Diane K. Jax

Increased Growth after Long-Term Oral 1α,25-Vitamin D3 in Childhood Renal Osteodystrophy

We evaluated oral 1,25-vitamin D3 for as long as 26 months in six prepubescent children with renal osteodystrophy previously treated with vitamin D2. Therapy was given at 14 to 41 ng per kilogram per day to correct hypocalcemia and reverse bone disease. Serum levels of 1,25-vitamin D3 were initially reduced at 15 +/- 5 pg per milliliter (mean +/- S.E.M.) and after treatment rose to 54 +/- 13. Serum calcium rose from 7.5 +/- 1.6 mg per deciliter (mean +/- S.D.) to 9.8 +/- 0.6 after one month (P less than 0.02). Alkaline phosphatase activity fell from 536 +/- 298 to 208 +/- 91 IU per liter after 12 months (P less than 0.05). Serum immunoreactive parathyroid levels fell from 900 +/- 562 microliter eq per milliliter 411 +/- 377. Healing of rickets and subperiosteal erosions was found. Remineralization of bone was demonstrated by the photon absorption technic. In four patients growth velocity, evaluated for 12 months before and after therapy, increased from 2.6 +/- 0.8 to 8.0 +/- 3.2 cm per year. Growth velocity per year increased from less than third percentile in each to the 10th to 97th percentile after therapy. Height increment ranged from 27 to 113 per cent of that expected for change in chronologic age and 40 to 114 per cent expected for change in bone age after therapy. This trial demonstrates that oral 1,25-vitamin D3 can reverse renal bone disease and increase growth in uremic children.

Developmental aspects of renal beta-amino acid transport I. Ontogeny of taurine reabsorption and accumulation in rat renal cortex.

Summary: Newborn Sprague-Dawley rat pups were found to have reduced net tubular reclamation of the β-amino acid taurine in vivo. The reabsorption of this compound increased between the 2nd and 4th week of life and the excreted taurine fell despite a rise in glomerular filtration rate indicative of increased transport with maturation.Kidney cortex in vivo accumulated taurine from plasma against a large chemical gradient. Newborn cortex slices in vitro accumulated taurine to higher levels at steady state at <0.4 mM, but uptake was less efficient at higher taurine concentrations. Further metabolism of taurine by cortex slices was not found at any age. Accumulation in adult and newborn cortex was greatest at 10–15 mEq/liter K in the external medium. Slices from younger animals had slightly higher pH optima for uptake and temperature elevation increases uptake more in neonatal than in adult kidney.Speculation: If the newborn rat is in part dependent on the transfer of taurine from mother to pup via milk, the process of weaning may require greater renal tubular reabsorption in order to maintain tissue and plasma pools of taurine. The improved reabsorption of taurine found in 4-week-old and adult animals may reflect maturational changes brought about by the reduction in dietary taurine (and other sulfur containing amino acids) after weaning.

Developmental Aspects of Renal β-Amino Acid Transport II. Ontogeny of Uptake and Efflux Processes and Effect of Anoxia

Summary: Renal cortex slices from newborn, 2-week, and 4-week-old Sprague-Dawley rats had reduced initial rates of taurine uptake compared to adult slices after short (< 30 min) incubation periods. From birth onward, steady-state accumulation occurred by at least two sodium-dependent uptake systems. The first system had an “apparent Km1” = 0.1 mM and a Vmax varying from 1.8 to 5.1 μmoles/ml ICF/120 min at four ages. The second uptake mode had an apparent Km2 = 12–16 mM and a Vmax of 45 μmoles/ml ICF/120 min. Efflux of taurine was reduced in slices from younger animals possibly accounting for taurinuria. Only other β-amino acids inhibited accumulation.Anoxia inhibited uptake at high concentrations (> 1.0 mM) at each age, but taurine accumulation at low concentrations (< 0.4 mM) was relatively protected from anoxia in neonatal (< 36 hr of age) tissue. Preincubation in taurine-free medium for 120 min enhanced low concentration, but not high concentration uptake in neonatal and 2-week slices.After preincubation in dibutyryl cyclic AMP (dbcAMP) enhanced uptake of taurine was found in adult cortex, but not in neonatal cortex.The ontogeny of renal taurine transport in cortex slices appeared to involve faster initial uptake rates and faster efflux as well as greater dependence on aerobic metabolism with maturation. Age-related differences in the response to preincubation and cyclic nucleotides were also indicative of maturational events in renal tubular amino acid transport.Speculation: Taurinuria found in the immature rat may involve decreased initial uptake rates and/or decreased efflux, but heterogeniety of transport processes across the antiluminal membrane was apparent at birth. Preincubation in taurine-free medium increased the rate of taurine accumulation by immature cortex, but changes in transport processes across the brush border membrane may account for the decrease in taurinuria found after the age of 2 weeks.

