Inhibition of Notch Signaling Ameliorates Acute Kidney Failure and Downregulates Platelet-Derived Growth Factor Receptor ß in the Mouse Model.

Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor ß (Pdgfrb). Treatment of ischemic mice with the x03B3;-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis.

Erythropoietin-enhanced endothelial progenitor cell recruitment in peripheral blood and renal vessels during experimental acute kidney injury in rats.

Beneficial effects of erythropoietin (EPO) have been reported in acute kidney injury (AKI) when administered prior to induction of AKI. We studied the effects of EPO administration on renal function shortly after ischemic AKI. For this purpose, rats were subjected to renal ischemia for 30 min and EPO was administered at a concentration of 500 U/kg either i.v. as a single shot directly after ischemia or with an additional i.p. dose until 3 days after surgery. The results were compared with AKI rats without EPO application and a sham-operated group. Renal function was assessed by measurement of serum biochemical markers, histological grading, and using an isolated perfused kidney (IPK) model. Furthermore, we performed flow cytometry to analyze the concentration of endothelial progenitor cells (EPCs) in the peripheral blood and renal vessels. Following EPO application, there was only a statistically non-significant tendency of serum creatinine and urea to improve, particularly after daily EPO application. Renal vascular resistance and the renal perfusion rate were not significantly altered. In the histological analysis, acute tubular necrosis was only marginally ameliorated following EPO administration. In summary, we could not demonstrate a significant improvement in renal function when EPO was applied after AKI. Interestingly, however, EPO treatment resulted in a highly significant increase in CD133- and CD34-positive EPC both in the peripheral blood and renal vessels.

Urodilatin and pentoxifylline prevent the early onset of Escherichia coli-induced acute renal failure in a model of isolated perfused rat kidney.

BACKGROUND/AIMS: Raised cytokine levels and a hypoperfusion-associated decrease in glomerular filtration rate (GFR) are hallmarks of the genesis of septic acute renal failure (ARF). Therefore, anti-inflammatory as well as renal vasodilating therapeutic strategies may afford renal protection during septic ARF. The present study was designed to determine the effects of administration of urodilatin, pentoxifylline and theophylline to improve renal function in an ex-vivo model of 'septic renal injury'. METHODS: Eight series of experiments were performed: no intervention, perfusion with a suspension containing Escherichia coli bacteria (strain 536/21); E. coli + 10 microg/l urodilatin, E. coli + 20 microg/l urodilatin, E. coli + 100 microM theophylline, E. coli + 100 microM pentoxifylline and E. coli + URO 20 microg/l given 90 min after start of perfusion. Renal vascular and glomerular functional parameters as well as TNF-alpha release were analyzed up to 180 min. RESULTS: Perfusion with E. coli caused an acute deterioration of renal vascular and glomerular function. URO 20 microg/l and PTX decreased renal vascular resistance (RVR) from 83.7 +/- 18.4 to 9.2 +/- 1.1 and 8.6 +/- 2.2 mm Hg/ml/min/g kidney and increased renal perfusion flow rate (PFR) from 8.2 +/- 1.5 to 14.6 +/- 0.8 and 14.1 +/- 2.2 ml/min/g kidney. As a result, GFR improved from 102.1 +/- 15.6 to 442 +/- 48.3 and 525.8 +/- 57 microl/min/g kidney during treatment with URO 20 microg/l and PTX, respectively. Renal TNF-alpha release was significantly reduced by URO 20 microg/l (from 178 +/- 23 to 45.2 +/- 2 and 47 +/- 3 pg/ml) in the E. coli + URO 20 microg/l and by PTX in the E. coli + PTX group if added to the perfusion medium upon start of perfusion. Interestingly, URO 20 microg/l also decreased RVR significantly from 62.2 +/- 6.1 to 35.9 +/- 6.0 mm Hg/ml/min/g kidney, improved PFR from 5.4 +/- 1.0 to 8.7 +/- 1.0 ml/min/g kidney, increased GFR from 160 +/- 43.3 to 280.7 +/- 27.9 microl/min/g kidney, and decreased TNF-alpha release to 122 +/- 18 pg/ml if applied 90 min after induction of septic ARF. In contrast, URO 10 microg/l did not significantly increase urine flow and did not appear to significantly improve renal perfusion. Theophylline showed no beneficial effects at all. CONCLUSION: This suggests that urodilatin and pentoxifylline might be useful to protect renal function if given before a septic renal insult. Additionally, treatment with urodilatin is capable of restoring renal function in early Gram-negative sepsis-induced ARF even if given after the septic insult.

