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.

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 51Cr‐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.

, one-clip (2K1C) is a model of renovascular hypertension where we previously found an exaggerated intracellular calcium (Ca i 2ϩ ) response to ANG II in isolated afferent arterioles (AAs) from the clipped kidney (Helle F, Vagnes OB, Iversen BM. Am J Physiol Renal Physiol 291: F140 -F147, 2006). To test whether nitric oxide (NO) ameliorates the exaggerated ANG II response in 2K1C, we studied ANG II (10 Ϫ7 mol/l)-induced calcium signaling and contractility with or without the NO synthase (NOS) inhibitor N G -nitro-L-arginine methyl ester (L-NAME). In AAs from the nonclipped kidney, L-NAME increased the ANG II-induced Ca i 2ϩ response from 0.28 Ϯ 0.05 to 0.55 Ϯ 0.09 (fura 2, 340 nm/380 nm ratio) and increased contraction from 80 Ϯ 6 to 60 Ϯ 6% of baseline (P Ͻ 0.05). In vessels from sham and clipped kidneys, L-NAME had no effect. In diaminofluorescein-FM diacetateloaded AAs from the nonclipped kidney, ANG II increased NOderived fluorescence to 145 Ϯ 34% of baseline (P Ͻ 0.05 vs. sham), but not in vessels from the sham or clipped kidney. Endothelial NOS (eNOS) mRNA and ser-1177 phosphorylation were unchanged in both kidneys from 2K1C, while eNOS protein was reduced in the clipped kidney compared with sham. Cationic amino acid transferase-1 and 2 mRNAs were increased in 2K1C, indicating increased availability of L-arginine for NO synthesis, but counteracted by decreased scavenging of the eNOS inhibitor asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolase 2. In conclusion, the Ca i 2ϩ and contractile responses to ANG II are blunted by NO release in the nonclipped kidney. This may protect the nonclipped kidney from the hypertension and elevated ANG II levels in 2K1C.two-kidney; one-clip; renovascular hypertension TWO-KIDNEY, ONE-CLIP (2K1C) has been studied as an ANG II-dependent model of renovascular hypertension. Earlier studies have demonstrated elevated circulating levels of ANG II, with high ANG II concentration in the cortical tissue of the clipped and nonclipped kidney (20).Renal blood flow (RBF) and intrarenal vascular resistance are controlled by autocrine and paracrine factors as well as myogenic and tubuloglomerular feedback responses. The afferent arteriole (AA) is the main site for RBF and glomerular filtration rate (GFR) regulation and plays an important role during development of 2K1C hypertension (12). The clipped kidney has reduced RBF and GFR (18), impaired autoregulation, and dilated AAs (14). Although RBF autoregulation is reset to higher perfusion pressures, the nonclipped kidney has well-maintained RBF and GFR (6) despite high levels of circulating and cortical tissue ANG II.Previously, we found that the ANG II dose-response curve in AAs from clipped kidneys did not saturate at high doses (10 Ϫ6 M), while those from sham and nonclipped kidneys did (5). The effect of cyclooxygenase (COX) inhibition, together with mRNA and protein (1) expression for the AT 1a receptor, was similar in the nonclipped and clipped kidneys. This indicated that the changed dose-response relationship was...

Background. Hypertensive renal damage starts in the juxtamedullary cortex (JMC) and gradually extends towards the outer cortex (OC). The intention of the study was to examine if the increase of fibrous tissue in the JMC of the spontaneously hypertensive rat (SHR) is dependent on an increase of collagen synthesis or a decreased collagen breakdown compared to the normotensive control (WKY). Methods and results. The renal damage was evaluated by light microscopy, and the amount of fibrosis was quantified using Sirius red staining. Real-time RT-PCR was used to quantify mRNA for: collagen-type-1-alpha-1 (col1a1), procollagen-n-and -c-proteinase, matrix metalloproteases, MMP-2 and MMP-9, tissue inhibitor of metalloproteases, TIMP-1 and TIMP-2. Western blot was used to quantify the proteins of MMP-2, MMP-9, TIMP-1 and TIMP-2. The relative activities of MMP-2 and MMP-9 were assayed by zymography. The JMC in SHR had an increased amount of collagen as measured by Sirius red, and a 15-fold increase in the mRNA for col1a1. The gene expression of procollagen-c-proteinase was unchanged while procollagen-n-proteinase was increased in SHR and had the highest expression in the JMC. The mRNA for MMP-2 and MMP-9 showed increased expression in SHR, but not specifically in the JMC. Protein analysis showed increased expression for MMP-2 in SHR and in the JMC. MMP-9 protein was lower in SHR. TIMP-1 was increased in SHR at both mRNA and protein level and more so in the JMC. The mRNA and protein analysis of TIMP-2 showed small differences between SHR and WKY.Conclusion. An imbalance of collagen metabolism featuring increased synthesis and inhibition of breakdown favours renal interstitial fibrosis in SHR.

Fibrosis in the nonclipped kidney of renal hypertensive rats starts around the juxtamedullary resistance vessels and then progresses in the JMC, whereas the outer cortex is protected. This suggests that pressure-induced injury to the vasculature attracts or activates fibroblasts in the perivascular area, which may allow damage to progress by impairing vessel function.