Developmental aspects of renal β-amino acid transport. III. ontogeny of transport in isolated renal tubule segments

Summary: Isolated renal tubules were prepared from newborn and adult Sprague-Dawley rats. They were used to study the uptake and accumulation of the β-ammo acid, taurine, by renal epithelium. Initial rate as well as steady-state kinetics were studied. Initial rate studies revealed heterogeneity of uptake in newborn and adult tubules. Slower uptake was present in the newborn in the low-affinity system. Slowed efflux was found in neonatal tissue. Newborn tubules in contrast to adult tubules demonstrated uptake under anaerobic conditions. Adult and newborn tissues showed decreased uptake of taurine when incubated with β-alanine. Physiologic taurinuria, be it in the rat or man, may be due to less rapid initial uptake and/or slowed efflux from renal epithelium.Speculation: β-Amino acid transport by newborn rat renal epithelium varies from that of adult rat renal epithelium. Although heterogeneity of uptake exists io both, newborn uptake by Pad efflux from renal epithelium is slower, aod iotracellular levels are higher in the newborn. Uptake differences may be exunined by luminal membraoe preparations; however, slowed efflux aod higher iotraceliular levels suggest studies on intracellular components and basal lateral membraoes are warraoted.

Heterogeneity of the β-amino-preferring transport system in rat kidney cortex. Differential influence of glutathione oxidation

Taurine, a naturally found beta-amino acid, is inert in rat renal cortex slices. Its active accumulation by slices is abolished by anaerobiosis, a strongly acidic media or the removal of Na+. Concentration-dependent uptake studies reveal more than one taurine carrier: the apparent Km value for uptake below 1.1 mM is 0.4 mM and the apparent Km value above 1.1 mM is 14.5 mM. Of all amino acids tested only beta-alanine, another beta-compound, inhibited uptake. The oxidizing agent diamide was used to lower the concentration of GSH in rat cortex slices. The ability to accumulate taurine by the low Km system was decreased in diamide-treated slices, but not by the high Km system. Diamide was found to greatly augment efflux of taurine taken up from lower concentrations but not from higher concentrations. GSH in the media prevented this diamide-induced inhibition of uptake and enhanced efflux at lower taurine concentrations. A possible mechanism of diamide inhibition of uptake is that intracellular GSH depletion leads to greatly enhanced efflux of taurine, thus preventing uptake.

The influence of glutathione oxidation on renal cortex taurine transport

Abstract The interaction of diamide, a rapidly reversible thioloxidizing re reagent, with the taurine accumulation system was examined in rat kidney cortex slices from animals of different ages. Diamide at 10 mM lowered renal cortex glutathione content by 80% at a time that taurine accumulation was inhibited by 65%. Although the addition of equimolar GSH overcame diamide inhibition of taurine uptake, GSH per se inhibited taurine accumulation at 0.01 mM, but not at 0.2 or 1.0 mM. Dithiothreitol (DTT) also overcame diamide inhibition of uptake. As previously shown by Pillion et al (Eur. J. Biochem. 79 , 73, 1977) diamide inhibited gluconeogenesis by cortex slices. Diamide inhibited taurine accumulation by 85% by the low Km taurine transport site in cortex from newborn, 2 week, 4 week and adult animals, but only 50% at the high Km site. In contrast to the situation in adult tissue, efflux of taurine from preloaded slices of immature animals was not increased by diamide. Accordingly, one maturational event identified by these studies is that diamide-enhanced efflux was found only in mature cortex.

Studies on the Renal Handling of Taurine: Changes during Maturation and After Altered Dietary Intake

The main route for the removal of taurine from the body is by urinary excretion. As a consequence, the renal tubular transport and handling of taurine is important in regulating the taurine content in the remainder of the body. In our previous report, we have described the use of stop-flow, free-flow micropuncture, continuous microperfusion and endogenous clearance as techniques to examine the renal handling of taurine (6). This chapter also covered our studies on the accumulation of taurine by thin cortex slices of mouse (4) and rat (5) kidney. These studies indicate that under normal conditions taurine excretion is 4 to 10% of the filtered load and that uptake by slices occurs by two processes: a low-Km, high-affinity uptake system and a high-Km, low-affinity uptake system. The kinetics of taurine accumulation are determined by Lineweaver-Burk analysis and the Eadie-Augustinson transformation. Other characteristics of taurine accumulation include transport by the β-amino acid transport system (such that β-amino acids but not α-amino acids will block uptake), sodium dependency of uptake, dependency on oxidative metabolism for active accumulation and enhanced efflux of taurine when incubated in a solution containing other β-amino acids.

