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1.
Science ; 358(6368): 1332-1336, 2017 12 08.
Article in English | MEDLINE | ID: mdl-29217578

ABSTRACT

Progressive kidney diseases are often associated with scarring of the kidney's filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases.


Subject(s)
Glomerulosclerosis, Focal Segmental/drug therapy , Hypertension, Renal/drug therapy , Indazoles/pharmacology , Proteinuria/drug therapy , TRPC Cation Channels/antagonists & inhibitors , Animals , Disease Models, Animal , Glomerulosclerosis, Focal Segmental/genetics , Mutation , Podocytes/drug effects , Rats , Rats, Inbred Dahl , Rats, Transgenic , Small Molecule Libraries , TRPC Cation Channels/pharmacology , rac1 GTP-Binding Protein/genetics
2.
JCI Insight ; 2(20)2017 10 19.
Article in English | MEDLINE | ID: mdl-29046476

ABSTRACT

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.


Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Regeneration/immunology , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Cartilage, Articular/cytology , Cell Differentiation , Cells, Cultured , Disease Models, Animal , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Injections, Intra-Articular , Islets of Langerhans Transplantation , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Mice , Mice, Transgenic , Rats , Rats, Transgenic , Skin Transplantation
3.
J Histochem Cytochem ; 65(12): 743-755, 2017 12.
Article in English | MEDLINE | ID: mdl-29058957

ABSTRACT

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.


Subject(s)
Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Gene Expression Regulation , Polycystic Kidney, Autosomal Dominant/metabolism , Polycystic Kidney, Autosomal Dominant/pathology , Animals , Disease Models, Animal , Fibrosis , Kidney/metabolism , Kidney/pathology , Male , Polycystic Kidney, Autosomal Dominant/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
4.
Int J Mol Sci ; 17(6)2016 May 24.
Article in English | MEDLINE | ID: mdl-27231899

ABSTRACT

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.


Subject(s)
Cell Adhesion Molecules/metabolism , Kidney Tubules, Proximal/metabolism , Polycystic Kidney Diseases/metabolism , Polycystic Kidney Diseases/pathology , Animals , Biomarkers/metabolism , Cell Dedifferentiation , Disease Models, Animal , Kidney Tubules, Proximal/pathology , Male , Organ Specificity , Rats , Up-Regulation
5.
Nephrol Dial Transplant ; 28(8): 2045-58, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23543593

ABSTRACT

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.


Subject(s)
Benzophenones/pharmacology , Catechol O-Methyltransferase Inhibitors , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Nitrophenols/pharmacology , Polycystic Kidney Diseases/drug therapy , Animals , Apoptosis/drug effects , Biomarkers/metabolism , Blotting, Western , Cell Proliferation/drug effects , Disease Progression , Female , Gene Expression Profiling , Humans , Immunoenzyme Techniques , Male , Oligonucleotide Array Sequence Analysis , Polycystic Kidney Diseases/pathology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tolcapone
6.
Am J Physiol Endocrinol Metab ; 302(9): E1044-54, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22318954

ABSTRACT

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.


Subject(s)
Aldosterone/physiology , Models, Animal , Rats, Transgenic , Receptor, Angiotensin, Type 2/metabolism , Zona Glomerulosa/physiology , Angiotensin II/physiology , Animals , Cell Proliferation , Gene Expression Regulation/physiology , Rats , Up-Regulation , Zona Glomerulosa/cytology
7.
Am J Pathol ; 177(6): 3000-9, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21119215

ABSTRACT

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.


Subject(s)
Nuclear Proteins/genetics , Polycystic Kidney Diseases/genetics , Amino Acid Substitution/genetics , Animals , Arginine/genetics , Gene Expression/physiology , Genetic Predisposition to Disease , Male , Mutant Proteins/genetics , Polycystic Kidney Diseases/pathology , Polymorphism, Single Nucleotide/physiology , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Tryptophan/genetics , Up-Regulation/physiology
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