The Aachen Minipig: Phenotype, Genotype, Hematological and Biochemical Characterization, and Comparison to the Göttingen Minipig.

BACKGROUND: The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. OBJECTIVE: In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. METHODS: Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. RESULTS: The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). CONCLUSION: The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Renal gene and protein expression signatures for prediction of kidney disease progression.

Although chronic kidney disease (CKD) is common, only a fraction of CKD patients progress to end-stage renal disease. Molecular predictors to stratify CKD populations according to their risk of progression remain undiscovered. Here we applied transcriptional profiling of kidneys from transforming growth factor-beta1 transgenic (Tg) mice, characterized by heterogeneity of kidney disease progression, to identify 43 genes that discriminate kidneys by severity of glomerular apoptosis before the onset of tubulointerstitial fibrosis in 2-week-old animals. Among the genes examined, 19 showed significant correlation between mRNA expression in uninephrectomized left kidneys at 2 weeks of age and renal disease severity in right kidneys of Tg mice at 4 weeks of age. Gene expression profiles of human orthologs of the 43 genes in kidney biopsies were highly significantly related (R(2) = 0.53; P < 0.001) to the estimated glomerular filtration rates in patients with CKD stages I to V, and discriminated groups of CKD stages I/II and III/IV/V with positive and negative predictive values of 0.8 and 0.83, respectively. Protein expression patterns for selected genes were successfully validated by immunohistochemistry in kidneys of Tg mice and kidney biopsies of patients with IgA nephropathy and CKD stages I to V, respectively. In conclusion, we developed novel mRNA and protein expression signatures that predict progressive renal fibrosis in mice and may be useful molecular predictors of CKD progression in humans.

The death ligand TRAIL in diabetic nephropathy.

Apoptotic cell death contributes to diabetic nephropathy (DN), but its role is not well understood. The tubulointerstitium from DN biopsy specimens was microdissected, and expression profiles of genes related to apoptosis were analyzed. A total of 112 (25%) of 455 cell death-related genes were found to be significantly differentially regulated. Among those that showed the greatest changes in regulation were two death receptors, OPG (the gene encoding osteoprotegerin) and Fas, and the death ligand TRAIL. Glomerular and proximal tubular TRAIL expression, assessed by immunohistochemistry, was higher in DN kidneys than controls and was associated with clinical and histologic severity of disease. In vitro, proinflammatory cytokines but not glucose alone regulated TRAIL expression in the human proximal tubular cell line HK-2. TRAIL induced tubular cell apoptosis in a dosage-dependant manner, an effect that was more marked in the presence of high levels of glucose and proinflammatory cytokines. TRAIL also activated NF-kappaB, and inhibition of NF-kappaB sensitized cells to TRAIL-induced apoptosis. It is proposed that TRAIL-induced cell death could play an important role in the progression of human DN.

Maternal environment interacts with modifier genes to influence progression of nephrotic syndrome.

Mutations in the NPHS2 gene, which encodes podocin, are responsible for some cases of sporadic and familial autosomal recessive steroid-resistant nephrotic syndrome. Inter- and intrafamilial variability in the progression of renal disease among patients bearing NPHS2 mutations suggests a potential role for modifier genes. Using a mouse model in which the podocin gene is constitutively inactivated, we sought to identify genetic determinants of the development and progression of renal disease as a result of the nephrotic syndrome. We report that the evolution of renal disease as a result of nephrotic syndrome in Nphs2-null mice depends on genetic background. Furthermore, the maternal environment significantly interacts with genetic determinants to modify survival and progression of renal disease. Quantitative trait locus mapping suggested that these genetic determinants may be encoded for by genes on the distal end of chromosome 3, which are linked to proteinuria, and on the distal end of chromosome 7, which are linked to a composite trait of urea, creatinine, and potassium. These loci demonstrate epistatic interactions with other chromosomal regions, highlighting the complex genetics of renal disease progression. In summary, constitutive inactivation of podocin models the complex interactions between maternal and genetically determined factors on the progression of renal disease as a result of nephrotic syndrome in mice.

A complex pattern of chemokine receptor expression is seen in osteosarcoma.

BACKGROUND: Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. METHODS: The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. RESULTS: Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. CONCLUSION: Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology of osteosarcoma, but also contradict aspects of previous reports describing the expression of these receptors in this tumor.

