Adam Vannay

Divergence of renal vascular endothelial growth factor mRNA expression and protein level in post-ischaemic rat kidneys

Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis and vascular protection. Synthesis of VEGF is induced by hypoxia and different cytokines including interleukin‐6 (IL‐6) and interleukin‐1β (IL‐1β). However, post‐ischaemic alterations of this growth factor in the kidney are incompletely known. To determine VEGF synthesis in renal ischaemia/reperfusion (I/R) injury unilateral warm ischaemia was induced by cross‐clamping the left renal pedicle for 55 min followed by 2 and 24 h of reperfusion (T2 and T24 kidneys; n= 6 in each group). Sham‐operated, non‐clamped animals served as controls (n= 6). Renal VEGF, IL‐6 and IL‐1β mRNA expression were determined by reverse transcription‐polymerase chain reaction (RT‐PCR). VEGF protein level and distribution were determined by Western blot and immunohistochemical analysis. Immunohistochemistry revealed prominent VEGF staining in the outer medulla of control, T2 and T24 kidneys. VEGF immunoreactivity accumulated at the basolateral area of tubular epithelial cells in T2 kidneys, while it was diffuse in control and T24 kidneys. VEGF protein levels were increased 2‐ to 3‐fold in T2 and T24 kidneys (both P < 0.01 versus controls), while VEGF mRNA expression remained unchanged. IL‐6 mRNA expression was increased (P < 0.01 versus controls) in T2 kidneys, while IL‐1β mRNA expression remained unchanged. Increased VEGF protein levels but not mRNA expression suggests that during renal I/R injury VEGF synthesis in kidneys – unlike in other organs – is primarily regulated at a post‐transcriptional level. As IL‐6 mRNA expression increased simultaneously with VEGF protein levels, the post‐ischaemic regulation of IL‐6 and VEGF synthesis might be interrelated in rat kidney.

Immune Phenotype of Children with Newly Diagnosed and Gluten-Free Diet-Treated Celiac Disease

BackgroundRecent data suggest the involvement of both the adaptive and the innate immune system in celiac disease (CD). However, little is known about the immune phenotype of children with CD and its alteration upon dietary intervention.AimsWe characterized the prevalence of major interacting members of the adaptive and innate immune system in peripheral blood of newly diagnosed children with CD and tested its alteration with the improvement of clinical signs after the introduction of gluten-free diet (GFD).MethodsPeripheral blood was taken from ten children with biopsy-proven CD at the time of diagnosis and after the resolution of clinical symptoms following GFD. As controls, 15 children with functional abdominal pain were enrolled. The prevalence of the cells of adaptive and innate immunity was measured with labeled antibodies against surface markers and intracellular FoxP3 using a flow cytometer.ResultsPatients with CD were found to have lower T helper, Th1 and natural killer (NK), NKT and invariant NKT cell prevalence and with higher prevalence of activated CD4+ cells, myeloid dendritic cells (DC) and Toll-like receptor (TLR) 2 and TLR-4 positive DCs and monocytes compared to controls. After resolution of symptoms on GFD, the majority of these changes normalized, although the prevalence of NK and NKT cell, DC and TLR-2 expressing DCs and monocytes remained abnormal.ConclusionsThe immune phenotype in childhood CD indicates the implication of both adaptive and innate immune system. The normalization of immune abnormalities occurs on GFD, but the kinetics of this process probably differs among different cell types.

Decreased mucosal expression of intestinal alkaline phosphatase in children with coeliac disease

A major function of the enzyme intestinal alkaline phosphatase (iAP) is the detoxification of lipopolysaccharide (LPS), the ligand of Toll-like receptor 4 (TLR4). Hence, iAP has a role in the defence of maintaining intestinal barrier integrity. As intestinal barrier integrity is impaired in coeliac disease (CD), we tested the expression and localization of iAP in duodenal mucosa specimens from children with newly diagnosed CD (n = 10), with CD on gluten-free diet (GFD) (n = 5) and compared to those from ten healthy children. The mRNA and protein expression was determined by RT-PCR and Western blot analysis, respectively. Tissue localization of iAP and TLR4 was determined by immunofluorescence staining. iAP protein expression level was significantly lower than normal in newly diagnosed CD, while it was normalised in children on GFD. iAP and TLR4 colocalized at the epithelial surface of duodenal mucosa in each group of subjects enrolled. The finding of decreased iAP protein levels in newly diagnosed CD is consistent with its role in decreased intestinal barrier integrity. The latter may be the result of decreased LPS-detoxifying ability.

