Increased urinary excretion of endothelin during hypothermic perfusion preservation in kidneys subjected to preretrieval warm ischemic injury.

The purpose of the study was two-fold: 1) to determine whether endothelin (ET) levels could be detected in the ureteral effluent during hypothermic perfusion preservation (HPP) and; 2) to determine whether preretrieval warm ischemic (WI) injury is associated with increased ureteral excretion of ET. In situ pre-WI injury was induced in Lewis rats (n=10) by a 30-min extrinsic occlusion of the suprarenal aorta. The left kidney underwent 16 hr of HPP, and ureteral effluent (UE) from ischemic and control kidneys (n=10) was collected over 16 hr of HPP. The UE ET concentration and total ET excretion over 16 hr of HPP were significantly higher in kidneys subjected to pre-WI injury compared with nonischemic controls. Kidneys subjected to pre-WI injury can be distinguished from nonischemic control kidneys during HPP by a significantly higher concentration of ET in the UE and a higher overall excretion of ET during HPP.

Identification of kidneys subjected to preretrieval warm ischemic injury by simultaneous monitoring of glomerular filtration and perfusate flow during hypothermic perfusion preservation.

BACKGROUND: Historically, ex vivo physiological evaluation of cadaveric renal allografts has been limited to assessing perfusate flow (PF) during hypothermic perfusion preservation (HPP). Using a small animal model, we have previously described a method for continuous monitoring of glomerular filtration rate (GFR) during HPP. Our study was undertaken to determine if monitoring GFR and PF during HPP distinguished kidneys subjected to preretrieval warm ischemic (WI) injury more reliably than PF alone. METHODS: In situ WI was induced in Lewis rats (n=10) by extrinsic occlusion of the suprarenal aorta for 30 min. After in situ cold perfusion and retrieval, the left kidney underwent 16 hr of HPP. Nonischemic (NI) control kidneys (n=10) were retrieved in the absence of suprarenal aortic occlusion. Longitudinal changes in PF, GFR, and filtration fraction (FF) during HPP were compared in WI versus NI kidneys (FF=GFR/PF x 100%). RESULTS: PF remained the same in both cohorts throughout HPP. GFR, however, increased to a significantly greater degree in WI versus NI kidneys during the first 4 hr of HPP (713+/-401 vs. 26+/-23%, respectively) (P<0.05). The increase in FF at 4 hr was 1203+/-696% in the WI kidneys versus 83+/-46% in the NI controls (P<0.05). CONCLUSIONS: In contrast to PF alone, measurement of both PF and GFR distinguished kidneys subjected to pre-WI from NI controls. The data provide a means to determine if monitoring of both GFR and PF during HPP will predict short- and long-term renal allograft function more reliably than PF alone.

Measurement of renin secretion in single perfused rabbit glomeruli.

A new technique is presented that allows the measurement of the renin secretion rate of single rabbit glomeruli during in vitro perfusion at controlled afferent arteriolar perfusion pressure. Rabbit glomeruli with intact afferent arteriole and Bowman's capsule are obtained by microdissection and cannulated with a pipette system that allows continuous afferent arteriolar pressure measurement. The renin secretion rate of 10 glomeruli, perfused at 40 mmHg, was measured in 15-min intervals with an antibody-trapping microassay. Renin secretion rate was low relative to total renin content (1.2-2.0% of content/perfusion h) and increased three- to fivefold in response to isoproterenol (10(-5) M). The afferent arteriole contracted to norepinephrine (10(-5) M) in each instance. This novel, although difficult, technique allows the study of renin release in vitro at controlled perfusion pressure, without the interfering effects of the macula densa, arterial angiotensin II, and the adrenergic nervous system. It should allow a new perspective on issues such as the pressure-flow dependence of renin release and the interaction of the afferent arteriolar endothelium with the renin-secreting juxtaglomerular cells.

Tubular site(s) of action of atrial natriuretic peptide in the rat.