Modular activation of nuclear factor-kappaB transcriptional programs in human diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure and a major risk factor for cardiovascular mortality in diabetic patients. To evaluate the multiple pathogenetic factors implicated in DN, unbiased mRNA expression screening of tubulointerstitial compartments of human renal biopsies was combined with hypothesis-driven pathway analysis. Expression fingerprints obtained from biopsies with histological diagnosis of DN (n = 13) and from control subjects (pretransplant kidney donors [n = 7] and minimal change disease [n = 4]) allowed us to segregate the biopsies by disease state and stage by the specific expression signatures. Functional categorization showed regulation of genes linked to inflammation in progressive DN. Pathway mapping of nuclear factor-kappaB (NF-kappaB), a master transcriptional switch in inflammation, segregated progressive from mild DN and control subjects by showing upregulation of 54 of 138 known NF-kappaB targets. The promoter regions of regulated NF-kappaB targets were analyzed using ModelInspector, and the NF-kappaB module NFKB_IRFF_01 was found to be specifically enriched in progressive disease. Using this module, the induction of eight NFKB_IRFF_01-dependant genes was correctly predicted in progressive DN (B2M, CCL5/RANTES, CXCL10/IP10, EDN1, HLA-A, HLA-B, IFNB1, and VCAM1). The identification of a specific NF-kappaB promoter module activated in the inflammatory stress response of progressive DN has helped to characterize upstream pathways as potential targets for the treatment of progressive renal diseases such as DN.

Early glomerular filtration defect and severe renal disease in podocin-deficient mice.

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.

Stereospecific and redox-sensitive increase in monocyte adhesion to endothelial cells by homocysteine.

OBJECTIVE: Previous studies have shown that elevated homocysteine (Hcy) levels promote the development of atherosclerotic lesions in atherosclerosis-prone animal models. There is evidence that oxidant stress contributes to Hcy's deleterious effects on the vasculature. The accumulation and adhesion of monocytes to the vascular endothelium is a critical event in the development of atherosclerosis. We investigated the effects of Hcy on the interaction between human endothelial cells (EC) (EC line EA.hy 926 and primary human umbilical vein EC [HUVEC]) and the monocytic cell line THP-1, and the impact of vascular oxidant stress and redox-sensitive signaling pathways on these events. METHODS AND RESULTS: L-Hcy, but not D-Hcy, increases the production of reactive oxygen species inside EC, enhances nuclear factor(NF)-kappaB activation, and stimulates intercellular adhesion molecule-1 (ICAM-1) RNA transcription and cell surface expression. This leads to a time- and dose-dependent increase in monocyte adhesion to ECs. Pretreatment of ECs with superoxide scavengers (MnTBAP and Tiron) or with an inhibitor of NF-kappaB activation abolished Hcy-induced monocyte adhesion, ICAM-1 expression, and nuclear translocation of NF-kappaB. CONCLUSIONS: These findings suggest that reactive oxygen species produced under hyperhomocysteinemic conditions may induce a proinflammatory situation in the vessel wall that initiates and promotes atherosclerotic lesion development.

Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4.

The homing and tissue-specific recruitment of bone marrow-derived progenitor cells is a major issue in stem cell research and therapy. Chemokine biology plays a central role in the homing and trafficking of leukocytes. Here we show functional expression of the chemokine receptors CCR1, CCR4, CCR7, CCR10, and CXCR5 on primary isolates of CD34- mesenchymal progenitor cells as well as immortalized mesenchymal stem cell (MSC) lines. Although mRNA expression of CXCR4 was detected in both primary cells and immortalized clones, the receptor was not expressed on the cell surface. On the basis of this expression profile, the MSC could potentially home to secondary lymphatic organs (CCR7, CXCR5), skin (CCR4, CCR10), small intestine (CCR10), and salivary glands (CCR10). To study tissue-specific homing, murine CD34- MSC lines showing concordant chemokine receptor expression were either transiently labeled with CMFDA, or were stably transfected with green fluorescent protein (GFP) expression plasmids. The MSC were then injected into syngeneic healthy mice, and the distribution of the cells determined. The injected cells efficiently homed to spleen, thymus, and lymph nodes. In addition, cells were found in the mucosa of the small intestine, skin, and salivary gland. No significant recruitment to bone marrow, liver, or kidney was seen. Chemokine biology may play an important role in the homeostasis and potentially tissue recruitment of early adult progenitor cells.

Polyomavirus DNA and RNA detection in renal allograft biopsies: results from a European multicenter study.

