Renal Inner Medulla Research Articles

Effect of sevoflurane on aquaroprins 3 after the renal ischemia-reperfusion injury in rat model

Objective To investigate the protective effect of inhaled sevoflurane on renal ischemia-reperfusion injury (IRI) and the its relationship with aquaporins-3 (AQP3) in rat model. Methods SD rats (n=8) were allocated randomly and equally to operating group (group C), ischemia reperfusion group (group I/R), sevoflurane postconditioning group (group Spo). IRI models were established by 45 min of renal ischemia and 2 h of reperfusion. All animals were killed 24 h after IRI. Serum urea nitrogen (BUN) and creatinine (Cr) were detected. 24 h urine was collected to observe the change of the urine volume and urine specific gravity. The quantity of AQP3 in renal inner medulla was detected by immunohistochemistry and Western blotting. Results (1) 24 h urine volume was significantly increased in group I/R [(16.3±9.1) ml] and group Spo [(10.8±5.6) ml] as compared with group C [(5.4±0.2) ml] (Group I/R: F=11.472, P<0.01, Group Spo: F=7.429, P<0.05). The 24 h urine specific gravity was significantly reduced in group I/R (3.014±0.012) and group Spo (3.034±0.022) as compared with group C (3.074±0.003) (Group I/R: F=188.235, P<0.01, Group Spo: F=25.964, P<0.01). (2) BUN in group I/R [(20.530±1.690) mmol/L] and group Spo [(12.300±2.370) mmol/L] was significantly increased as compared with group C [(7.033±0.440) mmol/L] (Group I/R: F=477.867, P<0.01, Group Spo: F=38.195, P<0.01). Cr level in group I/R [(122.3±9.5) μmol/L] and group Spo [(82.0±13.2) μmol/L] was significantly increased as compared with group C [(28.7±4.3) μmol/L] (Group I/R: F=644.543, P<0.01, Group Spo: F=117.922, P<0.01). The renal function changes in BUN and Cr were significantly improved in group Spo as compared with group I/R (F=63.952, 49.124, P<0.01). (3) As compared with group C (861.6±85.4), the protein expression of AQP3 in group I/R (138.4±31.4) and group Spo (419.8±74.2) (F=505.385, P<0.05) was significantly down-regulated (P<0.05). There protein expression of AQP3 was significant higher in group Spo than group I/R (F=97.586, P<0.01). Conclusion Sevoflurane postconditioning could effectively protect renal function against IRI, which was significantly correlated with the up-regulation of of AQP3. Key words: Sevoflurane; Kidney; Ischemia-reperfusion injury; Aquaporins 3

Oxidative stress induces BH4 deficiency in male, but not female, SHR.

We previously published that female spontaneously hypertensive rats (SHR) have significantly greater nitric oxide (NO) bioavailability and NO synthase (NOS) enzymatic activity in the renal inner medulla (IM) compared with age-matched males, although the mechanism responsible remains unknown. Tetrahydrobiopterin (BH4) is a critical cofactor required for NO generation, and decreases in BH4 as a result of increases in oxidative stress have been implicated in the pathogenesis of hypertension. As male SHR are known to have higher levels of oxidative stress compared with female SHR, we hypothesized that relative BH4 deficiency induced by oxidative stress in male SHR results in lower levels of NOS activity in renal IM compared with females. Twelve-week-old male and female SHR were randomized to receive tempol (30 mg/kg/day via drinking water) or vehicle for 2 weeks. Tempol treatment did not affect blood pressure (BP) in either sex, but reduced peroxynitrite levels only in males. Females had more total biopterin, dihydrobiopterin (BH2), and BH4 levels in renal IMs than males, and tempol treatment eliminated these sex differences. Females had greater total NOS activity in the renal IM than males, and adding exogenous BH4 to the assay increased NOS activity in both sexes. This sex difference in total NOS and the effect of exogenous BH4 were abolished with tempol treatment. We conclude that higher oxidative stress in male SHR results in a relative deficiency of BH4 compared with females, resulting in diminished renal NOS activity in the male.

Abstract P082: Mitochondrial Gene Expression is Decreased in Renal Inner Medulla of Non-human Primates with Spontaneous Hypertension

