Differences in junction-associated gene expression changes in three rat models of diabetic retinopathy with similar neurovascular phenotype.

Diabetic retinopathy, also defined as microvascular complication of diabetes mellitus, affects the entire neurovascular unit with specific aberrations in every compartment. Neurodegeneration, glial activation and vasoregression are observed consistently in models of diabetic retinopathy. However, the order and the severity of these aberrations varies in different models, which is also true in patients. In this study, we analysed rat models of diabetic retinopathy with similar phenotypes to identify key differences in the pathogenesis. For this, we focussed on intercellular junction-associated gene expression, which are important for the communication and homeostasis within the neurovascular unit. Streptozotocin-injected diabetic Wistar rats, methylglyoxal supplemented Wistar rats and polycystin-2 transgenic (PKD) rats were analysed for neuroretinal function, vasoregression and retinal expression of junction-associated proteins. In all three models, neuroretinal impairment and vasoregression were observed, but gene expression profiling of junction-associated proteins demonstrated nearly no overlap between the three models. However, the differently expressed genes were from the main classes of claudins, connexins and integrins in all models. Changes in Rcor1 expression in diabetic rats and Egr1 expression in PKD rats confirmed the differences in upstream transcription factor level between the models. In PKD rats, a possible role for miRNA regulation was observed, indicated by an upregulation of miR-26b-5p, miR-122-5p and miR-300-3p, which was not observed in the other models. In silico allocation of connexins revealed not only differences in regulated subtypes, but also in affected retinal cell types, as well as connexin specific upstream regulators Sox7 and miR-92a-3p. In this study, we demonstrate that, despite their similar phenotype, models for diabetic retinopathy exhibit significant differences in their pathogenic pathways and primarily affected cell types. These results underline the importance for more sensitive diagnostic tools to identify pathogenic clusters in patients as the next step towards a desperately needed personalized therapy.

MicroRNA-124 Alleviates Retinal Vasoregression via Regulating Microglial Polarization.

Microglial activation is implicated in retinal vasoregression of the neurodegenerative ciliopathy-associated disease rat model (i.e., the polycystic kidney disease (PKD) model). microRNA can regulate microglial activation and vascular function, but the effect of microRNA-124 (miR-124) on retinal vasoregression remains unclear. Transgenic PKD and wild-type Sprague Dawley (SD) rats received miR-124 at 8 and 10 weeks of age intravitreally. Retinal glia activation was assessed by immunofluorescent staining and in situ hybridization. Vasoregression and neuroretinal function were evaluated by quantitative retinal morphometry and electroretinography (ERG), respectively. Microglial polarization was determined by immunocytochemistry and qRT-PCR. Microglial motility was examined via transwell migration assays, wound healing assays, and single-cell tracking. Our data showed that miR-124 inhibited glial activation and improved vasoregession, as evidenced by the reduced pericyte loss and decreased acellular capillary formation. In addition, miR-124 improved neuroretinal function. miR-124 shifted microglial polarization in the PKD retina from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by suppressing TNF-α, IL-1ß, CCL2, CCL3, MHC-II, and IFN-γ and upregulating Arg1 and IL-10. miR-124 also decreased microglial motility in the migration assays. The transcriptional factor of C/EBP-α-PU.1 signaling, suppressed by miR-124 both in vivo (PKD retina) and in vitro (microglial cells), could serve as a key regulator in microglial activation and polarization. Our data illustrate that miR-124 regulates microglial activation and polarization. miR-124 inhibits pericyte loss and thereby alleviates vasoregression and ameliorates neurovascular function.

The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein-coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy.

Kidney Injury Molecule-1 Is Specifically Expressed in Cystically-Transformed Proximal Tubules of the PKD/Mhm (cy/+) Rat Model of Polycystic Kidney Disease.

Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.

The SAM domain of ANKS6 has different interacting partners and mutations can induce different cystic phenotypes.

The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. METHODS: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. RESULTS: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. CONCLUSIONS: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.

A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex.

This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

Alzheimer's disease and retinal neurodegeneration share a consistent stress response of the neurovascular unit.

