VEGF-B hypertrophy predisposes to transition from diastolic to systolic heart failure in hypertensive rats.

AIMS: Cardiac energy metabolism is centrally involved in heart failure (HF), although the direction of the metabolic alterations is complex and likely dependent on the particular stage of HF progression. Vascular endothelial growth factor B (VEGF-B) has been shown to modulate metabolic processes and to induce physiological cardiac hypertrophy; thus, it could be cardioprotective in the failing myocardium. This study investigates the role of VEGF-B in cardiac proteomic and metabolic adaptation in HF during aldosterone and high-salt hypertensive challenges. METHODS AND RESULTS: Male rats overexpressing the cardiac-specific VEGF-B transgene (VEGF-B TG) were treated for 3 or 6 weeks with deoxycorticosterone-acetate combined with a high-salt (HS) diet (DOCA + HS) to induce hypertension and cardiac damage. Extensive longitudinal echocardiographic studies of HF progression were conducted, starting at baseline. Sham-treated rats served as controls. To evaluate the metabolic alterations associated with HF, cardiac proteomics by mass spectrometry was performed. Hypertrophic non-treated VEGF-B TG hearts demonstrated high oxygen and adenosine triphosphate (ATP) demand with early onset of diastolic dysfunction. Administration of DOCA + HS to VEGF-B TG rats for 6 weeks amplified the progression from cardiac hypertrophy to HF, with a drastic drop in heart ATP concentration. Dobutamine stress echocardiographic analyses uncovered a significantly impaired systolic reserve. Mechanistically, the hallmark of the failing TG heart was an abnormal energy metabolism with decreased mitochondrial ATP, preceding the attenuated cardiac performance and leading to systolic HF. CONCLUSIONS: This study shows that the VEGF-B TG accelerates metabolic maladaptation which precedes structural cardiomyopathy in experimental hypertension and ultimately leads to systolic HF.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension.

BACKGROUND: Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. METHODS: Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. RESULTS: In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. CONCLUSIONS: Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

Na+ is shifted from the extracellular to the intracellular compartment and is not inactivated by glycosaminoglycans during high salt conditions in rats.

Recently, studies have emerged suggesting that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. We investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. Na+ accumulation was induced in rats by a high salt diet (HSD) (8% NaCl and 1% saline to drink) or by implantation of a deoxycorticosterone acetate (DOCA) tablet (1% saline to drink) using rats on a low salt diet (LSD) (0.1% NaCl) on tap water as control. Na+ and K+ were assessed by ion chromatography in tissue eluates, and the extracellular volume by equilibration of 51 Cr-EDTA. By tangential sectioning of the skin, we found a low Na+ content and extracellular volume in epidermis, both parameters rising by ∼30% and 100%, respectively, in LSD and even more in HSD and DOCA when entering dermis. We found evidence for an extracellular Na+ gradient from epidermis to dermis shown by an estimated concentration in epidermis ∼2 and 4-5 times that of dermis in HSD and DOCA-salt. There was intracellular storage of Na+ in skin, muscle, and myocardium without a concomitant increase in hydration. Our data suggest that there is a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. Salt stress results in intracellular storage of Na+ in exchange with K+ in skeletal muscle and myocardium that may have electromechanical consequences. KEY POINTS: Studies have suggested that Na+ can be retained or removed without commensurate water retention or loss, and that the skin plays a role as major Na+ reservoir via regulation of the content of glycosaminoglycans and osmotic gradients. In the present study, we investigated whether there were electrolyte gradients in skin and where Na+ could be stored to be inactivated from a fluid balance viewpoint. We used two common models for salt-sensitive hypertension: high salt and a deoxycorticosterone salt diet. We found a hydration-dependent high interstitial fluid Na+ concentration that will contribute to the skin barrier and thus be a mechanism for limiting water loss. There was intracellular Na+ storage in muscle and myocardium without a concomitant increase in hydration, comprising storage that may have electromechanical consequences in salt stress.

Pharmacokinetics and Pharmacodynamics of T-Cell Bispecifics in the Tumour Interstitial Fluid.

The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies.

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction.

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfß), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.

Time course of decompensation after angiotensin II and high-salt diet in Balb/CJ mice suggests pulmonary hypertension-induced cardiorenal syndrome.

