itself did not lead to elevation of plasma ADMA levels. Diabetic mice of both genotypes developed albuminuria (27 and 25 vs. 9 and 6 µg albumin/mg creatinine) (p < 0.01). There were no changes in glomerular matrix expansion, podocyte injury, inflammatory or proliferative response. CONCLUSIONS: STZ-induced diabetes led to the development of early features of diabetic nephropathy. Overexpression of DDAH1 and lowering of systemic ADMA levels did not prevent these changes, indicating that ADMA is not the major mediator of the early diabetic changes reflected by this experimental model.

Interference with Gsα-Coupled Receptor Signaling in Renin-Producing Cells Leads to Renal Endothelial Damage.

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

Cold Shock Proteins Mediate GN with Mesangioproliferation.

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

Extrarenal Progenitor Cells Do Not Contribute to Renal Endothelial Repair.

Endothelial progenitor cells (EPCs) may be relevant contributors to endothelial cell (EC) repair in various organ systems. In this study, we investigated the potential role of EPCs in renal EC repair. We analyzed the major EPC subtypes in murine kidneys, blood, and spleens after induction of selective EC injury using the concanavalin A/anti-concanavalin A model and after ischemia/reperfusion (I/R) injury as well as the potential of extrarenal cells to substitute for injured local EC. Bone marrow transplantation (BMTx), kidney transplantation, or a combination of both were performed before EC injury to allow distinction of extrarenal or BM-derived cells from intrinsic renal cells. During endothelial regeneration, cells expressing markers of endothelial colony-forming cells (ECFCs) were the most abundant EPC subtype in kidneys, but were not detected in blood or spleen. Few cells expressing markers of EC colony-forming units (EC-CFUs) were detected. In BM chimeric mice (C57BL/6 with tandem dimer Tomato-positive [tdT+] BM cells), circulating and splenic EC-CFUs were BM-derived (tdT+), whereas cells positive for ECFC markers in kidneys were not. Indeed, most BM-derived tdT+ cells in injured kidneys were inflammatory cells. Kidneys from C57BL/6 donors transplanted into tdT+ recipients with or without prior BMTx from C57BL/6 mice were negative for BM-derived or extrarenal ECFCs. Overall, extrarenal cells did not substitute for any intrinsic ECs. These results demonstrate that endothelial repair in mouse kidneys with acute endothelial lesions depends exclusively on local mechanisms.

Inducible glomerular erythropoietin production in the adult kidney.

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

Platelets are relevant mediators of renal injury induced by primary endothelial lesions.

Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown.

Renin lineage cells repopulate the glomerular mesangium after injury.

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

A new apparatus for standardized rat kidney biopsy.

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

Platelets are not critical effector cells for the time course of murine passive crescentic glomerulonephritis.

Although platelets are well-known effector cells of inflammatory renal disease, clinical studies were not able to establish platelet inhibition as an effective therapy. Our previous studies using Vasodilator stimulated Phosphoprotein- and P2Y1-deficient mice suggested some early, but no long-term effects of platelets in passive crescentic glomerulonephritis. To define the role of platelets for this disease model, passive crescentic glomerulonephritis was induced in 72 C57Bl/6 mice by intraperitoneal injection of sheep anti-rabbit glomerular basement membrane antibody on 2 consecutive days. Platelets were depleted using anti-glycoprotein Ibα antibodies (p0p3/p0p4) every 4th day. Mice treated with equal amounts of sterile Phosphate buffered solution or rat-IgG served as controls. Blood, urine, and tissues were harvested on days 3 and 28. Renal tissue sections were evaluated after immunostaining using (semi)quantitative and computer-assisted image analysis. Compared to controls, efficient depletion was achieved as indicated by a markedly prolonged bleeding time and a more than 90% reduction in platelet counts (800/nl vs. 42/nl; P < 0.001). Functional (creatinine-clearance and proteinuria) parameters demonstrated no significant differences between the groups. Neither parameters of renal injury (glomerulosclerosis and fibrosis) nor glomerular/tubulointerstitial matrix expansion (by collagen IV staining), glomerular capillary rarefaction (lectin staining), and the glomerular/tubulointerstitial proliferative response (proliferating cell nuclear antigen) demonstrated any differences between platelet-depleted mice and PBS- or rat-IgG-treated nephritic mice at any time point. Despite effective platelet inhibition/depletion, neither the short- nor long-term course of passive crescentic nephrotoxic nephritis was affected. These data indicate that platelets play a minor role during the time course of this disease model in the mouse.

PDGF-C mediates glomerular capillary repair.

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.

Autocrine VEGF-VEGF-R loop on podocytes during glomerulonephritis in humans.

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

Enhanced progenitor cell recruitment and endothelial repair after selective endothelial injury of the mouse kidney.

Primary and/or secondary injury of the renal microvascular endothelium is a common finding in various renal diseases. Besides well-known endothelial repair mechanisms, including endothelial cell (EC) proliferation and migration, homing of extrinsic cells such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC) has been shown in various organs and may contribute to microvascular repair. However, these mechanisms have so far not been studied after selective microvascular injury in the kidney. The present study investigated the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation. Erythropoietin was used to stimulate progenitor cells in a therapeutic approach. We found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors to orchestrate microvascular repair. Angiogenic factors started to increase as early as 30 min after disease induction. Progenitor cells could be first detected in the circulation and the spleen before they selectively homed to the diseased kidney. Injection of a high dose of erythropoietin 2 h after disease induction markedly attenuated vascular injury through nonhemodynamic mechanisms, possibly involving vascular endothelial growth factor release.

Correlation of enhanced thrombospondin-1 expression, TGF-beta signalling and proteinuria in human type-2 diabetic nephropathy.

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

A murine model of site-specific renal microvascular endothelial injury and thrombotic microangiopathy.

Despite the importance of endothelial injury and healing for primary and secondary renal disease and the availability of genetically engineered mouse models, to date no generally applicable murine disease model with site-specific renal endothelial injury has been established. We induced specific microvascular renal injury via selective renal arterial perfusion of the lectin concanavalin A (Con A) followed by sheep anti-concanavalin A and harvested tissues after 4 h, 24 h, days 3 and 7. Compared to control kidneys, histological evaluation demonstrated endothelial cell injury with subsequent complement, and platelet activation and thrombosis by light and electron microscopy. Mouse kidneys showed histologic evidence of severe glomerular and peritubular microvascular thrombosis with acute tubular necrosis, proteinuria, increased BUN and presence of schistocytes. Initial cell death of intrinsic renal cells resulted in a decrease of the glomerular cell count by 50% after 4 h followed by a proliferative response of glomerular (day 3, P < 0.05), interstitial (day 3, P < 0.05) and tubular cells leading to increased total glomerular cell count by day 7. This study establishes the Con A anti-Con A model as specific endothelial injury model in the mouse kidney providing a novel tool for investigating endothelial injury and repair mechanisms as well as elucidating the role of platelets in genetically engineered mice.

Analysis of NO-synthase expression and clinical risk factors in human diabetic nephropathy.

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

P2Y1 gene deficiency protects from renal disease progression and capillary rarefaction during passive crescentic glomerulonephritis.

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