Clarification and confocal imaging of the nonhuman primate placental micro-anatomy.

Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta.

Dialysis in the 1960s and the first hollow fiber dialyzer.

The history of the development of the first hollow fiber dialyzer as part of a federally funded project at Dow Chemical in Walnut Creek, California is interesting, as this project represented an initial step in the technical advances that dialysis has experienced over the last 40 years. The project, important in its own right, was revolutionary; the predominant design of dialyzers in use at the time employed either flat membranes or collapsed large cellulose tubing. The hollow fiber dialyzer project, in addition to being technologically important, brought together several individuals who over the intervening four decades have launched careers and collaborations that have had a profound impact, as well as resulting in major advances and contributions to a greater understanding of the dialysis process and adequate delivery of care.

Whither goest Kt/V?

Uremia is characterized by gross contamination of body water with a wide spectrum of retained solutes normally excreted by the kidney. The rationale for dialysis therapy is that these retained solutes have concentration-dependent toxicity, which can be ameliorated through removal by dialysis. Apart from the well-established clinical consequences of abnormalities in fluid, electrolyte, acid base metabolism, and retained beta 2-microglobulin (beta 2 m), there is very little understanding of solute-specific uremic toxicity. Evidence is reviewed to demonstrate the following: (1) Many aspects of the uremic syndrome are controlled by adequate dialysis of low molecular weight solutes. (2) Urea can serve as a generic molecule to quantitate the fractional clearance of body water by dialysis (Kt/V) of retained low molecular weight solutes. (3) Urea has no concentration-dependent toxicity, and the generation rate of putative toxic low molecular weight solutes is not proportional to urea generation. The major clinical consequences and controversies stemming from these interrelationships are reviewed. Kinetic approaches to determine Kt/V dose equivalency between intermittent and continuous dialysis therapy are reviewed. We conclude that Kt/V can and will be generalized to describe the kinetics of other solutes such as beta2m as our knowledge of uremic toxicity grows, and hence, it is predicted that it will goeth and goeth and goeth.

Is hematologic response to iron and erythropoietin in hemodialysis patients affected by other factors?

Multiple factors have been implicated in the hematologic response to erythropoietin (EPO). The authors studied 54 hemodialysis patients; 44 received 1.5 g of iron intravenously, 16 received oral iron for 12 weeks, and 24 were treated with EPO. Some patients received these treatments in sequence. The factors evaluated were serum albumin, protein catabolic rate, serologic evidence of hepatitis B or C, parathormone (PTH), and aluminum levels. Red cell production was expressed as milliliters of red blood cell increase per day per kilogram of body weight. For patients receiving EPO, hematologic response was normalized to 50 U/kg/dialysis. Of the patients on oral iron, 31% had a good response (hematocrit greater than or equal to 30%). Of the patients who received iron intravenously, 50% had a good response (hematocrit greater than or equal to 30%). All patients treated with EPO responded well, except for one patient who did not respond to doses of EPO up to 200 U/kg/dialysis. The response to intravenous iron dextran was more rapid than the response to oral iron or EPO. Nutritional factors (serum albumin and protein catabolic rate), serologic evidence of hepatitis, elevated PTH levels, or elevated aluminum levels did not significantly affect the response to iron supplementation or EPO treatment.

Shortfalls in the delivery of dialysis.

We investigated the extent to which dialysis prescription is achieved in the United States and the reasons for failure to do so. Dialysis treatment data (blood urea nitrogen [BUN], duration [T], and dialyzer urea clearance [K]), for 297 patients treated in 48 dialysis units in the United States were used to calculate the urea kinetic modeling parameter, V (urea distribution). V was also calculated for 5,650 treatments of 357 patients in five representative units starting or continuing to kinetically model patients monthly over the past 2 years. V was estimated in patients from height, weight, and sex (Vest). If V differed from Vest by 20% or more, we concluded that a decrease in BUN was inconsistent with expected therapy and represented "nondelivery" of dialysis (ie, KT is decreased by approximately 20% or more). Half the US units studied had more than 35% nondelivery of therapy; more than 50% of treatments were not delivered in 44% of the units. The reasons for nondelivery could not be determined directly in the US study because we had limited contact with their dialysis programs. Common causes are treatment delivery errors (K and T), access problems, recirculation due to rapid blood flow, and dialyzers that deviate from the manufacturers' specifications. The long-term (five-unit) study showed the same percent of nondelivery of dialysis in start-up units as in the US study (40% to 50%). Long-term, nondelivery decreases to 10% to 20% in these units. For these units, nondelivery tends to occur for the same patients month to month; a decrease in nondelivery of treatment from start-up to lower long-term levels is due to more deliberate delivery of dialysis therapy. The persistence of a 10% to 20% shortfall in therapy stems from either access or clotting problems, many of which cannot be corrected. We conclude that widespread nondelivery of dialysis exists in US dialysis facilities and probably world-wide. If optimal (minimal) dialysis prescription is the therapy goal of a dialysis unit, the deliberate delivery of the desired treatment should be routinely checked using quantitative assessment techniques such as urea kinetic modeling. If this is not done, optimal treatment may not be achieved and inadequate treatment may result.