BACKGROUND: The pathogenesis of Alzheimer's disease (AD) is characterized by neuronal injury, activation of microglia and astrocytes, deposition of amyloid-ß and secondary vessel degeneration. In the polycystic kidney disease (PKD) rat model, we observed neuronal injury, microglial activation and vasoregression. We speculated that this neuroretinal degeneration shares important pathogenetic steps with AD. Therefore, we determined the activation of astrocytes and the accumulation of amyloid-ß in PKD retinae. METHODS: Immunohistochemistry of PKD retinae for vimentin, carboxymethyllysin, beta-Amyloid 1-42, High-Mobility-Group- Protein B1 and amyloid protein precursor was performed. RESULTS: Adjunct to astrocyte activation, accumulation of beta-Amyloid 1-42 and High-Mobility-Group-Protein B1 in astrocytes and around vessels of the superficial network was found in PKD retinae prior to the onset of vasoregression. Amyloid precursor protein was localized adjacent to the outer segment of photoreceptors in PKD and control rats. The parallel appearance of AD-related peptides indicates an alarmine based response to photoreceptor degeneration and secondary vasoregression. CONCLUSION: The model has broad overlap with AD and may be suitable to study beneficial pharmacological concepts.

Downregulation of the antioxidant protein peroxiredoxin 2 contributes to angiotensin II-mediated podocyte apoptosis.

Podocytes have a significant role in establishing selective permeability of the glomerular filtration barrier. Sustained renin-angiotensin-aldosterone system activation is crucial to the pathogenesis of podocyte injury, but the mechanisms by which angiotensin II modulates podocyte survival due to physiological or injurious stimuli remain unclear. Here, we used proteomic analysis to find new mediators of angiotensin II-induced podocyte injury. Antioxidant protein peroxiredoxin 2 expression was decreased in cultured podocytes stimulated with angiotensin II. Peroxiredoxin 2 was found to be expressed in podocytes in vivo, and its expression was decreased in the glomeruli of rats transgenic for angiotensin II type 1 receptors in a podocyte-specific manner, or in rats infused with angiotensin II. Downregulation of peroxiredoxin 2 in podocytes resulted in increased reactive oxygen species release, protein overoxidation, and inhibition of the Akt pathway. Both treatment with angiotensin II and downregulation of peroxiredoxin 2 expression led to apoptosis of podocytes. Thus, peroxiredoxin 2 is an important modulator of angiotensin II-induced podocyte injury.

Transgenic overexpression of Anks6(p.R823W) causes polycystic kidney disease in rats.

The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.

Microglial Activation Is Associated With Vasoprotection in a Rat Model of Inflammatory Retinal Vasoregression.

Vascular dysfunction and vasoregression are hallmarks of a variety of inflammatory central nervous system disorders and inflammation-related retinal diseases like diabetic retinopathy. Activation of microglia and the humoral innate immune system are contributing factors. Anti-inflammatory approaches have been proposed as therapies for neurovascular diseases, which include the modulation of microglial activation. The present study aimed at investigating the effects of microglial activation by clodronate-coated liposomes on vasoregression in a model of retinal degeneration. Clodronate treatment over 5 weeks led to an increase in activated CD74+ microglia and completely prevented acellular capillaries and pericyte loss. Gene expression analyses indicated that vasoprotection was due to the induction of vasoprotective factors such as Egr1, Stat3, and Ahr while expression of pro-inflammatory genes remained unchanged. We concluded that activated microglia led to a shift toward induction of pleiotropic protective pathways supporting vasoprotection in neurovascular retinal diseases.

Alternative endogenous protein processing via an autophagy-dependent pathway compensates for Yersinia-mediated inhibition of endosomal major histocompatibility complex class II antigen presentation.

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.

Protective effect of Soluble Epoxide Hydrolase Inhibition in Retinal Vasculopathy associated with Polycystic Kidney Disease.