β-Amino Acid Transport in Isolated Tubule Segments

Using taurine as a model substrate, β-amino acid transport in isolated rat renal cortical tubule segments as studied. Heterogeneity of taurine accumulation was found in tubule segments and has previously been measured in renal cortex slices. In contrast to studies in cortical slices, initial rate kinetics could also be determined, and demonstrated uptake by two systems. Efflux of taurine from tubule cells was measured and showed 50% remaining in tubule segments after 30 min incubation in taurine-free medium. Concentrative uptake was prevented by NaCN and iodoacetate. Incubation with other β-amino acids resulted in decreased taurine accumulation. An important although unexpected finding was decreased taurine uptake by isolated tubules in the presence of Krebs-Ringer phosphate and Tris-HCl buffer as compared to Krebs-Ringer bicarbonate buffer. Isolated tubule preparations provide a rapid means of assessing β-amino acid transport including initial rate kinetics. However, when using tubules to study uptake, the buffer medium may play a critical role.

ONTOGENY OF RENAL β-AMINO ACID TRANSPORT IN RAT KIDNEY CORTEX

Taurine (T), a β-amino add characteristic of rodent urine, is taken up across the antiluminal membrane into adult cortex slices by 2 uptake processes that require oxidative metabolism and the presence of sodium. This non-metaboHzable compound is not incorporated into TCA precipitable material (<.01%) and thus is an ideal agent to directly examine transport. It can be used to explore the ontogeny of peritubular transport processes. Uptake of T is slower in newborn (N.B.), 2 week and 4 week old rat cortex slices than in the adult at 0.1, 1.0 and 20 mM T, but faster in NB at 0.01 mM T. Moreover, NB tissue is far more capable of T uptake in the presence of N2 (anoxia). Concentration-dependent uptake studies at all ages reveal at least 2 uptake processes: a high-affinity site with Km1=0.1 to 0.5 mM and a low-affinity site with Km2=12 to 15 mM. However maximal uptake at both sites proceeds adult > 4 week > 2 week > NB and initial uptake rate is greater in adult slices. B-alanine shares both uptake sites at all ages and both sites are Na-dependent. Efflux of T from preloaded slices is slower in NB, 2 week and 4 week slices as a further indication of the ontogenic maturation of peritubular transport processes with aging.These studies provide a setting against which such manipulations as dietary alteration or the use of hormonal agents can be used to attempt to augment the rate of uptake and efflux of T in immature slices.

ORAL 1α, 25-DIHYDROXYCHOLECALCIFEROL (1,25 DHCC) IN THE TREATMENT OF CHILDHOOD RENAL OSTEODYSTROPHY

Central to the development of renal osteodystrophy is the failure of the kidney to produce sufficient amounts of the most active natural vitamin D metabolite 1,25 DHCC. We have initiated oral therapy with 1,25 DHCC at 10-30 ngm/kg daily in 6 uremic children attempting to reverse bone disease or prevent further change. Patients with hypocalcemia or X-ray evidence of osteodystrophy while on high dose vitamin D2 or DHT were included in this study; none had primary glomerular disease. An improvement in gait and subjective improvement in muscle strength was noted in 4 patients. Mean serum calcium concentration rose from 7.5 ± 1.6 to 10 4 ± 3 mg/dl (P<.01) in all; serum phosphate rose from 5.1 ± 2.6 to 6.8 ± 2.0 mg/dl (N.S.) and serum alkaline phosphatase fell from 527 ± 214 to 250 ± 57 IU/L (P<.02). Serum irimunoreactive PTH levels fell from 864 ± 525 to 320 ± 51 pEq/ml (P<.05). X-ray evidence of osteodystrophy improved in 5. Bone mineral content, measured by single photon absorptiometry, improved by 10% in 3 patients. Serum levels of 1,25 DHCC rose in 3/3 patients. Although the follow-up period has been short, there has been an increase in growth rate. The complications found were hypercalcemia in younger patients, mild hypermagnesemia and hyperphosphatemia possibly related to the reduction in circulating PTH levels. The oral administration of 1,25 DHCC appears to be useful in the treatment of childhood renal osteodystrophy.