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.

σ1-Receptor Agonism Protects against Renal Ischemia-Reperfusion Injury

Mechanisms of renal ischemia-reperfusion injury remain unresolved, and effective therapies are lacking. We previously showed that dehydroepiandrosterone protects against renal ischemia-reperfusion injury in male rats. Here, we investigated the potential role of σ1-receptor activation in mediating this protection. In rats, pretreatment with either dehydroepiandrosterone or fluvoxamine, a high-affinity σ1-receptor agonist, improved survival, renal function and structure, and the inflammatory response after sublethal renal ischemia-reperfusion injury. In human proximal tubular epithelial cells, stimulation by fluvoxamine or oxidative stress caused the σ1-receptor to translocate from the endoplasmic reticulum to the cytosol and nucleus. Fluvoxamine stimulation in these cells also activated nitric oxide production that was blocked by σ1-receptor knockdown or Akt inhibition. Similarly, in the postischemic rat kidney, σ1-receptor activation by fluvoxamine triggered the Akt-nitric oxide synthase signaling pathway, resulting in time- and isoform-specific endothelial and neuronal nitric oxide synthase activation and nitric oxide production. Concurrently, intravital two-photon imaging revealed prompt peritubular vasodilation after fluvoxamine treatment, which was blocked by the σ1-receptor antagonist or various nitric oxide synthase blockers. In conclusion, in this rat model of ischemia-reperfusion injury, σ1-receptor agonists improved postischemic survival and renal function via activation of Akt-mediated nitric oxide signaling in the kidney. Thus, σ1-receptor activation might provide a therapeutic option for renoprotective therapy.

Expression of PARK7 is increased in celiac disease

Recently, it has been suggested that the gene called Parkinson’s disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies (p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis.

Increased synthesis of vascular endothelial growth factor in allergic airway inflammation in histidine decarboxylase knockout (HDC(-/-)) mice.

ABSTRACT Histamine and vascular endothelial growth factor (VEGF) have been implicated in the pathogenesis of allergic asthma; they enhance inflammation, vascular permeability, and mucus secretion. Histamine was suggested to alter the level of VEGF via the H2 receptors. Here the authors have applied histidine decarboxylase gene-targeted (HDC−/−) mice, lacking histamine, to investigate the effect of histamine deficiency on VEGF expression in an animal model of asthma. HDC−/− and wild-type (WT) mice were sensitized and challenged with ovalbumin (OVA). VEGF mRNA expression and protein level were determined in the lung. Number of VEGF-positive immune cells of bronchoalveolar lavage (BAL) and their intracellular VEGF content were measured by flow cytometry. VEGF protein level in the lung and in the BAL cells was increased in OVA treated (HDC−/−ova as well as in WTova) animals compared to their controls. However, there was no difference in the VEGF levels between HDC−/− or WT animals, either in the lung or in the BAL cells. In conclusion, increased VEGF production of the lung or BAL immune cells can be induced by allergen provocation independently from the genetic background of the animals. These data suggest that VEGF-mediated allergic processes can persist in the absence of histamine.

The role of Sigma-1 receptor in sex-specific heat shock response in an experimental rat model of renal ischaemia/reperfusion injury

We previously showed that female rats are more protected against renal ischaemia/reperfusion (I/R) injury than males, which is partly attributed to their more pronounced heat shock response. We recently described that Sigma‐1 receptor (S1R) activation improves postischaemic survival and renal function. 17β‐estradiol activates S1R, thus here we investigated the role of sex‐specific S1R activation and heat shock response in severe renal I/R injury. Proximal tubular cells were treated with 17β‐estradiol, which caused direct S1R activation and subsequent induction of heat shock response. Uninephrectomized female, male and ovariectomized female (Ovx) Wistar rats were subjected to 50‐min renal ischaemia followed by 2 (T2) and 24 (T24) hours of reperfusion. At T24 renal functional, impairment was less severe and structural damage was less prominent in females versus males or Ovx. Postischaemic increase in S1R, pAkt, HSF‐1, HSP72 levels were detected as early as at T2, while pHSP27 was elevated later at T24. Abundance of heat shock proteins was higher in healthy female rats and remained higher at T2 and T24 (female versus male or Ovx; resp.). We propose a S1R‐dependent mechanism, which contributes to the relative renoprotection of females after I/R injury by enhancing the heat shock response.