The genetic background of a mouse strain determines its susceptibility to disease. C57BL/6J and Balb/CJ are two widely used inbred mouse strains that we found react dramatically differently to angiotensin II and high-salt diet (ANG II + Salt). Balb/CJ show increased mortality associated with anuria and edema formation while C57BL/6J develop arterial hypertension but do not decompensate and die. Clinical symptoms of heart failure in Balb/CJ mice gave the hypothesis that ANG II + Salt impairs cardiac function and induces cardiac remodeling in male Balb/CJ but not in male C57BL/6J mice. To test this hypothesis, we measured cardiac function using echocardiography before treatment and every day for 7 days during treatment with ANG II + Salt. Interestingly, pulsed wave Doppler of pulmonary artery flow indicated increased pulmonary vascular resistance and right ventricle systolic pressure in Balb/CJ mice, already 24 h after ANG II + Salt treatment was started. In addition, Balb/CJ mice showed abnormal diastolic filling indicated by reduced early and late filling and increased isovolumic relaxation time. Furthermore, Balb/CJ exhibited lower cardiac output compared with C57BL/6J even though they retained more sodium and water, as assessed using metabolic cages. Left posterior wall thickness increased during ANG II + Salt treatment but did not differ between the strains. In conclusion, ANG II + Salt treatment causes early restriction of pulmonary flow and reduced left ventricular filling and cardiac output in Balb/CJ, which results in fluid retention and peripheral edema. This makes Balb/CJ a potential model to study the adaptive capacity of the heart for identifying new disease mechanisms and drug targets.

Angiotensin II and salt-induced decompensation in Balb/CJ mice is aggravated by fluid retention related to low oxidative stress.

Balb/CJ mice are more sensitive to treatment with angiotensin II (ANG II) and high-salt diet compared with C57BL/6J mice. Together with higher mortality, they develop edema, signs of heart failure, and acute kidney injury. The aim of the present study was to identify differences in renal gene regulation that may affect kidney function and fluid balance, which could contribute to decompensation in Balb/CJ mice after ANG II + salt treatment. Male Balb/CJ and C57BL/6J mice were divided into the following five different treatment groups: control, ANG II, salt, ANG II + salt, and ANG II + salt + N-acetylcysteine. Gene expression microarrays were used to explore differential gene expression after treatment and between the strains. Published data from the Mouse Genome Database were used to identify the associated genomic differences. The glomerular filtration rate (GFR) was measured using inulin clearance, and fluid balance was measured using metabolic cages. Gene ontology enrichment analysis of gene expression microarrays identified glutathione transferase (antioxidant system) as highly enriched among differentially expressed genes. Balb/CJ mice had similar GFR compared with C57BL/6J mice but excreted less Na+ and water, although net fluid and electrolyte balance did not differ, suggesting that Balb/CJ mice may be inherently more prone to decompensation. Interestingly, C57BL/6J mice had higher urinary oxidative stress despite their relative protection from decompensation. In addition, treatment with the antioxidant N-acetylcysteine decreased oxidative stress in C57BL/6J mice, reduced urine excretion, and increased mortality. Balb/CJ mice are more sensitive than C57BL/6J to ANG II + salt, in part mediated by lower oxidative stress, which favors fluid and Na+ retention.

NFAT5 regulates renal gene expression in response to angiotensin II through Annexin-A2-mediated posttranscriptional regulation in hypertensive rats.

The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma.

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R² = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.

Lymphangiogenesis Facilitates Initial Lymph Formation and Enhances the Dendritic Cell Mobilizing Chemokine CCL21 Without Affecting Migration.

OBJECTIVE: Lymphatic vessels play an important role in body fluid, as well as immune system homeostasis. Although the role of malfunctioning or missing lymphatics has been studied extensively, less is known on the functional consequences of a chronically expanded lymphatic network or lymphangiogenesis. APPROACH AND RESULTS: To this end, we used K14-VEGF-C (keratin-14 vascular endothelial growth factor-C) transgenic mice overexpressing the vascular endothelial growth factor C in skin and investigated the responses to inflammatory and fluid volume challenges. We also recorded interstitial fluid pressure, a major determinant of lymph flow. Transgenic mice had a strongly enhanced lymph vessel area in skin. Acute inflammation induced by lipopolysaccharide and chronic inflammation by delayed-type hypersensitivity both resulted in increased interstitial fluid pressure and reduced lymph flow, both to the same extent in wild-type and transgenic mice. Hyperplastic lymphatic vessels, however, demonstrated enhanced transport capacity after local fluid overload not induced by inflammation. In this situation, interstitial fluid pressure was increased to a similar extent in the 2 strains, thus, suggesting that the enhanced lymph vessel area facilitated initial lymph formation. The increased lymph vessel area resulted in an enhanced production of the chemoattractant CCL21 that, however, did not result in augmented dendritic cell migration after induction of local skin inflammation by fluorescein isothiocyanate. CONCLUSIONS: An expanded lymphatic network is capable of enhanced chemoattractant production, and lymphangiogenesis will facilitate initial lymph formation favoring increased clearance of fluid in situations of augmented fluid filtration.

Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing.

OBJECTIVE: A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set). MATERIALS AND METHODS: Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor). RESULTS: The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R² = .95 and R² = .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial-mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-ß1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets. CONCLUSIONS: This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.

Stromal Integrin α11ß1 Affects RM11 Prostate and 4T1 Breast Xenograft Tumors Differently.

PURPOSE: It has been implied that the collagen binding integrin α11ß1 plays a role in carcinogenesis. As still relatively little is known about how the stromal integrin α11ß1 affects different aspects of tumor development, we wanted to examine the direct effects on primary tumor growth, fibrosis, tumor interstitial fluid pressure (PIF) and metastasis in murine 4T1 mammary and RM11 prostate tumors, using an in vivo SCID integrin α11-deficient mouse model. METHODS: Tumor growth was measured using a caliper, PIF by the wick-in-needle technique, activated fibroblasts by α-SMA immunofluorescence staining and fibrosis by transmission electron microscopy and picrosirius-red staining. Metastases were evaluated using hematoxylin and eosin stained sections. RESULTS: RM11 tumor growth was significantly reduced in the SCID integrin α11-deficient (α11-KO) compared to in SCID integrin α11 wild type (WT) mice, whereas there was no similar effect in the 4T1 tumor model. The 4T1 model demonstrated an alteration in collagen fibril diameter in the integrin α11-KO mice compared to WT, which was not found in the RM11 model. There were no significant differences in the amount of activated fibroblasts, total collagen content, collagen organization or PIF in the tumors in integrin α11-deficient mice compared to WT mice. There was also no difference in lung metastases between the two groups. CONCLUSION: Deficiency of stromal integrin α11ß1 showed different effects on tumor growth and collagen fibril diameter depending on tumor type, but no effect on tumor PIF or development of lung metastasis.

Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

Proteomic Analysis of Minimally Damaged Renal Tubular Tissue from Two-Kidney-One-Clip Hypertensive Rats Demonstrates Extensive Changes Compared to Tissue from Controls.

BACKGROUND: Tubular atrophy and interstitial fibrosis mark the final stage in most forms of progressive kidney diseases. Little is known regarding changes in the tubular proteome. In this study, we investigated changes in the tubular proteome of normal or minimally damaged tubular tissue in the non-clipped kidney from rats with two-kidney one-clip (2K1C) hypertension. METHODS: Formalin-fixed paraffin-embedded kidney sections from four 2K1C rats with hypertensive kidney damage and 6 sham rats were used. Tubulointerstitial tissue without discernable interstitial expansion or pronounced tubular alterations was microdissected and this was assumed to represent an early stage of chronic tubular damage in 2K1C. Samples were analyzed by mass spectrometry and relative protein abundances were compared between 2K1C and sham. RESULTS: A total of 1,160 proteins were identified with at least 2 unique peptides, allowing for relative quantitation between samples. Among these, 151 proteins were more abundant, and 192 proteins were less abundant in 2K1C compared with sham. Transgelin, vimentin and creatine kinase B-type were among the proteins that were most increased in 2K1C. Ingenuity Pathway Analysis showed increased abundance of proteins related to Rho signaling and protein turnover (eIF2 signaling and protein ubiquitination), and decreased abundance of proteins related to fatty acid ß-oxidation. CONCLUSION: Tubular tissue from normal or minimally damaged hypertensive kidney damage demonstrate extensive proteomic changes with upregulation of pathways associated with progressive kidney damage, such as Rho signaling and protein turnover. Thus, proteomics presents itself to be a promising tool for the discovery of early damage markers from not yet morphologically visible tubular damage.

The Effect of Stromal Integrin ß3-Deficiency on Two Different Tumors in Mice.

