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1.  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.
PMCID: PMC4926336  PMID: 27231899
Kim-1; polycystic kidney; rat model; proximal tubule; dedifferentiation
2.  ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice 
PLoS ONE  2015;10(9):e0136781.
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.
PMCID: PMC4556665  PMID: 26327442
3.  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 
Nephrology Dialysis Transplantation  2013;28(8):2045-2058.
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.
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.
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.
Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.
PMCID: PMC3983428  PMID: 23543593
catechol-O-methyltransferase; gene expression profiling; modifier; polycystic kidney disease; tolcapone
4.  Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration 
PLoS ONE  2014;9(7):e102013.
Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.
PMCID: PMC4094460  PMID: 25013951
5.  Müller Cell Reactivity in Response to Photoreceptor Degeneration in Rats with Defective Polycystin-2 
PLoS ONE  2013;8(6):e61631.
Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2.
Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution.
Müller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators.
Cellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.
PMCID: PMC3670868  PMID: 23755094
6.  Alternative Endogenous Protein Processing via an Autophagy-Dependent Pathway Compensates for Yersinia-Mediated Inhibition of Endosomal Major Histocompatibility Complex Class II Antigen Presentation▿  
Infection and Immunity  2010;78(12):5138-5150.
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.
PMCID: PMC2981299  PMID: 20876292
7.  Gene Expression Profiling of Vasoregression in the Retina—Involvement of Microglial Cells 
PLoS ONE  2011;6(2):e16865.
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
PMCID: PMC3040753  PMID: 21379381
8.  Vasoregression Linked to Neuronal Damage in the Rat with Defect of Polycystin-2 
PLoS ONE  2009;4(10):e7328.
Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.
At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.
Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.
PMCID: PMC2752170  PMID: 19806208
9.  Pericyte Migration 
Diabetes  2008;57(9):2495-2502.
OBJECTIVE— The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration.
RESEARCH DESIGN AND METHODS— Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice.
RESULTS— Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (−20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates.
CONCLUSIONS— Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.
PMCID: PMC2518502  PMID: 18559662
10.  A renin transcript lacking exon 1 encodes for a non-secretory intracellular renin that increases aldosterone production in transgenic rats 
Renin transcripts lacking exon 1 and thus the signal sequence for co-translational transport to the endoplasmatic reticulum encode for a protein (exon[2-9]renin), that is confined to the cytoplasm. The function of exon(2-9)renin is currently unknown. Mitochondrial renin increases under conditions which stimulate aldosterone production. We hypothesized that exon(2-9)renin (1) is translated into a functionally active protein in vivo, (2) is not secreted but remains within the cytoplasm and (3) stimulates aldosterone production. To test these hypotheses we generated transgenic rats overexpressing exon(2-9)renin. Four transgenic lines were obtained expressing the transcript in various tissues including the heart and the adrenal gland. Renin was enriched particularly in the cytoplasm of transgenic rats. Renin was not elevated in plasma, indicating that exon(2-9)renin is produced but not secreted. The ratio of aldosterone to renin concentrations in plasma (PAC/PRC) was elevated in all transgenic lines except line 307, which also did not exhibit elevated cytoplasmatic renin levels in the adrenal gland (PAC/PRC in controls: 2.8±2.3; line 307: 1.9±0.8; n. s.; line 284: 5.8±1.9; P<0.02; line 294: 5.0±2.3; P<0.001; line 276: 10.3±5.1; P<0.001). We conclude that the exon(1A-9) renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX-(exon2-9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A-9) transcripts.
PMCID: PMC3865667  PMID: 18782187
renin-angiotensin system; cytoplasmatic renin; adrenal gland; aldosterone; transgenic rats
11.  Faster rates of post-puberty kidney deterioration in males is correlated with elevated oxidative stress in males vs females at early puberty 
BMC Genomics  2007;8:221.
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.
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.
Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.
PMCID: PMC1934371  PMID: 17620128
12.  Downregulation of the antioxidant protein peroxiredoxin 2 contributes to angiotensin II–mediated podocyte apoptosis 
Kidney International  2011;80(9):959-969.
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.
PMCID: PMC3257043  PMID: 21814176
Akt; angiotensin II; apoptosis; peroxiredoxin 2; podocyte; reactive oxygen species

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