With micropuncture techniques, the present study examined the delivery of chloride to the superficial late distal tubule and the base and tip of the papillary collecting duct in rats treated with either Wy 47663, a synthetic analogue of atrial natriuretic peptide (ANP), or vehicle alone. Whole kidney glomerular filtration rate (GFR) and single-nephron glomerular filtration rate were not significantly different between the two groups. Late distal tubule chloride delivery was also not different between ANP- (5.71 +/- 1.15%) and vehicle- (6.28 +/- 1.12%) treated animals. However, fractional delivery to the base of the papillary collecting duct was significantly greater in the ANP-treated rats (14.37 +/- 1.98%) compared with vehicle-treated rats (7.32 +/- 1.47%). Tip papillary collecting duct delivery was also significantly greater in the ANP-treated rats (1.97 +/- 1.96 vs. 3.09 +/- 0.60%). In addition, the percent of chloride delivered that was reabsorbed along the papillary collecting duct was significantly less in the ANP-treated rats. In conclusion, ANP inhibits reabsorption in some tubular segments between the superficial late distal tubule and papillary collecting duct base as well as in the accessible portion of the papillary collecting duct.

Effect of atriopeptin II on determinants of glomerular filtration rate in the in vitro perfused dog glomerulus.

Atrial natriuretic factor (ANF) is a peptide originally found to be present in extracts of mammalian atria that possess marked natriuretic and diuretic qualities. A number of mechanisms have been suggested to explain these properties. Recently, it has been suggested that ANF may enhance glomerular filtration. In this report, we describe a series of experiments designed to investigate if atriopeptin II, a 23-amino acid synthetic analogue of ANF, increases glomerular filtration rate (GFR) and, if so, the mechanism for this increase. We used the isolated perfused glomerulus technique (n = 6), which allows a single isolated glomerular unit to be perfused and the determinants of single-nephron GFR (SNGFR) to be measured. Two periods were performed in each experiment, the control followed by the experimental. The only difference between the two periods was the addition of atriopeptin II to the experimental perfusate at a final concentration of 5 X 10(-7) M. There was indeed a significant increase in the SNGFR (78 +/- 27 to 108 +/- 29 nl/min, P less than 0.01). This increase was associated with a significant increase in the glomerular capillary hydrostatic pressure (PGC) from 31 +/- 3 to 35 +/- 3 mmHg (P less than 0.05). The filtration fraction also increased in each experiment (from 0.16 +/- 0.3 to 0.25 +/- 0.03, P less than 0.005). Neither the afferent flow nor the efferent arteriolar flow changed, although there was a tendency for both to decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of albumin on glomerular ultrafiltration coefficient in isolated perfused dog glomerulus.

To evaluate the direct effect of albumin concentration on the glomerular capillary ultrafiltration coefficient (Kf), we compared the effect of "normal" (3.4 g/100 ml), "low" (0.1 g/100 ml), and "no albumin" (less than 0.005 g/100 ml) concentration on the determinants of single-nephron glomerular filtration rate (SNGFR) as measured with the isolated perfused glomerulus technique. When the albumin concentration was decreased from normal to low concentrations, the afferent flow rate increased from 318 +/- 147 (mean +/- SE) to 450 +/- 174 nl/min, the filtration fraction increased from 0.19 +/- 0.04 to 0.35 +/- 0.08, and the SNGFR increased from 49 +/- 21 to 126 +/- 34 nl/min. These changes were associated with a small though significant increase in Kf from 2.79 +/- 1.01 to 3.74 +/- 0.98 nl/(min X mmHg) (P less than 0.05). When the albumin concentration was decreased from low to no albumin the filtration fraction and SNGFR increased even further and were associated with a marked increase in Kf to a value of 27.04 +/- 11.43 nl/min X mmHg). We conclude that there is very little effect of decreases in albumin concentration on Kf until extremely low levels are reached, and at that point there is a marked increase in the ultrafiltration coefficient. Furthermore, when these extremely low concentrations of albumin are reached an important role for albumin in the basic function of the ultrafiltration barrier is demonstrable.

The role of complement in the pathogenesis of postischemic acute renal failure.

One hour occlusion of total renal blood flow results in oliguric acute renal failure as defined by an abrupt and severe diminution in glomerular filtration rate. In rats, after 1-2 h of such ischemia, the acute renal failure which follows is characterized by decreased renal blood flow and by intratubular obstruction with necrotic cellular debris. The present study has examined the possible role of the complement system in the development of this model of acute renal failure. Immunoreactive C3 was extensively localized within necrotic tubular epithelial cells and the walls of small muscular arteries in reperfused kidneys after 1 h of total renal ischemia. Depletion of complement by the administration of cobra venom factor 18 h prior to the induction of ischemia abrogated C3 localization and significantly attenuated the subsequent fall in renal blood flow following reperfusion but did not alter the oliguria or marked fall in glomerular filtration.