There is an increasing focus on the tumor microenvironment in carcinogenesis. Integrins are important receptors and adhesion molecules in this environment and have been shown to be involved in cell adhesion, proliferation, differentiation and migration. The present study aimed to evaluate the effect of stromal integrin ß3-deficiency on tumor growth, angiogenesis, interstitial fluid pressure (PIF), fibrosis and metastasis in a murine breast cancer (4T1) and a prostate tumor (RM11) model. We showed that stromal integrin ß3-deficiency led to an elevation in PIF that correlated to a shift towards thicker collagen fibrils in the 4T1 mammary tumor. In the RM11 prostate carcinoma model there was no effect of integrin ß3-deficiency on PIF and collagen fibril thickness. These findings support the notion that changes in the collagen scaffold influence PIF, and also indicate that there must be important crosstalk between the stroma and tumor cells, in a tumor cell line specific manner. Furthermore, stromal integrin ß3-deficiency had no effect on tumor growth or angiogenesis in both tumor models and no effect on lung metastasis in the 4T1 mammary tumor model. In conclusion, the stromal ß3 integrin influence PIF, possibly via its effect on the structure of the collagen network, in a tumor cell line dependent manner.

Matrix Metalloproteinase-2 Knockout and Heterozygote Mice Are Protected from Hydronephrosis and Kidney Fibrosis after Unilateral Ureteral Obstruction.

Matrix Metalloproteinase-2 (Mmp2) is a collagenase known to be important in the development of renal fibrosis. In unilateral ureteral obstruction (UUO) the obstructed kidney (OK) develops fibrosis, while the contralateral (CL) does not. In this study we investigated the effect of UUO on gene expression, fibrosis and pelvic remodeling in the kidneys of Mmp2 deficient mice (Mmp2-/-), heterozygous animals (Mmp2+/-) and wild-type mice (Mmp2+/+). Sham operated animals served as controls (Cntrl). UUO was prepared under isoflurane anaesthesia, and the animals were sacrificed after one week. UUO caused hydronephrosis, dilation of renal tubules, loss of parenchymal thickness, and fibrosis. Damage was most severe in Mmp2+/+ mice, while both Mmp2-/- and Mmp2+/- groups showed considerably milder hydronephrosis, no tubular necrosis, and less tubular dilation. Picrosirius red quantification of fibrous collagen showed 1.63±0.25% positivity in OK and 0.29±0.11% in CL (p<0.05) of Mmp2+/+, Mmp2-/- OK and Mmp2-/- CL exhibited only 0.49±0.09% and 0.23±0.04% (p<0.05) positivity, respectively. Mmp2+/- OK and Mmp2+/- CL showed 0.43±0.09% and 0.22±0.06% (p<0.05) positivity, respectively. Transcriptomic analysis showed that 26 genes (out of 48 examined) were differentially expressed by ANOVA (p<0.05). 25 genes were upregulated in Mmp2+/+ OK compared to Mmp2+/+ CL: Adamts1, -2, Col1a1, -2, -3a1, -4a1, -5a1, -5a2, Dcn, Fbln1, -5, Fmod, Fn1, Itga2, Loxl1, Mgp, Mmp2, -3, Nid1, Pdgfb, Spp1, Tgfb1, Timp2, Trf, Vim. In Mmp2-/- and Mmp2+/- 18 and 12 genes were expressed differentially between OK and CL, respectively. Only Mmp2 was differentially regulated when comparing Mmp2-/- OK and Mmp2+/- OK. Under stress, it appears that Mmp2+/- OK responds with less Mmp2 upregulation than Mmp2+/+ OK, suggesting that there is a threshold level of Mmp2 necessary for damage and fibrosis to occur. In conclusion, reduced Mmp2 expression during UUO protects mice against hydronephrosis and renal fibrosis.

Distinct protein signature of hypertension-induced damage in the renal proteome of the two-kidney, one-clip rat model.

BACKGROUND: Hypertensive nephrosclerosis is one of the most frequent causes of chronic kidney failure. Proteome analysis potentially improves the pathophysiological understanding and diagnostic precision of this disorder. In the present exploratory study, we investigated experimental nephrosclerosis in the two-kidney, one-clip (2K1C) hypertensive rat model. METHODS: The renal cortex proteome from juxtamedullary cortex and outer cortex of 2K1C male Wistar-Hannover rats (n = 4) was compared with the sham-operated controls (n = 6), using mass spectrometry-based quantitative proteomics. We combined a high abundant plasma protein depletion strategy with an extended liquid chromatographic gradient to improve peptide and protein identification. Immunohistology was used for independent confirmation of abundance. RESULTS: We identified 1724 proteins, of which 1434 were quantified with at least two unique peptides. Comparative proteomics revealed 608 proteins, including the platelet-derived growth factor receptor-ß signalling pathway, with different abundances between the non-clipped kidney of hypertensive 2K1C rats and the corresponding kidney of the normotensive controls (P < 0.05, absolute fold change ≥1.5). Among the most significantly altered proteins in the whole cortex were periostin, transgelin, and creatine kinase B-type. Relative abundance of periostin alone allowed clear classification of 2K1C and controls. Enrichment of periostin in 2K1C rats was verified by immunohistology, showing positivity especially around the fibrotic vessels. CONCLUSION: The proteome is altered in hypertension-induced kidney damage. We propose periostin, especially in combination with transgelin and creatine kinase B-type, as possible proteomic classifier to distinguish hypertensive nephrosclerosis from the normal tissue. This classifier needs to be further validated with respect to early diagnosis of fibrosis, prognosis, and its potential as a novel molecular target for pharmacological interventions.

