Regulation of ammonia production by mouse proximal tubules perfused in vitro. Effect of luminal perfusion.

Abstract

To investigate factors regulating ammonia (NH3) production by isolated defined proximal tubule segments, we examined the rates of total NH3 (NH3 + NH') production by individual proximal tubule segments perfused in vitro under a variety of perfusion conditions. Segments consisting of late convoluted and early straight portions of superficial proximal tubules were incubated at 370C in Krebs-Ringer bicarbonate (KRB) buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, pH 7.4. The rate of total ammonia production was calculated from the rate of accumulation of total NH3 in the bath. The total ammonia production rate by unperfused proximal segments was 6.0±0.2 (±SE) pmol/mm per minute, which was significantly lower than segments perfused at a flow rate of 22.7±3.4 nl/min with KRB buffer (21.5±1.4 pmol/mm per minute; P < 0.001) or with KRB buffer containing 0.5 mM L-glutamine (31.9±2.5; P < 0.001). The rate of NH3 production was higher in segments perfused with glutamine than in segments perfused without glutamine (P < 0.01).The perfusion-associated stimulation of NH3 production was characterized further. Analysis of collected luminal fluid samples revealed that the luminal fluid total NH3 leaving the distal end of the perfused proximal segment accounted for 91% of the increment in NH3 production observed with perfusion. Increasing the perfusion flow rate from 3.7±0.1 to 22.7±3.4 nl/min by raising the perfusion pressure resulted in an increased rate of total NH3 production in the presence or absence of perfusate glutamine (mean rise in rate of total NH3 production was 14.9±3.7 pmol/mm per minute in segments perfused with glutamine and 7.8±0.9 in those perfused without glutamine). In addition, increasing the perfusion flow rate at a constant perfusion pressure increased the rate of luminal output of NH3. Total NH3 production was not affected by reducing perfusate sodium concentration to 25 mM and adding 1.0 mM amiloride to the perfusate, a condition that was shown to inhibit proximal tubule fluid reabsorption. These observations demonstrate that the rate of total NH3 production by the mouse proximal tubule is accelerated by perfusion of the lumen of the segment, by the presence of glutamine in the perfusate,

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

“…Thus, the luminal total ammonia output rate represents the net rate of total ammonia secretion. In our previous studies, we demonstrated that most of the total ammonia produced by a perfused proximal tubule segment is secreted into the luminal fluid and enters the bath medium via the luminal fluid leaving the distal end of the perfused segment (8,9). In the present study, we examined the roles of the Na+-H+ exchanger and intraluminal pH on net luminal total ammonia secretion and total ammonia production rates.…”

“…Isolation andperfusion ofthe individualproximal tubule segments. This method has been described in detail (8). Briefly, a mouse was killed by cervical dislocation and a kidney rapidly removed and placed in iced Krebs-Ringer bicarbonate (KRB) buffer.…”

“…Total ammonia was measured using the method developed in this laboratory (8). This method was based upon a series of enzymatic reactions that were ultimately coupled with a bioluminescence reaction so that light emission was directly proportional to the amount of total ammonia in the sample.…”

“…production and net luminal total ammonia secretory rates may be determined in isolated perfused mouse proximal tubule segments (8,9). The total ammonia production rate represents the rate at which total ammonia leaves the perfused proximal tubule segment via the peritubular and luminal aspects of the tubule.…”

Contribution of individual superficial nephron segments to ammonium handling in chronic metabolic acidosis in the rat. Evidence for ammonia disequilibrium in the renal cortex.

“…(16) indicate that the ammonia permeability of the isolated perfused rabbit cortical collecting tubule is considerably less than that of proximal convoluted tubule. Further, the studies of Nagami and Kurokawa (17) indicate that the preferential entry of ammonia into proximal tubule fluid cannot be entirely explained by the peritubular and luminal differences in pH. Thus, the tubule fluid concentration of the free base, ammonia, may not exist at the diffusion equilibrium conditions in all nephron segments in the cortex.…”