Fresh Porcine Kidneys Research Articles

Evaluation of the mechanical properties of porcine kidney.

With the development of medical diagnosis and treatment, knowing the mechanical properties of living tissues becomes critical. The aim of this study was to investigation material properties of the fresh porcine kidney and the parametric characterization of its viscoelastic material behavior. The material investigation included uniaxial tension tests in different strain rates, relaxation tests, as well as hydrostatic compression tests on the samples extracted from the fresh porcine kidney cortex. Tension tests and relaxation tests were performed by a planar dog-bone specimen with a micron loading testing machine. Hydrostatic compression tests were performed on the kidney cylinder sample which was placed in a compression chamber. Furthermore, a nonlinear viscoelastic model recently proposed by us was employed to characterize the tension data at different strain rates and relaxation test data. The the experimental and numerical results show that the stress-strain relations of the porcine kidney cortex at different strain rates in tension are presented for the first time and a higher strain rate results in higher ultimate strength and initial Young modulus but a lower rupture strain. A damage-dependent visco-elastic model is employed to model the tension data at different strain rates and relaxation data and exhibits a good agreement with the experimental data, which also demonstrates that the damage has an obvious influence on the stress-strain relation. Through comparison with the existing reference covering the uniaxial compression data, it seems that the mechanical behavior of the porcine kidney cortex manifests a stress state-dependent mechanical behavior. The ultimate strength and rupture strain are larger in compression than that in tension.

Renal puncture access via a nonpapillary track in percutaneous nephrolithotomy: an in vitro porcine kidney experience.

Recently, an increasing number of investigators have debated the wide rule of the puncture to renal papilla in PCNL. We evaluated the effectof renal papillary and nonpapillary puncture on bleeding in an in vitro porcine kidney experience, with the aim of determiningthe safe puncture sites of collecting system in PCNL. A total of 70 fresh porcine kidneys were selected and subjected to nephrostomy. We performed a puncture through a renal papilla, infundibulum, renal column, or minor calyceal neck (including the front, back, up, and down). The primary outcome was the amountof bleeding. The results showed that the papillary puncture group yielded minimal bleeding (1.59 ± 1.01ml/min) compared with the infundibular puncture group (6.25 ± 4.46ml/min, P < .001), renal column puncture group (4.24 ± 3.79ml/min, P = 0.001), and minor calyceal neck puncture group (2.27 ± 1.35ml/min, P = 0.011). However, after stratifying by orientation, the up (1.75 ± 0.80ml/min, P = 0.501) or down (1.77 ± 0.72ml/min, P = 0.437) minor calyceal neck puncture group and papillary puncture group yielded comparable bleeding. In summary, nonpapillary puncture must be carefully considered. Infundibular and renal column punctures were inferior to papillary puncture, and up or down minor calyceal neckpuncture may be a prudent choice in specific situations.

Mechanical and functional validation of a perfused, robot-assisted partial nephrectomy simulation platform using a combination of 3D printing and hydrogel casting.

There is a scarcity of high-fidelity, life-like, standardized and anatomically correct polymer-based kidney models for robot-assisted partial nephrectomy (RAPN) simulation training. The purpose of this technical report is to present mechanical and functional testing data as evidence for utilizing a perfused hydrogel kidney model created utilizing 3D printed injection casts for RAPN simulation and training. Anatomically correct, tumor-laden kidney models were created from 3D-printed casts designed from a patient's CT scan and injected with poly-vinyl alcohol (PVA). A variety of testing methods quantified Young's modulus in addition to comparing the functional effects of bleeding and suturing among fresh porcine kidneys and various formulations of PVA kidneys. 7% PVA at three freeze-thaw cycles (7%-3FT) was found to be the formula that best replicates the mechanical properties of fresh porcine kidney tissue, where mean(± SD) values of Young's modulus of porcine tissue vs 7%-3FT samples were calculated to be 85.97(± 35) kPa vs 80.97(± 9.05) kPa, 15.7(± 1.6) kPa vs 74.56(± 10) kPa and 87.46(± 2.97) kPa vs 83.4(± 0.7) kPa for unconfined compression, indentation and elastography testing, respectively. No significant difference was seen in mean suture tension during renorrhaphy necessary to achieve observable hemostasis and capsular violation during a simulated perfusion at 120mmHg. This is the first study to utilize extensive material testing analyses to determine the mechanical and functional properties of a perfused, inanimate simulation platform for RAPN, fabricated using a combination of image segmentation, 3D printing and PVA casting.