Polyomavirus mediated nephropathy is an increasingly recognized complication in renal transplant recipients. In all, 362 renal biopsies collected from 15 European transplant centers were analyzed for presence of Polyomavirus nucleic acid (BK virus [BKV] and JC virus [JCV]). We evaluated 302 biopsies of patients with renal allograft dysfunction, including three with known BKV allograft nephropathy (BKVAN), and 60 native kidney biopsies. BKV DNA was detected in 8 of the 302 (2.6 %) biopsies obtained for transplant dysfunction, but in none of the controls. BKV RNA, indicating active viral replication, was found in all BKV DNA positive biopsies available for mRNA expression studies. Retrospective immunohistochemical staining was positive for SV40 large T antigen in all seven evaluated biopsies. BKV DNA and RNA were detected in biopsy tissues from patients with inconspicuous light microscopy for BKVAN. Further studies will evaluate the potential of intrarenal viral BKV RNA as an early predictor for BKVAN.

Expression of gremlin, a bone morphogenetic protein antagonist, in human diabetic nephropathy.

BACKGROUND: We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo. METHODS: Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease. RESULTS: Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score. CONCLUSION: In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.

CD20-positive infiltrates in human membranous glomerulonephritis.

Membranous glomerulonephritis (MGN), histologically defined by subepithelial immune deposits, is the most common cause of nephrotic syndrome in Caucasian adults. The current hypothesis of the underlying disease mechanism postulates production of antibodies against podocyte-derived antigens. Respective antigens could be demonstrated in different animal models and recently in human neonatal MGN. Further support for this hypothesis was generated by the response of human MGN to therapeutic B cell depletion by rituximab. However, the role of B cells in this disease is not well defined. In this study, the interstitial expression of CD20 mRNA was determined in 31 MGN patients and controls (tumor nephrectomies (n = 4), minimal change disease (MCD, n = 10) and focal segmental glomerulosclerosis (n = 6)). CD20 mRNA expression was significantly higher in MGN patients compared to controls. By immunohistochemistry, a focal or diffuse interstitial B cell infiltration could be detected in MGN patients (n = 63), which was absent or minimal in patients with MCD (n = 11). These data suggest an involvement of B cells in the pathogenesis of MGN, possibly as antigen-presenting cells. Further studies should investigate the potential to predict the response to therapeutic B cell depletion by intrarenal CD20 quantification, a potential diagnostic basis for the selection of a specific therapy currently evolving for renal disease.

A molecular signature of proteinuria in glomerulonephritis.

Proteinuria is the most important predictor of outcome in glomerulonephritis and experimental data suggest that the tubular cell response to proteinuria is an important determinant of progressive fibrosis in the kidney. However, it is unclear whether proteinuria is a marker of disease severity or has a direct effect on tubular cells in the kidneys of patients with glomerulonephritis. Accordingly we studied an in vitro model of proteinuria, and identified 231 "albumin-regulated genes" differentially expressed by primary human kidney tubular epithelial cells exposed to albumin. We translated these findings to human disease by studying mRNA levels of these genes in the tubulo-interstitial compartment of kidney biopsies from patients with IgA nephropathy using microarrays. Biopsies from patients with IgAN (n = 25) could be distinguished from those of control subjects (n = 6) based solely upon the expression of these 231 "albumin-regulated genes." The expression of an 11-transcript subset related to the degree of proteinuria, and this 11-mRNA subset was also sufficient to distinguish biopsies of subjects with IgAN from control biopsies. We tested if these findings could be extrapolated to other proteinuric diseases beyond IgAN and found that all forms of primary glomerulonephritis (n = 33) can be distinguished from controls (n = 21) based solely on the expression levels of these 11 genes derived from our in vitro proteinuria model. Pathway analysis suggests common regulatory elements shared by these 11 transcripts. In conclusion, we have identified an albumin-regulated 11-gene signature shared between all forms of primary glomerulonephritis. Our findings support the hypothesis that albuminuria may directly promote injury in the tubulo-interstitial compartment of the kidney in patients with glomerulonephritis.

A pilot study of gene expression-based categorization of pancreas transplant biopsies.

Gene expression profiling has emerged as a powerful strategy to define transcriptional mechanism activated in organ transplantation. We performed a pilot feasibility study of mRNA-based pancreas transplant biopsy stratification. The mRNAs expression of 32 genes, observed in renal transplant dysfunction, and 10 pancreas-specific genes were evaluated in 26 pancreas transplant biopsy specimens by quantitative real-time polymerase chain reaction using TaqMan Low Density Array technology. Unsupervised 2D hierarchical clustering segregated the biopsies in two main cluster branches, A and B. Six of seven patients (85.7%) in cluster A and 6 of 19 (31.6%) in cluster B retained functioning pancreas allograft. CD20/MS4A1 mRNA and protein, in addition to CD 3 protein, were detected in four specimens in cluster B. Three of those four pancreas transplants were subsequently lost. Our study demonstrates the potential association of gene expression with clinical outcome of pancreas transplants and justifies further studies in an independent cohort.

