David Z. Levine

Role of angiotensin II in dietary modulation of rat late distal tubule bicarbonate flux in vivo.

We have reported that overnight fasting stimulates bicarbonate reabsorption (JtCo2) in rat distal tubules. The present in vivo microperfusion studies evaluated the hypothesis that endogenous angiotensin II (AII) mediates this response. Rat late distal (LD) tubules were perfused at 8 nl/min in vivo with a hypotonic solution containing 28 mM bicarbonate. In overnight-fasted rats, LD JtCO2 was significantly higher than in normally fed rats (50 +/- 4 vs. 16 +/- 6 pmol/min.mm, P < 0.05). When overnight-fasted rats were salt-loaded, JtCO2 fell significantly (38 +/- 3 pmol/min.mm, P < 0.05). Conversely, in fed rats ingesting a zero-salt diet, JtCO2 increased three-fold (45 +/- 5 pmol/min.mm, P < 0.05). Enalaprilat infusion (0.25 micrograms/kg body wt, intravenously), in these zero-salt and overnight-fasted rats, reduced LD JtCO2 values to normal. Further, infusion of losartan (5 mg/kg body wt, intravenously), the specific AII AT1 receptor blocker, reduced JtCO2 in overnight-fasted rats by two-thirds (16 +/- 4 pmol/min.mm, P < 0.05). Finally, we perfused 10(-11) M AII intraluminally with and without 10(-6) M losartan: AII increased JtCO2 to 45 +/- 6 pmol/min.mm, equal to the zero-salt flux. This was completely abrogated by simultaneous losartan perfusion. Therefore, these results suggest that AII is an in vivo stimulator of late distal tubule bicarbonate reabsorption.

In vivo evidence of impaired solute transport by the thick ascending limb in potassium-depleted rats.

The objective of this investigation was to determine if thick ascending limb (TAL) solute removal is impaired in potassium-depleted rats, in vivo. We estimated TAL NaCl concentration by measuring in situ conductivity of tubular fluid presented to the early distal site after stop-flow periods of 10-60 s, during which a proximal equilibrium solution remained in contact with the reabsorbing epithelium. This allowed us to calculate the rate constant of the decrease in tubular fluid NaCl concentration and to determine equilibrium values for control, potassium-depleted, and potassium-repleted rats. After 60 s of stop-flow, NaCl concentration of TAL fluid decreased to 18.3 +/- 2.73 mM in control rats, while potassium-depleted rats had values almost twice as high (36.5 +/- 2.97 mM, P less than 0.01). The amount of NaCl remaining after 60 s of stop-flow in K-depleted rats was highly correlated with the plasma K concentration. Calculated rates of NaCl efflux from the TAL appeared to be normal in K-depleted rats while the concentration of NaCl achieved at equilibrium was nearly twice that measured in control rats. Acute systemic administration of KCl by gavage or infusion in K-depleted rats was associated with a decrease in TAL NaCl concentration to normal values. Addition of K to the perfusate, however, did not repair the defect. Our results can best be explained by assigning a special role to the peritubular K concentration. We suggest that the defect in TAL solute removal in K-depletion can be rapidly reversed, because decreases in peritubular K concentration limit Na efflux across the peritubular membrane by decreasing the activity of the Na-K-ATPase pump. We recognize that factors such as regional renal blood flow, local angiotensin II levels, and products of the cyclo-oxygenase enzyme system may play a role.

Secretion of bicarbonate by rat distal tubules in vivo. Modulation by overnight fasting.

We have performed microperfusion studies on distal tubule bicarbonate reabsorption (JtCO2) of fed and fasted rats to extend our previous observations of in vivo bicarbonate secretion and to resolve certain discrepancies between free-flow and microperfusion data. When rats are fasted overnight, as in previous free-flow studies, distal tubule microperfusion with a 28-mM tCO2 solution results in significant JtCO2 (53 +/- 6 pmol.min-1.mm-1) at normal flow and increases briskly (91 +/- 16 pmol.min-1.mm-1) with bicarbonate load. This response is not influenced by the addition of other normal tubular fluid constituents. However, when normally fed rats are used, as in our previous microperfusion studies, distal tubule JtCO2 is not different from zero when a 28-mM tCO2 solution is perfused at normal flow rates but becomes negative (-54 +/- 13 pmol.min-1.mm-1) at high flow rates, which indicates the existence of bicarbonate secretion against a concentration gradient. Alkali loading of fasted rats also elicits bicarbonate secretion at high flow. These results demonstrate for the first time that normal feeding or alkali loading can induce bicarbonate secretion in a mammalian nephron segment in vivo, and resolves previous discrepancies between free-flow and microperfusion data.

Luminal chloride modulates rat distal tubule bidirectional bicarbonate flux in vivo.