Rationale: Vasoregression secondary to glial activation develops in various retinal diseases, including retinal degeneration and diabetic retinopathy. Photoreceptor degeneration and subsequent retinal vasoregression, characterized by pericyte loss and acellular capillary formation in the absence diabetes, are also seen in transgenic rats expressing the polycystic kidney disease (PKD) gene. Activated Müller glia contributes to retinal vasodegeneration, at least in part via the expression of the soluble epoxide hydrolase (sEH). Given that an increase in sEH expression triggered vascular destabilization in diabetes, and that vasoregression is similar in diabetic mice and PKD rats, the aim of the present study was to determine whether sEH inhibition could prevent retinal vasoregression in the PKD rat. Methods: One-month old male homozygous transgenic PKD rats were randomly allocated to receive vehicle or a sEH inhibitor (sEH-I; Sar5399, 30 mg/kg) for four weeks. Wild-type Sprague-Dawley (SD) littermates received vehicle as controls. Retinal sEH expression and activity were measured by Western blotting and LC-MS, and vasoregression was quantified in retinal digestion preparations. Microglial activation and immune response cytokines were assessed by immunofluorescence and quantitative PCR, respectively. 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) mediated Notch signaling, microglial activation and migration were assessed in vivo and in vitro. Results: This study demonstrates that sEH expression and activity were increased in PKD retinae, which led to elevated production of 19,20-DHDP and the depression of Notch signaling. The latter changes elicited pericyte loss and the recruitment of CD11b+/CD74+ microglia to the perivascular region. Microglial activation increased the expression of immune-response cytokines, and reduced levels of Notch3 and delta-like ligand 4 (Dll4). Treatment with Sar5399 decreased 19,20-DHDP generation and increased Notch3 expression. Sar5399 also prevented vasoregression by reducing pericyte loss and suppressed microglial activation as well as the expression of immune-response cytokines. Mechanistically, the activation of Notch signaling by Dll4 maintained a quiescent microglial cell phenotype, i.e. reduced both the surface presentation of CD74 and microglial migration. In contrast, in retinal explants, 19,20-DHDP and Notch inhibition both promoted CD74 expression and reversed the Dll4-induced decrease in migration. Conclusions: Our data indicate that 19,20-DHDP-induced alterations in Notch-signaling result in microglia activation and pericyte loss and contribute to retinal vasoregression in polycystic kidney disease. Moreover, sEH inhibition can ameliorate vasoregression through reduced activity of inflammatory microglia. sEH inhibition is thus an attractive new therapeutic approach to prevent retinal vasoregression.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy.

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes.

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin-angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of alpha actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of alpha actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis.

Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes.

Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.

Faster rates of post-puberty kidney deterioration in males is correlated with elevated oxidative stress in males vs females at early puberty.

BACKGROUND: Post-puberty deterioration of kidneys is more rapid in males than in females. To reveal the underlying molecular mechanisms for this difference, we analyzed gender-dependent gene expression in kidneys of three groups of 36 day-old rats. RESULTS: The number of genes exhibiting gender-dependent expression was highly influenced by the genetic background of the rat group examined. 373, 288 and 79 genes showed differential gene expression between males and females (p = 0.001) in US, Mhm and Mhm*BN rats, respectively. Of all gender dependently expressed genes, only 39 genes were differentially expressed in all tested groups and the direction of expression change was the same for those genes for all groups. The gene expression profile suggests higher metabolic and transport activities, enhanced cell proliferation, elevated oxidative stress, and altered vascular biology in males. Furthermore, elevated levels of superoxide anion (two- to three-fold) in males compared to females were detected at early puberty, but neither at pre-puberty nor at late puberty/early adulthood. CONCLUSION: Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.

Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression.

Angiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14%; p < 0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26%; p < 0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

ALCAM a novel biomarker in patients with type 2 diabetes mellitus complicated with diabetic nephropathy.

BACKGROUND & AIM: Activated leukocyte cell adhesion molecule (ALCAM/CD166) functions analogue to the receptor of advanced glycation end products, which has been implicated in the development of diabetic nephropathy (DN). We investigated the expression of ALCAM and its ligand S100B in patients with DN. METHODS: A total of 34 non-diabetic patients, 29 patients with type 2 diabetes and normal albuminuria and 107 patients with type 2 diabetes complicated with DN were assessed for serum concentration of soluble ALCAM (sALCAM) by ELISA. Expression of ALCAM and S100B in kidney histology from patients with DN was determined by immunohistochemistry. Cell expression of ALCAM and S100B was analyzed through confocal immunofluorescence microscopy. RESULTS: Serum concentration of sALCAM was increased in diabetic patients with DN compared to non-diabetic (59.85±14.99ng/ml vs. 126.88±66.45ng/ml, P<0.0001). Moreover sALCAM correlated positively with HbA1c (R=0.31, P<0.0001), as well as with the stages of chronic kidney disease and negatively correlated with eGFR (R=-0.20, P<0.05). In diabetic patients with normal albuminuria sALCAM was increased compared to patients with DN (126.88±66.45ng/ml vs. 197.50±37.17ng/ml, P<0.0001). In diabetic patients, ALCAM expression was significantly upregulated in both the glomeruli and tubules (P<0.001). ALCAM expression in the glomeruli correlated with presence of sclerosis (R=0.25, P<0.001) and localized mainly in the podocytes supporting the hypothesis that membrane bound ALCAM drives diabetic nephropathy and thus explaining sALCAM decrease in diabetic patients with DN. The expression of S100B was increased significantly in the glomeruli of diabetic patients (P<0.001), but not in the tubules. S100B was as well localized in the podocytes. CONCLUSIONS: This study identifies for the first time ALCAM as a potential mediator in the late complications of diabetes in the kidney.

CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD).

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.