Pressure study of two miniaturised amplatz sheaths of 9.5 F and 12 F outer diameter for minimal invasive percutaneous nephrolithotomy (MIP): An ex vivo organ model measurement

The new devices were tested in a perfused organ model of fresh porcine kidneys with different irrigation pressures, applied either by gravitation or the use of a pressure pump (Uromat E.A.S.I. Pump, Karl Storz, Tuttlingen, Germany).In addition, the 9.5 F sheath was examined for active irrigation evacuation, i. e. suction of irrigation fluid through a mono-J-catheter. An urodynamic pressure probe measured intrapelvic pressure levels throughout the procedures. Regardless of the sheath diameters used, the intrapelvic pressure did not exceed 40 cmH2O (30 mmHg) when applying moderate irrigation pressure levels, either by pump or gravitation. The active suction of irrigation fluid from the kidney basin via the mono-J-catheter had no measurable impact on the detected intrarenal pressures. A crucial increase in the intrapelvic pressure was detected only when using the 9.5 F sheath in combination with applying high irrigation pressures. The newly designed miniaturised MIP sets maintain the favourable pressure features of the earlier 17.5 MIP sheath. Although the diameter has been reduced to 12F or 9.5 F, the intrapelvic pressures remained below 40 cmH2O when regular irrigation settings by gravitation or pump irrigation were used.

The anatomical structure of a fused renal pyramid and its clinical significance in the establishment of percutaneous renal access

Objective To explore the clinical significance of fused renal pyramid (FRP) structure in the establishment of percutaneous renal access. Methods From May 2017 to April 2018, 10 fresh porcine kidneys were selected to cast in blood vessels for grading the kidney artery. Then another 80 isolated porcine kidneys were used to simulate percutaneous renal puncture and dilatation to establish F24 operative access by the same surgeon. Under the endoscope and microscope, we compared the effects of four different puncture paths on the occurrence of renal vascular injury when respectively punctured through the normal renal pyramid (group A), the side of the FRP (group B), the centre of the FRP (group C) and the renal column (group D). Results The kidney arteries can be divided into six grades, there is grade Ⅳ branch-interlobar artery walking inside the FRP. The diameter of interlobar artery in the FRP was significantly smaller than that in the renal column (0.442±0.012) mm vs. (0.778±0.037) mm, (P<0.001). Endoscopic observation and pathological tissue section showed the following results. In group A, there was no injured blood vessel distributed along the access. There were six specimens with grade Ⅴ or Ⅵ arteries injury in the cortex. Owing to the small size of the renal pyramid and the inaccurate location of the puncture, there was also injury associated with a normal grade Ⅳ artery in the renal column. In group B, there was a certain distance between the tract and the grade Ⅳ artery that distributed in the FRP, injury was still noticed in four specimens. And six specimens have grade Ⅴ/Ⅵ arterial injury. As the distance between the tract and the renal column decreased, there was a case in which a simultaneously injury occurred to the extremity of a grade Ⅲ artery and a grade Ⅳ artery. In group C, there was a white thin strip of connective tissue exposed along the puncture tract. Ectopic grade Ⅳ artery injury occurred in fourteen specimens, and grade Ⅴ/Ⅵ artery injury occurred in seven specimens. In group D, there were grade Ⅲ to Ⅵ arteries distributed along the operational access, which was covered with white fat and connective tissue. The number of artery injury in grades Ⅲ, Ⅳ, and Ⅴ/Ⅵ were 4, 19, and 5, respectively. The mean ranks of artery injury degree in groups A (17.0), B (30.1), C (33.5) and D (41.5) gradually increased, and the difference was significant (P=0.006). There was a significant difference between group A and C (P=0.018), while no significant difference between group A and B (P=0.122), groups C and D (P=0.072). The proportion of grade Ⅳ artery injury in group A, B, and C was 5% (1/20), 25% (5/20), and 70% (14/20), respectively. There was a significant difference between group A and C (P=0.029), while no significant difference between group A and B (P=0.316). There was no significant difference in the injury of grade Ⅴ and Ⅵ artery in four groups (P=0.827). Conclusions When establishing a percutaneous renal access, vascular injury caused by puncturing through the FRP cannot be ignored. It is necessary to carefully identify and bypass the FRP when selecting the puncture path. If unavoidable, the puncture path should be on the centreline of one side pyramid of the FRP. Key words: Accurate puncture; Fused renal pyramid; Percutaneous nephrolithotomy; Vascular injury

