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Anette Melk

Publications and source records attributed to Anette Melk.

13 recordsLinked to original sources
PubMed2005

Transcriptional analysis of the molecular basis of human kidney aging using cDNA microarray profiling.

BACKGROUND: The molecular basis of renal aging is not completely understood. METHODS: We used global gene expression monitoring by cDNA microarrays to identify age associated genes in human kidney samples. Our samples included young (8 weeks-8 years, N= 4), adult (31-46 years, N= 7), and old kidneys (71-88 years, N= 9). RESULTS: Old kidneys had more glomerulosclerosis, tubular atrophy, interstitial fibrosis, and fibrous intimal thickening in small arteries. We identified approximately 500 genes that were differentially expressed among the three age groups. Old kidneys appeared to have increased extracellular matrix turnover and a nonspecific inflammatory response, combined with a reduction in processes dependent on energy metabolism and mitochondrial function. Quantitative supervised bioinformatics analyses of adult and old kidney expression data correlated the expression of 255 gene profiles with renal pathology scores. Microarray class prediction analysis (PAM) identified 50 unique genes that segregated old kidneys into two distinct clusters: those more similar within age class (OO, N= 5) versus old kidneys more similar to adult kidneys (OA, N= 4). The expression of six functionally significant genes was further validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) (FN1, MMP7, TNC, SERPIN3A, BPHL, CSPG2) in the experiment group and, subsequently, confirmed independently in 17 additional old and adult age-stratified test kidney samples. The p53 inducible gene, CSPG2, performed best in separating OO kidneys from adults and OA samples in this analysis. CONCLUSION: The method described in this study using independent validation samples can be envisioned to test utility of the identified genes in assessing age-related changes that contribute to decline in renal function.

Adult↗
PubMed2005

Epithelial to mesenchymal transition during late deterioration of human kidney transplants: the role of tubular cells in fibrogenesis.

The hallmark of failing renal transplants is tubular atrophy and interstitial fibrosis (TA/IF). Injury to tubular epithelial cells (TEC) could contribute to fibrogenesis via epithelial-mesenchymal transition (EMT). We examined the features of EMT in renal transplants that developed TA/IF. Biopsies from 10 allograft kidneys with impaired function and TA/IF and 10 biopsies from transplants with stable function were compared to their implantation biopsies. Relative to implantation biopsies, TEC in TA/IF kidneys showed loss of epithelial markers (E-cadherin, cytokeratin) with altered distribution. Some TEC also showed new cytoplasmic expression of mesenchymal markers vimentin, S100A4, and alpha smooth muscle actin (alpha-SMA) and collagen synthesis marker heat shock protein (HSP-47), both in deteriorating and atrophic tubules. Double immunostaining showed coexpression of cytokeratin and vimentin, S100A4 and HSP-47, indicating intermediate stages of EMT in TA/IF. These changes were absent or much less in transplants with stable function. EMT features in the TA/IF group correlated with serum creatinine (vimentin, S100A4, HSP-47), history of T-cell-mediated rejection (cytokeratin, S100A4) and proteinuria (cytokeratin). These findings support a model in which the TEC damage induces loss of epithelial features and expression of fibroblast features, as a common pathway of deterioration by either immunologic or nonimmunologic processes.

Actins↗
PubMed2005

Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney.

Some features of kidney transplants with dysfunction overlap the lesions of aging, such as tubular atrophy and interstitial fibrosis (TA/IF) without major glomerular abnormalities. Somatic cell limitations could contribute to deterioration in aging and disease states. Since expression of p16(INK4a), a cell cycle inhibitor associated with somatic cell senescence in vitro, is induced in aged kidney, we studied whether kidneys with dysfunction and TA/IF manifested increased p16(INK4a) expression. We performed p16(INK4a) immunostaining on transplanted kidneys and native kidneys with chronic renal diseases. At implantation, transplants manifested little TA/IF, and nuclear p16(INK4a) immunostaining was consistent with age. However, transplants biopsied for abnormal function displaying TA/IF showed strong nuclear and cytoplasmic p16(INK4a) staining, beyond the amount predicted for age. Both atrophic and non-atrophic nephrons displayed increased p16(INK4a), suggesting that it was not simply a feature of atrophy. Epithelial p16(INK4a) staining was not increased in transplants with good function, but was increased in diseased native kidneys. The finding of increased p16(INK4a) expression in renal transplants and diseased kidneys with TA/IF and impaired function supports the concept that some cell senescence changes that accompany aging are also induced by injury and disease stresses. Thus, aging, injury and disease may share common pathways involving somatic cell senescence.

Adult↗
PubMed2005

Expression of CTL associated transcripts precedes the development of tubulitis in T-cell mediated kidney graft rejection.