Enhanced expression of Janus kinase-signal transducer and activator of transcription pathway members in human diabetic nephropathy.

OBJECTIVE: Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure. RESEARCH DESIGN AND METHODS: Kidney biopsies were obtained from 74 patients (control subjects, early and progressive type 2 diabetic nephropathy). Glomerular and tubulointerstitial mRNAs were microarrayed, followed by bioinformatics analyses. Gene expression changes were confirmed by real-time RT-PCR and immunohistological staining. Samples from db/db C57BLKS and streptozotocin-induced DBA/2J mice, commonly studied murine models of diabetic nephropathy, were analyzed. RESULTS: In human glomeruli and tubulointerstitial samples, the Janus kinase (Jak)-signal transducer and activator of transcription (Stat) pathway was highly and significantly regulated. Jak-1, -2, and -3 as well as Stat-1 and -3 were expressed at higher levels in patients with diabetic nephropathy than in control subjects. The estimated glomerular filtration rate significantly correlated with tubulointerstitial Jak-1, -2, and -3 and Stat-1 expression (R(2) = 0.30-0.44). Immunohistochemistry found strong Jak-2 staining in glomerular and tubulointerstitial compartments in diabetic nephropathy compared with control subjects. In contrast, there was little or no increase in expression of Jak/Stat genes in the db/db C57BLKS or diabetic DBA/2J mice. CONCLUSIONS: These data suggest a direct relationship between tubulointerstitial Jak/Stat expression and progression of kidney failure in patients with type 2 diabetic nephropathy and distinguish progressive human diabetic nephropathy from nonprogressive murine diabetic nephropathy.

Modification of kidney barrier function by the urokinase receptor.

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Nitric oxide modulates expression of extracellular matrix genes linked to fibrosis in kidney mesangial cells.

Mesangial cells are thought to be important mediators of glomerular inflammation and fibrosis. Studies have established a direct role for nitric oxide (NO) in the regulation of gene expression in mesangial cells. Representational difference analysis was used to investigate changes in gene expression elicited by the treatment of S-nitroso-L-glutathione in rat mesangial cells. Seven upregulated and 11 downregulated genes were identified. Four out of 11 downregulated genes (connective tissue growth factor, thrombospondin-1, collagen type I alpha1 and collagen type I alpha2) are known to be linked to inflammation and fibrosis. Results were verified across species in mesangial cells treated with a series of NO donors using Northern blot analysis, quantitative real-time PCR and protein analysis methods. Induction of endogenous NO production by cytokine stimulation also triggered regulation of the genes. One example gene, connective tissue growth factor, was studied at the promoter level. Promoter-reporter gene studies in mesangial cells demonstrated that NO acts at the transcriptional level to suppress gene expression. Our results reveal a complex role of NO in regulating gene expression in mesangial cells and suggest an antifibrotic potential for NO.

Interstitial vascular rarefaction and reduced VEGF-A expression in human diabetic nephropathy.

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes. Although the majority of DN models and human studies have focused on glomeruli, tubulointerstitial damage is a major feature of DN and an important predictor of renal dysfunction. This study sought to investigate molecular markers of pathogenic pathways in the renal interstitium of patients with DN. Microdissected tubulointerstitial compartments from biopsies with established DN and control kidneys were subjected to expression profiling. Analysis of candidate genes, potentially involved in DN on the basis of common hypotheses, identified 49 genes with significantly altered expression levels in established DN in comparison with controls. In contrast to some rodent models, the growth factors vascular endothelial growth factor A (VEGF-A) and epidermal growth factor (EGF) showed a decrease in mRNA expression in DN. This was validated on an independent cohort of patients with DN by real-time reverse transcriptase-PCR. Immunohistochemical staining for VEGF-A and EGF also showed a reduced expression in DN. The decrease of renal VEGF-A expression was associated with a reduction in peritubular capillary densities shown by platelet-endothelial cell adhesion molecule-1/CD31 staining. Furthermore, a significant inverse correlation between VEGF-A and proteinuria, as well as EGF and proteinuria, and a positive correlation between VEGF-A and hypoxia-inducible factor-1alpha mRNA was found. Thus, in human DN, a decrease of VEGF-A, rather than the reported increase as described in some rodent models, may contribute to the progressive disease. These findings and the questions about rodent models in DN raise a note of caution regarding the proposal to inhibit VEGF-A to prevent progression of DN.

Induction of TRPC6 channel in acquired forms of proteinuric kidney disease.

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.