Mitochondrial gene expression may influence renal function and consequently, long-term blood pressure control. The African Green Monkey (Chlorocebus aethiops sabaeus; AGM) exhibits heritable, spontaneous hypertension and thus is a translational model for the study of human essential hypertension. We hypothesized that renal mitochondrial gene expression in hypertensive AGMs is decreased and may contribute to renal mitochondrial dysfunction in specific kidney regions. AGMs were phenotyped as normotensive (NT, systolic blood pressure; SBP &lt;120 mmHg) or hypertensive (HT, SBP &gt; 140 mmHg) by forearm plethysmography. Gene expression was determined using qRT-PCR with RNA extracted from renal cortex, outer medulla (OM), inner medulla (IM), and liver of 18 HT (mean SBP 166±7 mmHg) and 18 NT (mean SBP 98±3 mmHg) animals. In renal cortical and OM tissue of HT vervets, COX 3 (Complex IV), Cyt B (Complex III), NADH4 (Complex I) and ATP8 (Complex V) expression were similar in NT and HT AGMs. In IM of HT AGMs, COX3, NADH4, ATP8, and CYTB were downregulated by 4.7-fold (p=0.007), 4-fold (p=0.002), 4.1-fold (p=0.006), and 3-fold (p=0.018), respectively. In the liver, COX 3 expression was decreased 1.9-fold (p=0.04), 1.6-fold for ATP8 (p=0.03), and 2.0-fold for CYTB (p=0.01). Expression of SDH (Complex II) and COX4 (Complex IV), nuclear encoded subunits of the OXPHOS chain, was also assessed. SDH expression was up-regulated 8-fold in renal OM (p=0.005) but unchanged in liver, IM, and cortex. COX4 expression however, was down-regulated in OM by 8-fold (p=0.05), in IM by 5-fold (p=0.011) but unchanged in cortex and liver. Gene expression of the mitochondrial transcription factor TFAM was downregulated by 4-fold in renal OM, and unchanged in renal cortex and liver. Citrate synthase activity showed no difference in mitochondrial number between NT and HT AGMs (p=0.73). We suggest that reduced expression of mitochondrially encoded OXPHOS subunits in the renal IM may contribute to the development of hypertension in the AGM. Mitochondrial gene down-regulation in the liver may be a consequence of the systemic hypertension. We conclude that altered mitochondrial gene expression may be a cellular response to increased oxidative stress in the renal inner medulla of HT AGM.

Protein carbamylation is a physiological post‐translational modification in the renal inner medulla

We hypothesize that the high urea concentration in the renal inner medulla (IM) is associated with physiological carbamylation of proteins. Using a carbamylation‐specific antibody, immunoblotting revealed multiple bands in the kidney of antidiuretic rats with a gradient of carbamylation from cortex to IM, suggestive of large‐scale protein carbamylation. To test the specificity of the antibody, we incubated BSA with 1 M urea in PBS or PBS alone for 4 days. The antibody recognized a band consistent with carbamylated BSA in the presence but not the absence of urea. To confirm widespread protein carbamylation in rat IM, we carried out protein mass spectrometry. Proteins, processed in the absence of urea, were separated via SDS‐PAGE. Successive gel blocks were subjected to in‐gel trypsin digestion. Using LC‐MS/MS (Orbitrap) and a high‐stringency spectral search (SEQUEST), we identified 686 carbamylation sites in 579 proteins. Many of the identified sites were present on lysine side chains. The results show that extensive protein carbamylation occurs in rat renal IM, which could have an impact on cell signaling by blocking post‐translational modifications of lysines, e.g. acetylation and ubiquitylation.

Vasopressin increases ATF3 mRNA expression in mouse renal inner medulla

In renal inner medulla (IM) the concentration of urea is high and variable depending on the concentrating mechanism. During water restriction (WR), the level of vasopressin increases, followed by further increase of urea concentration in IM. Expression of activating transcription factor 3 (ATF3) is increased by high urea in vitro. To determine whether ATF3 is regulated by high urea in vivo, we water restricted mice for 48 hours and found that ATF3 mRNA increased by 3.81 ± 0.24 fold vs. control 1.00 ± 0.06 (p≤0.05). 7 day infusion of AVP increased ATF3 mRNA by 1.69 ± 019 fold vs. control 1.00 ± 0.15 (p≤0.05). However, 7 day infusion of dDAVP, an agonist of type 2 vasopressin receptor, did not cause significant change in the expression of ATF3 mRNA. ATF3 is an important component in the pathway of endoplasmic reticulum (ER) stress. We previously found that GCN2, a kinase in ER stress pathway, was involved in high urea associated increase of ATF3 in vitro. However, there was no difference in the basal level of ATF3 mRNA between GCN2 null and wild type mice. As in wild type mice, 48 hours of WR increased ATF3 mRNA in IM of GCN2 null mice by 4.14 ± 0.09 fold vs. control GCN2 null mice 1.00 ± 0.06 (p≤0.05). Knocking down GRP78, an important ER chaperone, in mIMCD3 cells did not affect high urea induced increase of ATF3 protein. We concluded that deficiency of ER stress genes does not affect vasopressin induced increase of ATF3 in renal IM.