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte-associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T-cell-mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.

Animals↗
PubMed2004

Longitudinal analysis of T-helper cell phenotypes in renal-transplant recipients undergoing growth hormone therapy.

BACKGROUND: Treatment with recombinant human growth hormone (rhGH) in growth-retarded children after renal transplantation is effective, but there have been concerns regarding the safety of rhGH because of its possible immunomodulatory actions. We therefore evaluated the immune phenotypes of pediatric renal-transplant recipients and controls in response to rhGH with regard to a possible shift toward a T-helper (TH)1-type response. METHODS: Intracellular cytokines, activation markers, costimulatory, and adhesion molecules were studied in 13 children after renal transplantation (Tx+GH). Children with chronic renal failure (CRF+GH, n=11) before and under rhGH therapy and pediatric renal-transplant recipients without rhGH therapy (Tx, n=33) served as controls. Measurements were performed by four-color flow cytometry before and 4, 12, 18 and 24 weeks after initiation of rhGH therapy. RESULTS: Under baseline conditions, Tx+GH patients did not differ from Tx patients. During rhGH therapy in children with transplants, interleukin (IL)-2 production increased threefold at 4 weeks, and IL-4 and IL-13 increased by 70% at 12 weeks. All three cytokines returned to baseline after 18 weeks. No patient experienced rejection. In CRF+GH patients, baseline values for all investigated cytokines were higher than in patients with transplants but did not change in response to rhGH therapy. CONCLUSION: Our data indicates that rhGH therapy in stable, pediatric renal-transplant recipients has a mild and transient immunostimulatory effect in vivo. Immunosuppression and graft function in patients with transplants undergoing rhGH treatment should therefore carefully be monitored.

Adolescent↗
PubMed2004

Expression of p16INK4a and other cell cycle regulator and senescence associated genes in aging human kidney.

BACKGROUND: Somatic cells in vitro have a finite life expectancy before entering a state of senescence. If this state has an in vivo counterpart, it could contribute to organ aging. We have previously shown that human kidney cortex displays telomere shortening with age. In the present study, we evaluated the relationship between renal age in humans and a number of phenomena associated with cellular senescence in vitro. METHODS: Human kidney specimens were obtained at 8 weeks to 88 years of age and were assessed for changes related to aging. RESULTS: We found that human kidneys expressed relatively constant levels of mRNAs for genes potentially related to senescence. Among the candidate genes surveyed, the cell cycle regulator p16INK4a emerged with the strongest association with renal aging for both mRNA and protein expression. Proliferation as measured by Ki-67 expression was inversely correlated with p16INK4a expression, compatible with a role for p16INK4a as an irreversible cell cycle inhibitor. Cyclooxygenase 1 and 2 (COX-1 and COX-2) mRNA expression was elevated in older kidneys, associated with increased protein expression. Comparison of gene expression with age-related histologic changes revealed that glomerulosclerosis correlated with p16INK4a and p53, whereas interstitial fibrosis and tubular atrophy were associated with p16INK4a, p53, COX-1, transforming growth factor-beta 1 (TGF-beta 1), and heat shock protein A5 (HSPA5). CONCLUSION: We conclude that some changes observed in cellular senescence in vitro do occur in human kidney with age, particularly in the renal cortex, in some cases correlating with histologic features. P16INK4a emerged with the most consistent correlations with age and histologic changes and inversely correlated with cell replication.

Adolescent↗
PubMed2004

Lesions of T-cell-mediated kidney allograft rejection in mice do not require perforin or granzymes A and B.

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.

Animals↗
PubMed2003

Cell senescence in rat kidneys in vivo increases with growth and age despite lack of telomere shortening.

BACKGROUND: Somatic cells in vitro have a finite life expectancy before entering a state of senescence, but it is unclear whether this state occurs in vivo in kidney development, growth, and aging. We previously showed that human kidney cortex displays telomere shortening with age. In this study, we compared the structural and functional changes in rat kidney with age to phenomena associated with cellular senescence in vitro. METHODS: We assessed the changes in Fischer 344 rat kidneys from age 1 to 9 months to define growth and development and from age 9 to 24 months to define aging. RESULTS: Rat kidney telomeres were approximately 35 to 40 kb long and did not shorten significantly. Expression of mRNA for p16INK4a, a characteristic senescence gene in vitro, was undetectable in most young rats but rose 27 fold during growth and a further 72-fold during aging. p16INK4a protein was localized to the nucleus and increased with age. p16INK4a mRNA also increased in other tissues. Lipofuscin and senescence-associated beta-galactosidase increased in epithelium with growth and aging and their occurrence was significantly associated with each other. Lipofuscin was particularly found in atrophic nephrons. CONCLUSION: We conclude that cell senescence occurs in both growth and aging in rat kidney and may contribute to the age-related pathology. These changes are not due to telomere shortening, but may reflect cumulative environmental stress.