The effects of replacing luminal chloride with gluconate on distal tubule bicarbonate transport were studied in vivo in normally fed rats, overnight-fasted rats, and rats made mildly alkalotic by administration of desoxycorticosterone acetate (DOCA). In paired microperfusions of the same tubule with 0 or 55 mM Cl at 25 nl/min, net secretion of bicarbonate by distal tubules of fed rats was inhibited by chloride replacement. Zero chloride perfusion in DOCA rats also resulted in an inhibition of net bicarbonate secretion at 25 nl/min. In contrast, replacement of 45 mM chloride also perfused at 25 nl/min in fasted rats caused an increase in net bicarbonate reabsorption. To further characterize the effects of changes in luminal chloride, experiments were undertaken in fasted rats with 0, 45, and 100 mM chloride-containing solutions perfused at 8 and 25 nl/min. Perfusion with zero Cl resulted in net bicarbonate reabsorption at 8 nl/min that increased markedly with high flow, whereas bicarbonate reabsorption did not change significantly during perfusion at high flow with a 45-mM Cl perfusate. In marked contrast, perfusion with a 100-mM Cl solution resulted in only minimal bicarbonate reabsorption at 8 nl/min with significant secretion observed at high flow. Thus, chloride-free perfusates inhibit bicarbonate secretion and enhance bicarbonate reabsorption, while high chloride perfusates elicit net bicarbonate secretion in usually reabsorbing distal tubules.

Can rodent models of diabetic kidney disease clarify the significance of early hyperfiltration?: recognizing clinical and experimental uncertainties

In the past, hyperfiltration and increased glomerular capillary pressure have been identified as important determinants of the development of DN (diabetic nephropathy). Recently, some basic research and clinical reviews on DN have omitted identifying hyperfiltration as an important risk factor. At the same time, different rodent models of DN have been described without and with documented hyperfiltration. In the present review, the importance of hyperfiltration is reassessed, reviewing key clinical and research studies, including the first single nephron studies in a mouse model of DN. From clinical studies of Type 1 and Type 2 diabetes mellitus, it is clear that many patients do not have early hyperfiltration and, even when present, its contribution to subsequent DN remains uncertain. Key mechanisms underlying hyperfiltration in rodent models are reviewed. Findings on intrarenal NO metabolism and the control of single-nephron GFR (glomerular filtration rate) in rodent models of DN are also presented. Characterization of valid experimental models of DN should include a careful delineation of the absence or presence of early hyperfiltration, with special efforts made to establish the specific role hyperfiltration may play in the emergence of DN.

DIFFERENTIATION OF RENAL INTERCALATED CELLS IN FETAL AND POSTNATAL RATS

SummaryAn ultrastructural study was conducted on the kidneys from rat fetuses and pups from ages ranging from birth to 8 weeks to identify the time of appearance of each of the two intercalated cell types. With transmission electron microscopy, A-intercalated cells were recognized by their large apical microvilli and microplicae as well as by the numerous subapical vesicles. Their identification was confirmed by the presence of typical studs at the cytoplasmic face of the apical plasma membrane. By scanning electron microscopy the cells were recognized by their typical microplicae at the apical surface. In 19-day-old fetuses and newborns, A-intercalated cells were numerous in the epithelium lining the renal pelvis and inner medullary intercalated ducts. Two weeks after birth they disappeared from these regions but became numerous at the outer medullary collecting ducts and also at the cortical collecting ducts although to a lesser degree. B-intercalated cells were recognized by the scarcity of microvilli, the absence of microplicae, and the large number of basal infoldings. Their identification was confirmed by the presence of studs at the cytoplasmic face of the basolateral membrane. B-cells started to appear 3 weeks after birth and increased thereafter. We speculate that the particular stages at which the two cell types differentiate might be related to changes in acid-base status.

Bicarbonate secretion in vivo by rat distal tubules during alkalosis induced by dietary chloride restriction and alkali loading.

To examine in vivo the separate effects on distal tubule JtCO2, of dietary chloride restriction, bicarbonate loading, and changes in luminal chloride concentration, we microperfused distal tubules at a physiologic flow rate (8 nl/min) with solutions containing either 45 or 0 mM chloride (after gluconate substitution). Rats were fed a diet containing zero, minimal, or normal amounts of chloride, while drinking either water or a solution of 0.15 M sodium bicarbonate. Neither extracellular fluid volume contraction nor negative chloride balance ensued. Analysis of covariance with repeated measures demonstrated that dietary chloride, drinking sodium bicarbonate, and perfusion with either 45 mM or zero chloride, each have separate and significant modulating effects on distal tubule bicarbonate secretion. During mild alkalemia, there is modest bicarbonate secretion which is significantly different from zero (-9.9 +/- 3.2 pmol.min-1.mm-1, P less than 0.01), and which is suppressed after perfusion with zero chloride. In contrast, during more pronounced metabolic alkalosis after supplemental bicarbonate drinking, the bicarbonate secretory flux is brisk (-26 +/- 3 pmol.min-1.mm-1) and significantly different from zero and persists (-11 +/- 3 pmol.min-1.mm-1) even during perfusion with zero luminal chloride. Accordingly, in this two-day model of alkalosis induced by dietary chloride restriction, there is regulatory secretion of bicarbonate by distal tubules in vivo which is modulated by luminal chloride concentration.