Architecture of inner medullary descending and ascending vasa recta: pathways for countercurrent exchange

Pathways and densities of descending vasa recta (DVR) and ascending vasa recta (AVR) in the outer zone of the inner medulla (IM) were evaluated to better understand medullary countercurrent exchange. Nearly all urea transporter B (UT-B)-positive DVR, those vessels exhibiting a continuous endothelium, descend with little or no branching exclusively through the intercluster region. All DVR have a terminal fenestrated (PV-1-positive) segment that partially overlaps with the UT-B-positive segment. This fenestrated segment descends a distance equal to approximately 15% of the length of the connecting UT-B-positive segment before formation of the first branch. The onset of branching is indicative of vessel entry into the intracluster region. The number density of UT-B-positive DVR at 3,000 mum below the OM-IM boundary is approximately 60% lower than the density at 400 mum below the OM-IM boundary, a result of DVR joining to fenestrated interconnecting vessels and an overall decline in UT-B expression. AVR that lie in the intercluster region (designated AVR(2)) lie distant from CDs and ascend to the OM-IM boundary with little or no branching. AVR(2a) represent a subcategory of AVR(2) that abut DVR. The mean DVR length (combined UT-B- and PV-1-positive segments) nearly equals the mean AVR(2a) length, implying a degree of overall equivalence in fluid and solute countercurrent exchange may exist. The AVR(2)/DVR ratio is approximately 2:1, and the AVR(2a)/DVR ratio is approximately 1:1; however, the AVR/DVR ratio determined for the full complement of fenestrated vessels is approximately 4:1. The excess fenestrated vessels include vessels of the intracluster region (designated AVR(1)). Countercurrent exchange between vasa recta occurs predominantly in the intercluster region. This architecture supports previous functional estimates of capillary fluid uptake in the renal IM.

Long term dDAVP increases the expression of urea transporters and ER stress pathway genes in kidneys of mice with urinary concentrating defect.

It is well‐know that both AQP2 and urea transporter A1 (UTA1) are vasopressin‐regulated genes in normal renal inner medullas. However, we previously reported that 7 day dDAVP regulated AQP2 expression is impaired in the renal inner medulla (IM) of AQP1 null mice with urinary concentrating defect. In the present study, we investigated the regulation of UTA1 and UTA3 in 7day dDAVP infused AQP1 null mice. We found that 7 day dDAVP increases the expression of UTA1 and UTA3 proteins to 212 ± 40.4% vs. control 100 ± 19.5% and 588 ± 93.7% vs. control 100 ± 34.4%, respectively, which are similar to the results from dDAVP infused wild type mice. 7 day dDAVP also increases the expression of endoplasmic reticulum (ER) stress genes in IM of AQP1 null mice. GRP78 mRNA increases by 1.74 fold and protein increases by 2 fold compared to that in control 7 days after dDAVP infusion. Immunohistochemistry demonstrates that 7 day dDAVP causes GRP78 protein localize on the apical membrane of collecting duct cells in the papillary tip of IM with AQP2. Electron microscopy of immuno‐gold staining confirms the apical location of GRP78. Moreover, 7 day dDAVP increases GADD153 mRNA to 2.93 ± 0.13 and ATF4 mRNA to 1.55 ± 0.06 compared to those in controls 1 ± 0.05 and 1 ± 0.06, respectively, in IM of AQP1 null mice. Conclusion: in renal IM of AQP1 null mice 1) the regulation of UTA1 and UTA3 by dDAVP is intact and 2) 7 day dDAVP activates ER stress pathway.

Microarray Analysis of Gene Expression in the Renal Inner Medulla of the Dahl Salt-Resistant and Salt-Sensitive Rat

49 The inner medulla contains several mechanisms that play a central role in fluid-volume homeostasis. We hypothesized that transcriptional regulation of the inner medulla in response to a high sodium diet differs between the Dahl salt-resistant (SR) and salt-sensitive (SS) rats. SR and SS (16 wk) were placed on either a 0.5% (low) or 4.0% (high) NaCl diet. After 5 weeks, total RNA from the renal inner medulla was isolated from two rats in each of the following groups: SR/low, SR/high, SS/low, and SS/high. Labeled cRNA was hybridized to microarrays representing 7000 genes (Affymetrix). In response to increased Na intake in the SR rat, 46 genes were up-regulated and 7 genes down-regulated (>2-fold changes). This adaptive response included the up-regulation of guanylin (3-fold;3X), cytochrome P 450 (8X), cyclooxygenase-2 (3X), kidney-specific androgen-regulated protein (8X) and the down regulation of Tamm-Horsfall protein (-39X). In contrast, high salt in SS rats revealed the differential expression of only one gene, the up-regulation of guanylin (3X). To investigate the general absence of a transcriptional response in SS rats, we found that, 23 genes were expressed at higher levels and 14 at lower levels in SS/low compared to SR/low. Interestingly, 7 of 23 genes that were elevated in the SS/low, were the same genes that were up-regulated in the SR/high. Thus, components of the adaptive response to high Na in SR rats are operating in SS rats prior to a Na challenge. Ten genes were increased and four were decreased in the SS/high when compared to the SR/high. The ROMK K + channel (3X), Na-K-2Cl cotransporter (6X), and an ATPase inhibitor (8X) were expressed at higher levels in the SS/high. A majority of the other genes not mentioned have not been studied in the context of sodium handling or hypertension. These findings reveal clear differences in the basal gene expression within the inner medulla between SR and SS rats. Furthermore, these results show that salt-sensitivity is associated with a diminished transcriptional response to a high salt diet which supports the hypothesis that this region of the kidney contributes to genetic salt-sensitive hypertension.