Horst Pagel

Quality of salivary tears following autologous submandibular gland transplantation for severe dry eye.

Abstract · Background: This study aimed to characterise the composition of the pre-ocular fluid after transplantation of the autologous submandibular gland (SMG) for patients with severe dry eye. · Methods: Stimulated and unstimulated pre-ocular fluid from 15 patients (17 eyes) with a viable SMG graft (“SMG-salivary tears”), as well as normal tears and SMG saliva (20 normal subjects/ 20 eyes), was sampled. As global tear parameters, fern pattern analysis and SDS gel electrophoresis were performed. As specific quality parameters, total protein content, secretory immunoglobulin A (SIgA), lysozyme, amylase, sodium, potassium and osmolality were measured using routine laboratory methods. The flow rate of SMG-salivary tears was determined in 5 patients by means of sequential scintillography. · Results: The fern pattern of SMG-salivary tears was coarse and thus more similar to normal SMG saliva than tears. SDS gel electrophoresis of the SMG-salivary tears showed albumin and two unidentified proteins in addition to the normal tear pattern. Osmolality and total protein content of SMG-salivary tears were higher than in normal SMG saliva, but still lower than in normal tears. High activities of normal tear antibacterial proteins (SIgA, lysozyme and amylase) were detected in the salivary tears. Stimulation of the secretion did not alter the composition of SMG-salivary tears. The flow rate of SMG-salivary tears was closer to that of normal tears than normal SMG saliva. · Conclusion: Salivary tears resulting from SMG-transplantation represent condensed SMG saliva. Thus their quality is intermediate between normal tears and normal SMG saliva. High levels of secretory proteins demonstrate that the gland maintains an active function. Surgical denervation and residual tear components from the ocular surface are the most likely factors to cause the complex differences between normal SMG saliva and SMG-salivary tears. The effects of this secretion on the ocular surface are currently being evaluated in a clinical and laboratory study.

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.

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 μg/l urodilatin, E. coli + 20 μg/l urodilatin, E. coli + 100 μM theophylline, E. coli + 100 μM pentoxifylline and E. coli + URO 20 μg/l given 90 min after start of perfusion. Renal vascular and glomerular functional parameters as well as TNF-α release were analyzed up to 180 min. Results: Perfusion with E. coli caused an acute deterioration of renal vascular and glomerular function. URO 20 μg/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 μl/min/g kidney during treatment with URO 20 μg/l and PTX, respectively. Renal TNF-α release was significantly reduced by URO 20 μg/l (from 178 ± 23 to 45.2 ± 2 and 47 ± 3 pg/ml) in the E. coli + URO 20 μg/l and by PTX in the E. coli + PTX group if added to the perfusion medium upon start of perfusion. Interestingly, URO 20 μg/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 μl/min/g kidney, and decreased TNF-α release to 122 ± 18 pg/ml if applied 90 min after induction of septic ARF. In contrast, URO 10 μg/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.

Inhibition of Notch Signaling Ameliorates Acute Kidney Failure and Downregulates Platelet-Derived Growth Factor Receptor β 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 β (Pdgfrb). Treatment of ischemic mice with the γ-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.

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.

Virulence Factors of Escherichia coli Contribute to Acute Renal Failure

Background: The development of acute renal failure (ARF) significantly enhances the mortality of patients with Gram-negative septic shock. The role of specific bacterial virulence factors different from lipopolysaccharides (LPS) in the deterioration of renal function in septic shock remains to be determined. Methods: An Escherichia coli wild-type strain (536/21 WT, O6:K15:H31) was isolated from a patient suffering from a urinary tract infection. The strain expresses various virulence factors (e.g. hemolysin, fimbriae) genetically encoded by pathogenicity islands. The spontaneous deletion mutant 536/21 Del lacks the expression of these virulence factors. Isolated rat kidneys were perfused with a suspension (5 × 104/ml) of the respective strain or control perfusion medium and the renal functional parameters were analyzed. Intrarenal deposition of E. coli was detected by immunohistology and Gram staining. Results: The perfusion of the isolated perfused rat kidney with a uropathogenic E. coli wild-type strain (536/21 WT) caused an acute deterioration of renal function which was not observed in kidneys exposed to a deletion mutant of E. coli 536/21 lacking the expression of virulence factors. The glomerular filtration rate and the urine flow rate significantly decreased only in kidneys perfused with the E. coli wild-type strain, while there was no change versus controls in kidneys perfused with the deletion mutant. Conclusions: Distinctive bacterial virulence factors different from LPS such as hemolysin and the presence of different fimbriae may contribute to the development of ARF in sepsis induced by E. coli. Anti-LPS strategies may not be sufficient to reduce the risk of ARF in Gram-negative septic shock.