Low temperature scanning electron microscopy: advantages and applications.

Cryo-preparation of specimens for scanning electron microscopy can be completed within a few minutes. Chemical fixation and contact with solvents is avoided, levels of specimen hydration are maintained, low melting-point materials are stabilized, volume changes are minimized and internal structure can be revealed by freeze-fracture. Elements are not lost or substantially relocated prior to X-ray microanalysis and specimen luminescence is enhanced. The displacement of internal structure and material subject to X-ray microanalysis by the growth of ice crystals in hydrated samples can be minimized by adopting fast freezing methods designed to limit ice crystal growth. The technique enables a wide range of industrial and biological materials to be examined rapidly and free from artifacts commonly associated with more conventional preparation methods.

The influence of glucocorticoid dose on protein catabolism after renal transplantation.

Protein catabolic rate (PCR) and protein balance were measured daily by computerized mass balance studies in 20 subjects during hospitalization after renal transplantation. All hospital courses were uncomplicated. Ten subjects received approximately 1 mg/kg/day prednisone, and ten subjects received 3-5 mg/kg/day prednisone on day 1 with a tapering dose to approximately 1 mg/kg/day by discharge. In both groups, PCR rose during the first 3-4 postoperative days then stabilized at an accelerated level. PCR was significantly greater in the higher prednisone group. Despite encouragement most subjects ate less protein than prescribed, and most were in negative protein balance. Mean daily and net protein deficits were more severe in the higher prednisone group. Higher protein intakes improved protein balance. The protein catabolic effects of the two regimens have been defined and a dose dependency demonstrated. In any therapeutic situation the use of the minimum effective dose of steroids seem advised, and high protein intake should be encouraged to improve protein balance. Some steroid morbidity might thus be avoided.

A mechanistic analysis of the National Cooperative Dialysis Study (NCDS).

The purpose of the NCDS was to determine the probability of clinical failure (PF) as a function of the level of dialysis and protein catabolic rate (pcr, g/kg/day). The level of dialysis prescribed in the NCDS was mechanistically defined as Kt/V (product of dialyzer urea clearance and treatment time divided by body urea volume), which exponentially determines decrease in BUN during dialysis and is also a mathematical analogue of pcr, BUN. Mechanistic analysis (MA) showed that PF was a discontinuous function of Kt/V as it was prescribed in the NCDS and that a dependence of PF on pcr could not be assessed because of the study design. The MA results were compared to those reported with statistical analysis (SA) that used BUN and pcr. The SA predicts PF is strongly dependent on pcr with nutrition-dependent high PF for pcr less than or equal to 0.8 and low PF with high pcr and intensive dialysis. The MA suggests SA results may not be valid because a continuous outcome function is assumed and, due to study design, Kt/V was a dependent variable of pcr and these two variables cannot be clearly separated by analysis of BUN and pcr alone.

Use of urea kinetics in the nutritional care of the acutely ill patient.

In acutely ill patients nitrogen balance is often assessed clinically from measurements of protein intake and urinary urea nitrogen. We have utilized urea kinetic modeling to measure urea generation rates, protein catabolic rates and nitrogen balance in 19 acutely ill patients with varying degrees of renal dysfunction and have studied the effect of varying caloric intake on protein balance during a period of fixed protein intake. In patients with measured creatinine clearances equal to or greater than 50 ml/min there was a highly significant correlation between nitrogen balance estimates derived from urea kinetic modeling and those obtained from urinary urea nitrogen (R = 0.939; p less than 0.001). When creatinine clearance measurements were between 20 to 50 ml/min the correlation between the two estimates was poorer (R = 0.337; p less than 0.001). In patients whose creatinine clearance was below 20 ml/min the correlation between measurements was worse still (R = 0.229; p less than 0.002). To determine the effects of increasing caloric intake on protein catabolic rate seven acutely ill patients were studied. When caloric intake was increased from 27.8 to 34.2 kcal/kg/day while on a fixed protein intake of 1.27 g/kg/day there was a significant fall in protein catabolic rate from 1.39 to 0.99 g/kg/day (p less than 0.002). As urea kinetic modeling takes into account changes in blood urea nitrogen, extrarenal losses of urea and the urinary urea pool, it is the preferred method for measuring protein balance in acutely ill patients particularly those with poor renal function. Serial monitoring of protein catabolic rates permits easy continuous assessment of the effect of increasing caloric intake on protein sparing during parenteral hyperalimentation.