Identification of a common molecular pathway in hypertensive renal damage: comparison of rat and human gene expression profiles.

BACKGROUND: There is a common structural progression in hypertensive renal damage with early arterial damage and fibrosis in the juxtamedullary cortex. METHOD: The present investigation identifies a common pathway using three-gene expression profiles from hypertensive rat models: 60-week-old spontaneously hypertensive rat (SHR), salt-loaded stroke-prone SHR (SHRSP), and the non-clipped kidney after 24 weeks of two-kidney, one-clip hypertension (2K1C). Kidney damage was scored using a specialized system. Gene-expression profiles were determined using microarrays and validated using a panel of 47 genes by quantitative real-time PCR. RESULTS: All groups showed kidney damage (SHRs: 0.32 ±â€Š0.09 vs. Wistar-Kyoto rats: 0.06 ±â€Š0.03; 2K1C: 0.27 ±â€Š0.13 vs. pooled controls: 0.01 ±â€Š0.01; SHRSP: 1.13 ±â€Š0.14 vs. WKY: 0.04 ±â€Š0.03; all P < 0.05). A total of 1614 genes were changed in the SHR experiment, 1323 in the SHRSP, and 576 in the 2K1C. Eighty-eight genes were similarly regulated in all three models. Gene ontology enrichment analysis identified 59 ontologies that were enriched in all three datasets. These included over-representation to extracellular matrix, response to oxidative stress, and immune system processes. Out of the 88 in-common genes, 40 could be connected in a common pathway that was compared to two gene-expression profiles from human kidneys with histologically verified fibrosis to identify a highly significant number of in-common genes that were also represented in the common genetic pathway. CONCLUSION: There is a common pathway during the development of hypertensive kidney damage in rats irrespective of model. Interestingly, large parts of this common pathway are conserved in human kidney damage, which may indicate a broader importance in the development of chronic kidney disease.

Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats.

BACKGROUND: It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. METHODS: In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography-tandem mass spectrometry. RESULTS: 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. CONCLUSIONS: Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats.

Nitric oxide in afferent arterioles after uninephrectomy depends on extracellular L-arginine.

Uninephrectomy (UNX) causes hyperperfusion of the contralateral remaining kidney via increased nitric oxide (NO) synthesis. Although the exact mechanism remains largely unknown, we hypothesize that this would be localized to the afferent arteriole and that it depends on cellular uptake of l-arginine. The experiments were performed in rats 2 days (early) or 6 wk (late) after UNX and compared with controls (Sham) to study acute and chronic effects on NO metabolism. Renal blood flow was increased after UNX (21 ± 2 ml·min(-1)·kg(-1) in sham, 30 ± 3 in early, and 26 ± 1 in late, P < 0.05). NO inhibition with N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) caused a greater increase in renal vascular resistance in early UNX compared with Sham and late UNX (138 ± 24 vs. 88 ± 10, and 84 ± 7%, P < 0.01). The lower limit of autoregulation was increased both in early and late UNX compared with Sham (P < 0.05). L-NAME did not affect the ANG II-induced contraction of isolated afferent arterioles (AA) from Sham. AA from early UNX displayed a more pronounced contraction in response to L-NAME (-57 ± 7 vs. -16 ± 7%, P < 0.05) and in the absence of L-arginine (-41 ± 4%, P < 0.05) compared with both late UNX and Sham. mRNA expression of endothelial NO synthase was reduced, whereas protein expression was unchanged. Cationic amino acid transporter-1 and -2 mRNA was increased, while protein was unaffected in isolated preglomerular resistance vessels. In conclusion, NO-dependent hyperperfusion of the remaining kidney in early UNX is associated with increased NO release from the afferent arteriole, which is highly dependent on extracellular L-arginine availability.