Effects of Angiotensin II and the AT1 Receptor Antagonist Losartan on the Renal Excretion of Urodilatin

Background: The precise mechanisms regulating the natriuretic peptide urodilatin (ANP-95-126) remain to be defined. Renal excretion of urodilatin (UUROV) has been shown to be modified by variations in plasma sodium and renal perfusion pressure. This suggests a relationship between urodilatin and the renin-angiotensin system. Methods: We investigated the effects of angiotensin II (AII, 0.1 nmol/l) and the AT1 receptor antagonist losartan (LS, 1 µmol/l) on UUROV and renal function in isolated rat kidneys perfused for 180 min in a closed circuit system. A further series employing a vasoconstricting concentration of endothelin-1 (ET-1, 0.01 nmol/l) was performed to explore the effects of vasoconstriction and glomerular filtration rate (GFR) on UUROV. Results: Urine flow (UV) and urinary sodium excretion (UNaV) decreased and renal vascular resistance (RVR) increased after treatment with AII (n = 5) in comparison with a control group (n = 6; p < 0.05). Treatment with LS (n = 5) and AII+LS (n = 5) had no significant effect on these parameters. GFR decreased after AII (p < 0.05) and was not significantly altered by other interventions. UUROV decreased after AII (p < 0.05) and was comparable to the control group after LS and AII+LS. ET-1 (n = 5) induced a significant increase in RVR and decreased UV and UNaV (p < 0.05). Point-to-point analysis revealed that the ET-1-induced vasoconstriction and the subsequent decrease in GFR had no effect on UUROV. Conclusions: This suggests that vasoconstrictory concentrations of AII decrease UUROV in the isolated perfused rat kidney. The lack of effect of ET-1 on UUROV suggests that the AII-induced alterations in urodilatin excretion cannot be explained by vasoconstriction per se.

Pentoxifylline improves circulatory and metabolic recovery after cardiopulmonary resuscitation

BACKGROUND To evaluate the effectiveness of a bolus application of pentoxifylline (PTXF) at the beginning of CPR in a standardized resuscitation animal model. METHODS AND RESULTS In a laboratory model of cardiac arrest, 12 Wistar rats (382-413 g) were randomized into two groups. Both groups underwent 4 min of cardiopulmonary arrest induced by a transthoracic application of a fibrillating current of 10 mA. At the beginning of CPR, group one (n=6) received a bolus injection of 10 mg kg(-1) body weight PTXF versus sodium chloride in group two (controls: n=6). All animals developed a severe lactate acidosis during and after CPR but in PTXF treated animals acid-base values returned to baseline pattern. During return of spontaneous circulation (ROSC) in the PTXF group lactate concentration decreased from 13.4+/-2.1 to 1.9+/-0.7 mmol l(-1) within 60 min (P<0.01). In control animals, lactate values remained high (10.8+/-3.5 by 60 min, P<0.01). After bolus injection of PTXF pH increased from 6.93+/-0.06 to 7.29+/-0.13 within 60 min of ROSC versus 6.85+/-0.05 to 6.97+/-0.23 in sodium chloride treated animals (P<0.01). Within 5 min of ROSC, PTXF treated animals achieved higher oxygenation values (PTXF P(a)O(2)=216.9+/-62.5 mmHg, control 132. 2+/-15.1 mmHg, P<0.01). CONCLUSIONS Administration of PTXF at the beginning of CPR improved macrocirculation, acid-base status and arterial oxygenation.

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.

A kidney cannot succeed days after cardiac surgery when on high-dose furosemide.

With great interest we read the study by Redfors et al (1) about the renal oxygen supply/demand relationship in patients with cardiac surgery-associated acute kidney injury and further congratulate the authors for this elegant experimental setup. The authors draw the conclusion that acute renal failure after cardiac surgery is NOT an “acute renal success” but caused by a combination of renal vasoconstriction and tubular sodium resorption at a high oxygen demand. However, we are not convinced that this is the only way to interpret the presented data. In contrast, we feel that these data are much more an excellent experimental proof that high-dose furosemide therapy should be avoided in patients with cardiac surgery-associated acute kidney injury.