Aging↗
PubMed2003

Cytokine single nucleotide polymorphisms and intrarenal gene expression in chronic allograft nephropathy in children.

BACKGROUND: Single nucleotide polymorphisms (SNPs) have been reported to influence cytokine production. Certain cytokine high producer genotypes have been associated with an increased risk for acute rejection and chronic allograft dysfunction (CAD) after transplantation. Our study evaluates SNP distribution for transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-alpha) in pediatric renal transplant recipients and control individuals and correlates them to corresponding intrarenal gene expression. METHODS: SNPs for TGF-beta1 (codon 10 and 25), IL-10 (positions -1082, -819, -592) and TNF-alpha (position -308) were determined in 30 patients with stable graft function, in 75 patients with CAD, and in 173 control individuals by allele-specific polymerase chain reaction (PCR). Intrarenal cytokine gene expression was studied in 25 biopsies with chronic allograft nephropathy (CAN) and 27 normal kidney specimens by real-time reverse transcription (RT)-PCR. RESULTS: No difference in allele and genotype frequency was detected in any of the investigated groups. No correlation between genotype and intragraft cytokine gene expression was recognized in CAN patients. However, in normal kidney specimens, the low producer TGF-beta1 genotype at codon 10 was associated with significant lower TGF-beta1 mRNA expression. This association was not found for IL-10 or TNF-alpha. CONCLUSION: Our results do not support the proposition that certain specific cytokine genotypes for TGF-beta1, IL-10, and TNF-alpha are associated with CAD. Intrarenal cytokine gene expression only correlated to one TGF-beta1 SNP in normal kidney specimens. Since overall TGF-beta1 expression was higher in transplanted patients compared to controls, this suggests that SNPs may not play a significant role once the immune system is activated.

Adolescent↗
PubMed2002

Quantitative gene expression of TGF-beta1, IL-10, TNF-alpha and Fas Ligand in renal cortex and medulla.

BACKGROUND: Inflammatory, fibrogenic or apoptotic processes in the kidney are regulated by intra- and intercellular mediators. Intrarenal upregulation of genes may precede structural changes by days and can be examined in extremely small amounts of tissue. With the advent of new quantitative PCR methods results of gene expression are available within few hours after kidney biopsies. METHODS: In order to establish reference values for intrarenal gene expression of TGF-beta1, IL-10, TNF-alpha and Fas Ligand in renal cortex and medulla, we analysed 28 histologically normal kidney samples available after tumour nephrectomy by quantitative real-time PCR. After reverse transcription of isolated RNA, cDNA aliquots were quantified for target genes using the threshold cycle (C(t)) method normalized for the house keeping gene GAPDH. RESULTS: Expression of target genes was lower in cortex as compared to medulla, but the differences were only significant for IL-10 (P=0.0125). TGF-beta1 was found with the highest gene expression about five PCR-cycles (delta C(t)) after GAPDH with markedly lower results for TNF-alpha (delta C(t) approximately 9), IL-10 (approximately 12) and Fas Ligand (approximately 14). CONCLUSIONS: These results are the first reported reference values for human intrarenal gene expression which should facilitate interpretation of data from native or transplant kidneys in future studies.

Adult↗
PubMed2000

Telomere shortening in kidneys with age.

The histology and function of the kidney deteriorates with age and age-related diseases, but the mechanisms involved in renal aging are not known. In vitro studies suggest that telomere shortening is important in replicative senescence, and is accelerated by stresses that increase replication. This study explored the relationship between age and telomere length in surgical samples from 24 human kidneys, which were either histologically normal (17) or displayed histologic abnormalities (7). Telomere loss was assessed by two independent methods: Southern blotting of terminal restriction fragments (TRF) and slot blotting using telomere-specific probes. The results of these methods correlated with each other. The mean TRF length determined by Southern blotting in cortex was about 12 kb pairs (kbp) in infancy and was shorter in older kidneys. The slope of the regression line was about 0.029 kbp (0.24%, P = 0.023) per year. Telomere DNA loss in cortex by the slot blot method was 0.25% per year (P = 0.011). By both methods, the telomere loss in medulla was not significant and was less than in cortex. Comparisons of TRF length from 20 paired samples from cortex and medulla showed that TRF was greater in cortex than medulla, with the differences being greater in young kidneys and lessening with age due to telomere loss in cortex. These findings indicate that telomeres shorten in an age-dependent manner in the kidney, either due to developmental factors or aging, particularly in renal cortex.

Adult↗