Protein catabolism during the postoperative course after renal transplantation.

Protein catabolic rate (PCR) was measured daily by computerized mass balance studies in 50 subjects during hospitalization after renal transplant. All subjects received 60 mg prednisone per day. PCR rose over the first 3 to 4 postoperative days and then stabilized at an accelerated level, which was sustained through the third posttransplant week. Rejection therapy with either 3 mg/kg/d prednisone or 15 mg/kg/d of methylprednisolone for 3 days further increased PCR, but there was no difference in PCR between these two regimens. Protein restriction did not decrease PCR and subjects offered a higher protein diet did not have further acceleration of PCR. We conclude that 60 mg/kg/d prednisone produces an obligatory acceleration of PCR that is further accentuated by higher steroid doses. The use of minimal maintenance doses of prednisone consistent with adequate immunosuppression seems wise. Protein balance may be improved if protein intake is increased to match individual rates of accelerated protein catabolism.

Hydrogen ion balance in dialysis therapy.

A model to describe hydrogen ion balance (H+B) in acetate and bicarbonate dialysis therapy was developed based on measurement of metabolic addition of hydrogen ion (H+) to the body between and during dialyses and measurement of net buffer repletion during dialysis. Metabolic H+ generation was shown to be equal to 0.77 times the protein catabolic rate plus the total net removal of lactate and beta-hydroxybutyrate ions during dialysis. Buffer repletion was calculated from total net flux of acetate and bicarbonate during dialysis. The model was used for eight paired studies of H+B on one week each of acetate and bicarbonate dialysis and showed that cumulative H+B with acetate was -7 +/- 28 (M +/- SEM) mmol/week compared to -175 +/- 45 mmol/week with bicarbonate (P less than 0.001). It is concluded that there is an initial, strongly negative H+B when patients on acetate dialysis are converted to bicarbonate. The possible physiologic significance of this is discussed.

Which mathematical model to study uremic toxicity? National Cooperative Dialysis Study.

Mathematical modelling advantages and limitations to study dialysis adequacy are evaluated, the use of the single pool urea model in the guidance of the National Cooperative Dialysis Study (NCDS) is described, and therapeutic control results from the Control phase of the NCDS are reported. The relevance of using urea as a target compound and the practicality of modelling its levels in clinical settings using a single pool model are discussed. The NCDS involves intensive participation of 8 geographically separate centers to control BUN at two weekly time averaged concentrations (50 +/- 5 and 100 +/- 5 mg/dl) using standard clinical dialyzers and two different lengths of dialysis (3 and 4.5 hr) in the presence of .8-1.4 g/kg/day protein intake. Control phase data on 195 patients indicates a remarkable level of clinical precision and method reproducibility as well as a high degree of patient compliance. Patient urea volumes averaged 39.8 +/- 8.9 liters, net rates of daily protein catabolism were 1.06 +/- .17 g/kg; daily weight gain: .96 +/0 .43 kg; and dialyzer clearances to maintain patients in the control phase for 3 to 6 months were 168 +/- 44 ml/min. Clearances required to randomize patients into four experimental groups ranged 40-250 ml/min. Less than 1/4 of dialyzers were larger than 1.8 m2 and were not specific to any experimental group.

Effect of the hemodialysis prescription on patient morbidity: report from the National Cooperative Dialysis Study.

This report summarizes morbidity in 151 patients in a cooperative trial designed to evaluate the clinical effects of different dialysis prescriptions. Four treatment groups were divided along two dimensions: dialysis treatment time (long or short), and blood urea nitrogen (BUN) concentration averaged with respect to time (TACurea) (high or low). Dietary protein was not restricted. There was no difference in mortality between the groups. Withdrawal of patients from the high-BUN groups for medical reasons was significantly greater than withdrawal from the low-BUN groups. Hospitalization was also greater in the high-BUN groups, but dialysis treatment time had no significant effects. The data indicate that the occurrence of morbid events is affected by the dialysis prescription. Increased morbidity appears to accompany prescriptions associated with a relatively high BUN. Conversely, morbidity may be decreased by prescriptions associated with more efficient removal of urea if the dietary intake of protein and other nutrients is adequate.