Experimental and finite element analysis for prediction of kidney injury under blunt impact

Kidneys are third most injured organs in abdominal trauma after liver and spleen; this study therefore is an attempt to understand the behaviour of kidneys under blunt trauma. Dynamic impact tests were performed on 20 fresh porcine kidneys to study the injury propagation in the organ, and the acceleration of the impactor was measured. A kidney model was developed with structural details like capsule and cortex. The kidney cortex was modelled with solid hexahedral elements and the capsule was modelled with quadratic shell elements. The material models for the capsule and cortex were used from the experimental data reported in our previous study. The developed model was calibrated using previous and current experimental results to reproduce the injuries of the organ in terms of acceleration of the impactor, and the injuries sustained by the organ during the experiments. The developed kidney model is observed to be robust and can be integrated with the available human body finite element models to simulate accidents and to predict or simulate injuries.

Investigation of Forces Involved in Closure of the Renal Remnant After Simulated Partial Nephrectomy

To investigate the potential biomechanical causes of the complications of partial nephrectomy (PN) in a preclinical model of sliding-clip renorrhaphy. PN is a reference standard for amenable small renal masses. One disadvantage of PN, however, is the risk of postoperative bleeding and/or urinary leak. Simulated tumor excision and reconstruction using sliding-clip renorrhaphy were performed on fresh porcine kidneys. Suture tension (newtons, N) was measured in nonperfused and perfused states. Mean suture tension initially applied during renorrhaphy was 2.8 ± 0.7 N. After simulated perfusion to 120 mm Hg, increased tension was necessary to control fluid extravasation (average, 3.2 ± 0.7 N). For intravascular pressures above 200 mm Hg, an average tension of 3.4 ± 0.7 N was necessary to prevent observable leakage. The increase in suture tension under normal and hypertensive states averaged 21 ± 28% and 29 ± 31%, respectively. In experiments examining maximum suture tension before suture tear through, failure of the anchored sutures varied with the width of incorporated renal capsule. Mean forces were 5.7 ± 3.2, 8.8 ± 5.7, and 14.0 ± 6.0 N with 0.5, 1.0, and 1.5 cm of capsule, respectively. Review of video footage indicated that acute angles appear to contribute to suture failure. This study demonstrates that the tension required to cause suture failure is only slightly higher than the tension typically applied during PN and necessary to control bleeding and urine leaks. After reperfusion of the kidney, the tension can increase by ≥ 29% under hypertensive conditions. Incorporation of sufficient (≥ 0.5 cm) capsule and avoidance of acute angles of entry or exit during closure of the kidney are likely to reduce suture failure.