Renal ischemic injury in the dog: characterization and effect of various pharmacologic agents.

Renal ischemia has been implicated as a major factor in the pathogenesis of acute renal failure. Despite several differences between the intrarenal norepinephrine infusion and renal artery occlusion models, they have been assumed to be prototypic models of ischemic renal injury. In our previous studies, an intrarenal infusion of norepinephrine caused a marked reduction in inulin clearance 3 hours after infusion. This reduction could be significantly attenuated by the concurrent infusion of mannitol, furosemide, or bradykinin. The effects of these three protective agents were evaluated before and after variable durations of renal artery occlusion to establish the similarities between the models and the magnitude of versatility of these protective agents. In the renal artery occlusion model, capsular fascia was stripped to eliminate collateral flow and ensure maximal renal ischemia. Three hours after 120 minutes of renal artery occlusion (n = 7), inulin clearance returned to 5.7% +/- 2.2% (SEM) of the control values and was not statistically different from that observed in the norepinephrine model. Intrarenal infusion of mannitol, furosemide, or bradykinin prior to and during the occlusion period, however, had no protective effect. Shorter durations of renal artery occlusion were evaluated to ensure an equivalent or decreased severity of acute renal failure compared with the norepinephrine model. After 90 or 60 minutes of renal artery occlusion, the clearance of inulin returned to 10.9% +/- 3.3% (n = 8) and 31.1% +/- 8.2% (n = 4) of control values, respectively. An intrarenal infusion of mannitol, furosemide, or bradykinin still had no significant protective effect, despite the decreased insult in the 60-minute renal artery occlusion studies. In summary, these findings demonstrate fundamental differences between renal artery occlusion and the norepinephrine model of renal functional impairment, and they suggest that the insult associated with norepinephrine infusion may involve factors other than cessation of blood flow.

Host-parasite interactions in the pathogenesis of experimental renal candidiasis.

To study the pathogenesis of renal candidiasis, viable Candida albicans blastospores were injected directly into the left renal artery of New Zealand white rabbits. The progression of the disease was followed by light and electron microscopy over a 6-day period. Within 5 minutes after injection of the yeasts, the organisms localized within glomerular and peritubular capillaries of the cortex. Localization of yeasts within the capillaries occurred through adherence, demonstrated by the presence of surface fibrils originating from the yeast cells. Two to 10 hours later, inflammatory nodules comprised of polymorphonuclear leukocytes formed within capillary lumina. Many of the entrapped yeasts remained viable and extended through adjacent endothelium and epithelium by the formation of germ tubes which penetrated between or directly through intact host cells. After 24 hours, numerous hyphal forms were observed within tubules of the cortex, and some necrotic host cells were noted at sites of penetration. Abscesses replaced renal parenchyma in focal areas during subsequent time intervals. These studies indicate that attachment of Candida albicans to endothelium within capillaries of the cortex is a key event in the disease process. Also, growth of germ tubes into renal tubules provides a temporal advantage for amplification of Candida organisms.

Alterations in vascular function and morphology in acute ischemic renal failure.

Left renal arteries of rats were clamped for 40 min, and the kidneys were studied 48 hr and 7 days following restoration of blood flow. At 48 hr, there was severe oliguria or anuria. Renal blood flow (RBF) was in the normal range, but there was a loss of RBF autoregulation between 95 to 120 mm of mercury in seven out of nine rats. Morphologically, arcuate and interlobular arteries and afferent arterioles showed focal, segmental necrosis of smooth muscle cells and diapedesis of red blood cells across their walls. At 7 days, renal function was still severely depressed. RBF showed a slight decrease that did not reach statistical significance, and RBF autoregulatory capacity was lost in 8 out of 11 rats. Morphologically, vascular lesions were characterized at this stage by marked thickening and fibrosis of the tunica adventitia of the interlobular arteries and afferent arterioles. Structural vascular alterations may impair smooth muscle contractile function and thus interfere with RBF autoregulatory function in this model of acute renal failure.

In vitro perfusion of the isolated dog glomerulus.