A Correlated Study of Kidney Function and Ultrastructure in Potassium-Depleted Rats

In order to provide an ultrastructural description of kaliopenic nephropathy in rats, potassium deficiency uncomplicated by chloride depletion or extracellular fluid volume expansion was induced by dietary means alone. After 12 days, serum and muscle analyses demonstrated severe potassium depletion, while urine concentrating ability was markedly impaired. Although micropuncture techniques demonstrated that net fluid transport in proximal tubules was normal, the epithelial cells showed the following morphological alterations: electron-lucent vacuoles and lysosome-like dense bodies were numerous, focal cytoplasmic degradation was present, and intercellular spaces were dilated in many proximal tubules. Medullary cell droplets were characterized by fusion, massive enlargement with sequestration of cytoplasmic components such as ribosomes, and occasional extrusion through ruptured plasma membranes--features not previously reported.

Repair of the myocardial lesion during potassium repletion of kaliopenic rats: an ultrastructural study.

The myocardial lesions of potassium-depleted rats heal with minimal scarring when the animals are repleted with potassium. To elucidate the ultrastructural features of this unique form of myocardial repair, the rats were first depleted of potassium by dietary restriction for 6 weeks. The lesion with myofibral degeneration consisted predominantly of mononuclear cells, many of which contained prominent lysosomal bodies. Interspersed among these cells were small, isolated myofibers which, probably in the process of degeneration, had lost the sarcomeric arrangement of myofilaments as well as most of the organelles. They were partly surrounded by an irregular basal lamina, and contained randomly distributed myofilaments. When the rats were repleted with potassium, the lesions showed a sudden increase of myofibers which resembled those seen during depletion but were in groups with intercellular attachments and encircled by a continuous basal lamina. The interstitial cells during the period of repletion were devoid of lysosomal bodies, and showed proliferation of cytoplasmic organelles. Some of the interstitial cells were partially surrounded by a basal lamina and contained prominent arrays of filaments. They did not, however, appear to be related to the myofibers because of their size and shape. It seemed likely that degenerative myofibers during depletion persisted in an immature form and acted as reserve cells which with repletion of potassium reverted to developing myofibers and ultimately reconstituted the myocardium.

Exploring Ethical, Moral, and Legal Issues Related to Kidney Diseases

SUSAN WAS a 12-yearold girl who stood out in the minds of many of her caregivers as one of the more tragic cases they could remember. As an otherwise healthy 3-year-old, she had developed meningococcemia and had been desperately ill in the pediatric intensive care unit for many weeks. Although her survival had been in doubt on several occasions, she eventually recovered with the loss of one hand, several fingers on the other hand, and partial loss of both feet. In addition, she sustained irreversible loss of renal function. Fortunately, however, her neurological function seemed to have been completely spared, and she began a regular regimen of dialysis and rehabilitative therapy. Her parents and 2 older siblings were completely devoted to her recovery. About 1 year after her primary illness, her father donated his kidney to her for transplantation. The procedure went well, and she maintained excellent renal function on a standard immunosuppressive regimen of cyclosporin, azathioprine, and prednisone. She returned to school at an age-appropriate grade level and, despite her disabilities, had an essentially normal childhood. Six years later, at the age of 10, she went to summer camp. While swimming in the lake, she disappeared for several minutes. When the lifeguards found her, Susan was unconscious and without pulse or respirations. They began cardiopulmonary resuscitation (CPR) and she recovered a spontaneous pulse. She was airlifted back to the children’s hospital where she had received her long-term care and was treated for hypoxic-ischemic multisystem organ failure. Although she initially had moderate renal dysfunction, mild acute respiratory distress syndrome (ARDS), and mild hepatic insufficiency, these resolved within several days. Unfortunately, she was left with severe hypoxic-ischemic encephalopathy. Again, she entered a rehabilitation program, but this time her progress was not as significant or satisfactory. After 4 months she was discharged to go home and had home-nursing assistance several hours a day. One year after her near-drowning experience, Susan remained nonverbal. Some of her caregivers, including her Principal Discussant: Robert D. Truog, MD Professor of Anesthesia, Pediatrics, and Medical Ethics Harvard Medical School Director, Multidisciplinary Intensive Care Unit, Children’s Hospital Boston, Massachusetts