Anchoring strength of V-LOCTM barbed suture and Hem-o-lok clips: experimental comparison of nephro sparing surgery in porcine kidney model

Objective To compare anchoring capabilities and holding strength of Hem-o-lok clips and barbed sutures on porcine renal parenchyma.Methods An ex-vivo model was used for the study by using fresh porcine kidneys with intact renal capsule.A longitudinal incision was made on the renal capsule and parenchyma to a depth of 0.5 cm.The following sutures were tested:a 2-0 Vicryl (Covidien) with one and two Hem-o-lok clips (Weck),2-0 V-loc (Covidien),and 4-0 V-loc.An interrupted suture was placed through the renal capsule and parenchyma and attached to a hand-held force tester to quantify the holding strength.The tearing strength was also measured with the force tester using a horizontal mattress suture of 2-0 V-loc and 2-0 Vicryl secured with one Hem-o-lok clip (suture passing either through the middle or the far edge of the clip).Then,a Nephromax balloon catheter (Boston Scientific) was placed into the parenchymal incision,which was closed by a horizontal mattress suture with sliding-clip technique or by using a V-loc suture.The balloon was dilated and the incision of renal parenchyma was observed.Every procedure was repeated five times.Finally,the laparoscopy partial nephrectomy was performed with 2-0 V-loc suture in the living porcine model.Results The mean forces required for suture slippage are 2.52 N (2-0 V-loc suture ),3.26 N (2-0 Vicryl suture with one Hem-o-lok clip) and 4.10 N (2-0 Vicryl suture with two Hem-o-lok clips).And in the 2-0 V-loc suture group,the forces to violate the renal capsule was lower than in the two Hem-o-lok clips group.Unlike the renorrhaphy using the sliding clip technique,the horizontal mattress closure with the V-loc suture had withstand balloon dilation.However,in the living animal lab,the V-loc suture can facilitate renorrhaphy safely during partial nephrectomy.Conclusions The V-loc barbed suture can provide a similar holding strength with the same size Vicryl suture with one Weck clip anchor.However,we need more study to show enough safety due to the tissue tearing effect. Key words: Partial nephrectomy; Renorrhaphy; Warm ischemia; Safety

The “Visual Dilator System”: Initial Experimental Evaluation of an Optical Tract Dilation Technique in Percutaneous Nephrolithotomy

Ascertaining the optimal depth of dilator progression is a crucial factor during tract dilation in percutaneous nephrolithotomy. Dilation is generally guided under fluoroscopy, ultrasonography, or combination of both techniques, and it is still difficult to prevent overdilation causing perforation and vascular injury or underdilation causing difficulty in establishment of access. Thus, improvements in dilation technique bears clinical significance. Our aim was to evaluate the feasibility in an animal model of using the "visual dilator system" for optimal percutaneous tract dilation under real-time visual guidance. The visual dilator system consisted of a transparent hollow dilator made of polyvinyl chloride and a rigid ureteroscope inserted within its lumen. The ureteroscope was connected to a standard endoscopic camera system. The dilator system backloaded with an access sheath was passed over a guidewire to dilate the tract and position the access sheath under visual guidance. Saline was used as irrigation to maintain clarity during dilation. The dilation technique was implemented to establish percutaneous access tract in six PCNL simulator models made from fresh porcine kidney placed inside an eviscerated chicken carcass and four female swine (eight kidneys) to experimentally evaluate its feasibility and efficacy. All tracts were successfully established with dilation through the intervening tissue layers, approach into the target calix, as well as access sheath placement being visually monitored through the dilator wall to confirm accuracy. All procedures were free of dilation-related complications such as collecting system perforation, excessive bleeding (in-vivo model), or loss of access. The limited number of animal models and lack of randomized comparative study with other dilator modalities were major drawbacks of the study. The study demonstrates feasibility of percutaneous access tract dilation using a Visual dilator system. This technique can provide visual confirmation of accuracy in dilation to improve safety and efficacy of the procedure.