The effects of angiotensin-converting enzyme inhibition on the urinary excretion of NTproBNP in male volunteers.

AIMS: This study was designed to test if the renal excretion of the N-terminal prohormone of the B-type natriuretic peptide (NTproBNP) is modulated by angiotensin-converting enzyme inhibition (ACE-I). METHODS: Following 7 days on a sodium-enriched diet and an induction period of 4 days with incremental dosages of enalapril (2.5, 5, 7.5, 10 mg) or placebo, 10 healthy subjects underwent crossover and double-blind treatment with 20 mg enalapril sodium or placebo at 8:00 h. After 4 h (at 12:00 h), 20 ml.kg(-1) NaCl 0.9% was infused over 60 min. Hemodynamics were determined and blood and urine were sampled at 8:00, 12:00, 13:00, 14:00, 16:00, and 18:00 h. Angiotensin II (AII), NTproANP, and NTproBNP were determined by radio- and electrochemiluminescence immunoassays. RESULTS: In the whole group, ACE-I led to a lower arterial blood pressure during the fourth day of induction and during the time from 8:00 to 16:00 h, a decrease in AII levels from 8:00 to 14:00 h (p < 0.05), and to a higher cumulative urine output (p < 0.05) in comparison with control. Neither cumulative sodium nor urinary NTproBNP/creatinine excretion were significantly increased after ACE-I. However, a subgroup of 6 volunteers - showing an increase in sodium excretion after ACE-I - also demonstrated lower AII levels at 13:00 h, a higher cumulative urine flow, and a higher urinary NTproBNP/creatinine excretion in comparison with control (all: p < 0.05). CONCLUSIONS: This suggests that the renal excretion of NTproBNP is modified by enalapril. However, it remains to be determined if this is a direct effect of ACE-I, the decrease in arterial blood pressure, or other potentially confounding variables like bradykinin or endopeptidase activity.

Is the renal production of erythropoietin controlled by the brain stem?

Although the structure and function of erythropoietin (Epo) are well documented, the mechanisms of the regulation of the renal synthesis of Epo are still poorly understood. Especially, the description of the localization and function of the O(2)-sensitive sensor regulating the renal synthesis of Epo is insufficient. A body of evidence suggests that extrarenal O(2)-sensitive sensors, localized particularly in the brain stem, play an important role in this connection. To support this concept, high cerebral pressure with consecutive hypoxia of the brain stem was generated by insufflation of synthetic cerebrospinal fluid into the catheterized cisterna magna of rats. When the cerebral pressure of the rats was above the level of their mean arterial blood pressure or the high cerebral pressure persisted for a longer period (>/=10 min), the Epo plasma concentration increased significantly. Bilateral nephrectomy or hypophysectomy before initiation of high intracranial pressure abolished this effect. Systemic parameters (heart rate, blood pressure, Pa(O(2)), Pa(CO(2)), arterial pH, renal blood flow, glucose concentration in blood) were not affected. Other stressors, like restricting the mobility of the rats, had no effect on Epo production. Hence, the effect of high cerebral pressure on renal synthesis of Epo seems to be specific. Increasing cerebral hydrostatic pressure leads to increased renal synthesis of Epo. Obviously, during hypoxia, cerebral O(2)-sensitive sensors release humoral factors, triggering the renal synthesis of Epo. The structure and function of these "Epo-releasing-factors" will have to be characterized in future experiments.

Effects of tilting and volume loading on plasma levels and urinary excretion of relaxin, NT-pro-ANP, and NT-pro-BNP in male volunteers.

The polypeptide relaxin (RLX) has been suggested to play a role in cardiorenal integration and to be related to the natriuretic peptide system. We hence examined the effects of variations in thoracic blood volume and intravenous volume loading on plasma and urinary RLX levels and associated changes in natriuretic peptide levels in healthy men. Two groups of eight subjects were randomly tilted into a 15 degrees feet-down or a 15 degrees head-down position. Ten volunteers were crossover subjected to an infusion of 15 ml/kg of 0.9% NaCl (over 60 min) or control during an observation period of 10 h. Blood and urine were sampled at timed intervals. RLX, NH(2)-terminal prohormones of atrial natriuretic peptide (NT-pro-ANP), and NH(2)-terminal prohormones of brain natriuretic peptide (NT-pro-BNP) were determined by enzyme, radio-, and electrochemoluminescence immunoassays, respectively. NT-pro-ANP levels (in percentage of baseline levels) were higher (P < 0.05) during the head-down (124 +/- 13%) than during the feet-down position (82 +/- 6%). NT-pro-BNP and RLX were not affected by tilting. Volume loading induced a short-lasting increase in plasma NT-pro-ANP, a delayed increase in plasma NT-pro-BNP, had no effect on plasma RLX, and induced a parallel increase in urine flow, renal excretion of sodium, RLX, and NT-pro-BNP. It is concluded that variations in thoracic blood volume in healthy men are not associated with variations in plasma RLX. Increased urinary RLX and NT-pro-BNP excretion during volume loading suggest renal production and a possible role of kidney-derived RLX and brain natriuretic peptide in sodium homeostasis in men.

