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1.  Genetic Analysis of Intracapillary Glomerular Lipoprotein Deposits in Aging Mice 
PLoS ONE  2014;9(10):e111308.
Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes.
Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0–4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping.
Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3.
By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponses.
PMCID: PMC4213026  PMID: 25353171
2.  Identification of Novel Genes Associated with Renal Tertiary Lymphoid Organ Formation in Aging Mice 
PLoS ONE  2014;9(3):e91850.
A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10−137) in male mice; Ctnnbip1 (P = 6.42×10−267) and Tnfrsf8 (P = 5.42×10−245) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.
PMCID: PMC3956762  PMID: 24637805
3.  Gaseous Hydrogen Sulfide Protects against Myocardial Ischemia-Reperfusion Injury in Mice Partially Independent from Hypometabolism 
PLoS ONE  2013;8(5):e63291.
Ischemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes. Gaseous hydrogen sulfide (H2S) is protective during IRI by inducing a hypometabolic state in mice which is associated with anti-apoptotic, anti-inflammatory and antioxidant properties. We investigated whether gaseous H2S administration is protective in cardiac IRI and whether non-hypometabolic concentrations of H2S have similar protective properties.
Male C57BL/6 mice received a 0, 10, or 100 ppm H2S-N2 mixture starting 30 minutes prior to ischemia until 5 minutes pre-reperfusion. IRI was inflicted by temporary ligation of the left coronary artery for 30 minutes. High-resolution respirometry equipment was used to assess CO2-production and blood pressure was measured using internal transmitters. The effects of H2S were assessed by histological and molecular analysis.
Treatment with 100 ppm H2S decreased CO2-production by 72%, blood pressure by 14% and heart rate by 25%, while treatment with 10 ppm H2S had no effects. At day 1 of reperfusion 10 ppm H2S showed no effect on necrosis, while treatment with 100 ppm H2S reduced necrosis by 62% (p<0.05). Seven days post-reperfusion, both 10 ppm (p<0.01) and 100 ppm (p<0.05) H2S showed a reduction in fibrosis compared to IRI animals. Both 10 ppm and 100 ppm H2S reduced granulocyte-influx by 43% (p<0.05) and 60% (p<0.001), respectively. At 7 days post-reperfusion both 10 and 100 ppm H2S reduced expression of fibronectin by 63% (p<0.05) and 67% (p<0.01) and ANP by 84% and 63% (p<0.05), respectively.
Gaseous administration of H2S is protective when administered during a cardiac ischemic insult. Although hypometabolism is restricted to small animals, we now showed that low non-hypometabolic concentrations of H2S also have protective properties in IRI. Since IRI is a frequent cause of myocardial damage during percutaneous coronary intervention and cardiac transplantation, H2S treatment might lead to novel therapeutical modalities.
PMCID: PMC3651205  PMID: 23675473
4.  CUBN as a Novel Locus for End-Stage Renal Disease: Insights from Renal Transplantation 
PLoS ONE  2012;7(5):e36512.
Chronic kidney disease (CKD) is a complex disorder. As genome-wide association studies identified cubilin gene CUBN as a locus for albuminuria, and urinary protein loss is a risk factor for progressive CKD, we tested the hypothesis that common genetic variants in CUBN are associated with end-stage renal disease (ESRD) and proteinuria. First, a total of 1142 patients with ESRD, admitted for renal transplantation, and 1186 donors were genotyped for SNPs rs7918972 and rs1801239 (case-control study). The rs7918972 minor allele frequency (MAF) was higher in ESRD patients comparing to kidney donors, implicating an increased risk for ESRD (OR 1.39, p = 0.0004) in native kidneys. Second, after transplantation recipients were followed for 5.8 [3.8–9.2] years (longitudinal study) documenting ESRD in transplanted kidneys – graft failure (GF). During post-transplant follow-up 92 (9.6%) cases of death-censored GF occurred. Donor rs7918972 MAF, representing genotype of the transplanted kidney, was 16.3% in GF vs 10.7% in cases with functioning graft. Consistently, a multivariate Cox regression analysis showed that donor rs7918972 is a predictor of GF, although statistical significance was not reached (HR 1.53, p = 0.055). There was no association of recipient rs7918972 with GF. Rs1801239 was not associated with ESRD or GF. In line with an association with the outcome, donor rs7918972 was associated with elevated proteinuria levels cross-sectionally at 1 year after transplantation. Thus, we identified CUBN rs7918972 as a novel risk variant for renal function loss in two independent settings: ESRD in native kidneys and GF in transplanted kidneys.
PMCID: PMC3344899  PMID: 22574174
5.  Differential Expression of Proteoglycans in Tissue Remodeling and Lymphangiogenesis after Experimental Renal Transplantation in Rats 
PLoS ONE  2010;5(2):e9095.
Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction.
Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence.
Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function.
Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.
PMCID: PMC2816722  PMID: 20140097
6.  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

Results 1-6 (6)