Recent technological advances allowing direct in vivo measurements of the determinants of glomerular ultrafiltration have greatly expanded our understanding of that process. In addition, these in vivo studies have clarified the dynamics of glomerular ultrafiltration in a number of physiologic and pathophysiologic conditions. Despite this progress, important issues remain unresolved and beyond the scrutiny of in vivo techniques. We have therefore devised a technique for in vitro glomerular perfusion of the isolated dog glomerulus. In eight glomeruli perfused at physiologic rates, the glomerular filtration rate averaged 39 nl/min and the filtration fraction was 0.19. Filtration pressure disequilibrium was observed in all studies and thus allowed calculation of a unique value for the ultrafiltration coefficient. That parameter averaged 2.34 nl/(min X mmHg). Morphologic studies employing transmission electron microscopy indicate that isolated perfused glomeruli remain ultrastructurally intact. The method for glomerular isolation and in vitro perfusion is presented in detail, the results obtained are compared with published in vivo results, and the advantages offered by the technique are discussed.

Methodologic considerations in the study of glomerular ultrafiltration.

Over the past decade numerous studies have detailed the dynamics of glomerular ultrafiltration. Observations in a unique strain of Munich-Wistar rats, which afford the direct measurement of intraglomerular capillary hydrostatic pressure, have determined that under normal physiologic conditions these animals are in a state of filtration equilibrium. Both mathematical models based on this observation and experimental evidence indicate that under these circumstances the primary determinant of glomerular filtration rate (GFR) is the rate of glomerular plasma flow. In addition, this state of filtration equilibrium provides a condition in which changes in the product of glomerular surface area and glomerular capillary hydraulic conductivity (ultrafiltration coefficient) have no influence on glomerular filtration rate. Similarly, changes in glomerular hydrostatic pressure have a relatively small influence on GFR. These conclusions represent a marked alteration of the previously held concepts of the determinants of GFR. Unfortunately, studies in other strains of rats and in other animal species, particularly the dog, have led to the conclusion that filtration disequilibrium is the normal physiologic condition. The validity of this conclusion, however, is made uncertain by methodologic necessities that may lead to erroneous physiologic measurements of the determinants of GFR. In the present Editorial Review we examine the data reflecting on the applicability of filtration equilibrium to mammalian species. In addition, we summarize two new in vitro techniques for the further study of the dynamics of glomerular ultrafiltration.

Further studies on segmental sodium transport in the rat kidney during expansion of the extracellular fluid volume.

The present studies were designed to further investigate the possibility of heterogeneity of nephron function during Ringer loading in the rat, and to determine the specific nephron segment responsible for this finding. As in previous studies from this laboratory with smaller rats (50-125 g), net addition of sodium between late distal tubule and papillary base (6.9 vs. 10.4% of the filtered load, respectively, P <0.005) was found in more mature rats (170-230 g). In contrast, there was net reabsorption of sodium between these two segments in nonvolume-expanded animals, 1.70 vs. 0.45% of the filtered sodium load, P <0.005. Because nephron heterogeneity of sodium transport during extracellular volume expansion is the most likely explanation for these findings, further studies were performed to determine the specific juxtamedullary nephron segment responsible for the net addition pattern between late distal tubule and papillary base in Ringer-loaded animals. First, a comparison was made of sodium delivery to the late proximal tubule of superficial nephrons vs. the delivery rate to the bend of Henle's loop of juxtamedullary nephrons in both hydropenia and Ringer loading. Fractional sodium delivery was quite comparable between the superficial and juxtamedullary nephrons in both hydropenia and Ringer loading although the absolute level was much greater in both groups of nephrons in the Ringer studies. Chlorothiazide (15 mg/kg loading and 15 mg/kg per h) given during Ringer loading markedly increased late distal sodium delivery, 19% of the filtered load, but did not prevent net addition of sodium at the papillary base. In contrast, furosemide (5 mg/kg loading and 5/mg/kg per h) given during Ringer loading completely reversed the segmental pattern, 35.5 and 28.8% at late distal tubule and papillary base, respectively, P <0.005. These studies demonstrate that the net addition of sodium between late distal tubule and papillary base during Ringer loading is not limited to immature rats and that the segmental pattern does not occur in non-volume-expanded animals. Further, the reversal of the net addition pattern with furosemide, but not chlorothiazide, and the comparable proximal nephron delivery rates in Ringer loading suggest that the loop of Henle of juxtamedullary nephrons reabsorbs less sodium than the same portion of superficial nephrons in this setting. A model is proposed to explain this finding.