Biochemical tissue monitoring during hypoxia and reoxygenation.

Oxygen deficiency during critical illness may cause profound changes in cellular metabolism and subsequent tissue and organ dysfunction. Clinical treatment in these cases targets rapid reoxygenation to avoid a prolonged impaired synthesis of cellular high-energy phosphates (ATP). However, the effect of this therapeutic intervention on tissue metabolism has not been determined yet. Thus the present study was designed to determine the effects of hypoxia and reoxygenation with either room air or 100% oxygen on variables of interstitial metabolism in different tissues using in vivo microdialysis. Twenty-seven adult, male CD-rats (407-487 g; Ivanovas, Kisslegg, Germany) were studied during general anesthesia. Following preparation and randomization, rats were normoventilated for 45 min (FiO(2) 0.21), followed by induction of hypoxia (FiO(2) 0.1, 40 min) and reoxygenated for 50 min either with FiO(2) 1.0 (group 1, n=10) or FiO(2) 0.21 (group 2, n=10). Control animals (n=7) were ventilated with 21% oxygen during the observation period. Additional to invasive haemodynamic parameters, biochemical tissue monitoring was performed using CMA 20 microdialysis probes, inserted into muscle, subcutaneous space, liver, and the peritoneal cavity allowing analyses of lactate and pyruvate at short intervals. Hypoxia induced a significant reduction in mean arterial pressure (MAP) in group 1 and 2 compared with the control group (P<0.05) without any significant differences between both treatment groups. This was accompanied by a significant increase in blood lactate (10.5+/-3.1 mM (group 1) and 12.3+/-4.1 mM (group 2) vs. 1.5+/-0.3 mM (control); P<0.05) and severe metabolic acidosis (base excess (BE): -18.3+/-5 mM (1) and -17.3+/-7 mM (2) vs. -2.6+/-1.8 mM (control), P<0.05). During hypoxia, the interstitial lacate/pyruvate ratio in groups 1 and 2 increased to 455+/-199% (muscle), 468+/-148% (intraperitoneal), 770+/-218% (hepatic) and 855+/-432% (subcutaneous) (P<0.05 vs. control, respectively). No significant inter-organ or inter-group differences in interstitial dialysates were observed in the treatment groups, neither during hypoxia nor during reoxygenation. Our data suggest, that hypoxia induces comparable metabolic alterations in various tissues and that reoxygenation with 100% oxygen is not superior to 21% oxygen in restoring tissue metabolism after critical hypoxia.

The antimycotic ciclopirox olamine induces HIF-1alpha stability, VEGF expression, and angiogenesis.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master regulator of oxygen homeostasis. Protein stability and transactivation function of the alpha subunit are controlled by iron- and oxygen-dependent hydroxylation of proline and asparagine residues. The anti-mycotic ciclopirox olamine (CPX) is a lipophilic bidentate iron chelator that stabilizes HIF-1alpha under normoxic conditions at lower concentrations than other iron chelators, probably by inhibiting HIF-1alpha hydroxylation. As shown by the inhibition of iron-dependent quenching of FITC-labeled deferoxamine (DFX) fluorescence, CPX appears to have an even higher affinity for iron than DFX. Initial observations that treatment with 1% CPX, but not with placebo, occasionally caused reddening of wound margins in a mouse skin wound model prompted us to investigate the capability of CPX to induce angiogenesis. CPX-induced HIF-1-mediated reporter gene activity and endogenous HIF-1 target gene expression, including elevation of transcription, mRNA, and protein levels of the vascular endothelial growth factor (VEGF). In the chick chorioallantoic membrane assay, inert polymer disks containing CPX but not the solvent alone induced angiogenesis. In summary, these results suggest that CPX induces angiogenesis in vivo via HIF-1 and VEGF induction. Therefore, CPX might serve as an alternative to recombinant VEGF treatment or to VEGF gene therapy for therapeutic angiogenesis.