MitoQ Blunts Mitochondrial and Renal Damage during Cold Preservation of Porcine Kidneys

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.

Real Time 3D Visualization of Intraoperative Organ Deformations Using Structured Dictionary

Restricted visualization of the surgical field is one of the most critical challenges for minimally invasive surgery (MIS). Current intraoperative visualization systems are promising. However, they can hardly meet the requirements of high resolution and real time 3D visualization of the surgical scene to support the recognition of anatomic structures for safe MIS procedures. In this paper, we present a new approach for real time 3D visualization of organ deformations based on optical imaging patches with limited field-of-view and a single preoperative scan of magnetic resonance imaging (MRI) or computed tomography (CT). The idea for reconstruction is motivated by our empirical observation that the spherical harmonic coefficients corresponding to distorted surfaces of a given organ lie in lower dimensional subspaces in a structured dictionary that can be learned from a set of representative training surfaces. We provide both theoretical and practical designs for achieving these goals. Specifically, we discuss details about the selection of limited optical views and the registration of partial optical images with a single preoperative MRI/CT scan. The design proposed in this paper is evaluated with both finite element modeling data and ex vivo experiments. The ex vivo test is conducted on fresh porcine kidneys using 3D MRI scans with 1.2 mm resolution and a portable laser scanner with an accuracy of 0.13 mm. Results show that the proposed method achieves a sub-3 mm spatial resolution in terms of Hausdorff distance when using only one preoperative MRI scan and the optical patch from the single-sided view of the kidney. The reconstruction frame rate is between 10 frames/s and 39 frames/s depending on the complexity of the test model.

Ex Vivo Thermoacoustic Imaging Over Large Fields of View With 108 MHz Irradiation

Thermoacoustic signals are generated over a large field of view by 900 ns TE10 pulses with 108 MHz carrier frequency. Test specimens selectively absorb the TE10 pulse energy producing rapid thermal expansions that generate ultrasonic pulses. 108 MHz irradiation provides excellent depth penetration in soft tissue, allowing blood and physiologic saline to generate strong signals. Thermoacoustic signal generation from a depth of several cm is well above our systems noise floor. Rotating the test specimen provides single-slice tomographic signal encoding. Filtered back-projection reconstruction yields images over a 6.4 cm field of view. Reconstructions of tissue mimicking prostate phantoms and fresh porcine kidney tissue are presented.

D-Aminoacid Oxidase-Fe&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O&amp;lt;sub&amp;gt;3&amp;lt;/sub&amp;gt; Nanoparticle Complex Mediated Antitumor Activity in Swiss Albino Mice

D-Aminoacid oxidase (DAO) was isolated from fresh porcine kidney; its cytotoxic potential was studied under in vitro and in vivo conditions. The isolated DAO was complexed with Fe2O3 nanoparticles and its potential as an oxidation therapeutic agent was analysed. The ability of the complex in eliciting H2O2 mediated cytotoxicity was studied on Dalton’s lymphoma ascites cells (DLA). The induction of apoptosis in DLA cells by Fe2O3-DAO complex was studied by morphological examination and alkaline single cell gel electrophoresis (comet assay). The antitumor activity of the complex was investigated by oral administration of the complex and the substrate D-alanine to tumor bearing Swiss albino mice and by targeting the complex to the tumor site, using an externally applied magnetic field. Fe2O3-DAO along with D-alanine showed remarkable cytotoxicity in a substrate concentration-dependent manner. Both morphological examination and comet assay revealed that Fe2O3-DAO/D-alanine induced apoptosis. Oral administration of Fe2O3-DAO and D-alanine along with magnetic targeting significantly suppressed tumor growth in mice. The present report provides the first evidence for the promising application of enzyme bound nanoparticles for targeted oxidation therapy.

A Novel Training Model for Laparoscopic Partial Nephrectomy Using Porcine Kidney

The purpose of this study was to create a simple model for teaching laparoscopic partial nephrectomy (LPN) for duplicating the blood supply setting and main technical points of the procedure. A fresh porcine kidney was placed on an unfolded metallic box to help trainees observe, practice, and develop LPN skills in both tumor incision and suturing. The metallic box was specially designed not only for fixing and positioning the kidney but also for storing the outflow of the kidney while its artery was kept artificially infused. A spherical renal parenchyma excision about 2 cm in diameter was initially made and the suture-repairing work followed. This model was used by trainees who had fair laparoscopic competency after completing dry laboratory training programs. During a 20-day training period the trainees were assessed with respect to the time needed to complete LPN. A mentor-trainee fellowship program was planned to guarantee the success of the operations and the quality of LPN. There was a good reduction in the total operative time from the fourth to the last attempt. There was also increasing improvement in the quality of the incision suturing during the attempts. By the end of 10 attempts all trainees were more confident in completing a good quality LPN in a mean time of 28.4 minutes. This model not only offers trainees the opportunities to acquire the skills necessary for LPN but also provides for more economical practice by using easily available porcine kidney.

A Biological Phantom for Contrast-Media-Based Perfusion Studies With CT

Perfusion computed tomography (PCT) is increasingly getting popular with the advent of computed tomography (CT) systems with adequate temporal resolution and spatial coverage. We sought to develop a biological phantom for perfusion measurements in CT to design, improve, and validate scan protocols and postprocessing algorithms in vitro. A special technique was applied to prepare and preserve a fresh porcine kidney. The kidney was connected to an open circuit driven by a peristaltic pump with the option to inject contrast material. We evaluated repeated dynamic contrast-enhanced CT acquisitions with different input flow rates and the relation to calculated parenchymal flow results of the phantom. Flow was calculated with 2 different algorithms. Identical scans were performed with a time interval of 1 year to check long-term stability of the phantom. Different bolus geometries were designed and bolus dispersion was measured for the setup using a tubing array. We found a linear relationship between the input flow rate of the circuit and the calculated phantom tissue flow with a correlation coefficient rr2 = 0.99 for both algorithms. Both algorithms resulted in very similar absolute values, the mean difference was 3.1 mL/100 mL/min. Perfusion measurements with contrast material injection and storage did not alter the phantom. The enhancement properties did not change over the time of 1 year. With our setup, it was possible to design typical bolus geometries as they occur in clinical practice. Bolus dispersion was small: peak enhancement and bolus width changed by about only 5% over 2-m tube length. A phantom for parenchymal flow measurements suitable for repeated measurements over a long period of time was developed. The setup allows the design of diverse bolus geometries with negligible dispersion.

Effects of urinary ouabain-like factors and vanadium diascorbate on calcium mobilization in porcine inner medullary collecting duct cells: Comparison with the effects of ouabain and vasopressin

It is speculated that ouabain-like factors (OLF) play a role in the pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. We subsequently identified the chemical structure of OLF-2 as vanadium (V IV ) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on free calcium concentration [Ca 2+] i, in cultured porcine IMCD cells. [Ca 2+] i was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10 −4 mol/L) produced a slow increase in [Ca 2+] i from 39 ± 10 to 169 ± 21 nmol/L (n = 7 ) after 4 min. Similarly, OLF-2 (10 −4 mol/L) resulted in an increase in [Ca 2+] i from 74 ± 20 to 216 ± 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (MW 403) dose-dependently increased [Ca 2+] i . At a concentration of 10 −6 mol/L it increased [Ca 2+] i from 46 ± 5 to 149 ± 9 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca 2+] i was found with ouabain (10 −6 mol/L), which increased [Ca 2+] i from 61 ± 22 to 180 ± 29 nmol/L (n = 5) after 4 min in contrast to AVP (10 −7 mol/L), which rapidly increased [Ca 2+] i from 48 ± 10 to 299 ± 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca 2+] i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.

Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.

1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)