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This study examines for the first time the effects of uninephrectomy (Nx) on modulation of whole kidney glomerular filtration rate (GFR), single-nephron GFR (SNGFR), and progression of diabetic nephropathy in the db/db mouse model of type 2 diabetes mellitus. To characterize SNGFR and tubuloglomerular feedback (TGF) responses to Nx and chronic neuronal nitric oxide synthase inhibition in the db/db mouse, we studied the effects of Nx on whole kidney GFR, SNGFR, and TGF characteristics in db/db and wild-type (WT) mice after Nx or sham Nx. We also documented progression of glomerular changes over a 6-mo period. Whole kidney GFR and SNGFR were significantly higher in db/db Nx than db/db sham mice, without change in proximal tubule reabsorptive rates. The TGF responses, determined as proximal-distal SNGFR differences, were brisk: 12.1 [+ or -] 1.0 vs. 8.4 [+ or -] 0.6 nl/min in WT sham (P < 0.05), 15.7 [+ or -] 1.0 vs. 12.0 [+ or -] 1.0 nl/min in WT Nx (P < 0.05), and 17.8 [+ or -] 1.3 vs. 14.3 [+ or -] 1.0 nl/min in db/db Nx (P < 0.05) mice. Chronic ingestion of the neuronal nitric oxide synthase inhibitor S-methylthiocitrulline for 2-3 wk after Nx had no effect on SNGFR or the TGF response. These studies show further elevations in whole kidney GFR and SNGFR in these hyperglycemic morbidly obese db/db mice, with an intact TGF system after Nx. In addition, in the db/db Nx mice, 4-6 mo after Nx, there was an exacerbation of the lesions of diabetic nephropathy, as quantified by a significant increase in the ratio of mesangial surface area to total glomerular surface area. hyperfiltration: tubuloglomerular feedback; neuronal nitric oxide synthase

Hyperfiltration has been implicated in the progression toward diabetic nephropathy in type 2 diabetes mellitus (DM2). This study focuses for the first time on the in vivo modulation of single-nephron GFR (SNGFR) in the classic B6.Cg-[m.sup.+/+][Lepr.sup.db]/J (db/db) mouse model of DM2. To obtain stable preparations, it was necessary to use a sustaining infusion of 3.3 ml x 100 g body [wt.sup.-1] x [h.sup.-t], or higher. SNGFR (measured both proximally and distally) was greater in db/db vs. heterozygote (db/m) mice (P < 0.05) but not vs. the wild-type (WT) mice. The tubuloglomerular feedback (TGF) responses, determined as free-flow proximal vs. distal SNGFR differences, were significant in db/db mice (11.6 [+ or -] 0.8 vs. 9.3 [+ or -] 1.0 nl/min, P < 0.01), in db/m mice (8.0 [+ or -] 0.8 vs. 7.2 [+ or -] 0.6 nl/min, P < 0.02), and WT mice (9.9 [+ or -] 0.6 vs. 8.9 [+ or -] 0.7 nl/min, P < 0.05). After increasing the sustaining infusion in the db/db mice, to offset glycosuric urine losses, the SNGFR increased significantly, and the TGF response was abolished. In these volume-replete db/db mice, absolute fluid reabsorption measured both at the late proximal and distal tubular sites were significantly increased vs. db/m mice infused at 3.3 ml x 100 g body [wt.sup.-t] x [h.sup.-1]. After infusion of the neuronal nitric oxide synthase (nNOS) inhibitor S-methylthiocitrulline, SNGFR fell in both db/db and db/m mice. These studies show that SNGFR is elevated in this mouse model of DM2, is suppressed by nNOS inhibition, and is modulated by TGF influences that are altered by the diabetic state and responsive to changes in extracellular fluid volume. hyperfiltration; tubuloglomerular feedback; neuronal nitric oxide synthase

Five-sixths nephrectomized rats were used in the evaluation of the effects of potassium depletion on bicarbonate reabsorption in the distal tubules left behind. The regulatory mechanism involved is also examined with the use of proton secretion inhibitors. Results reveal that potassium depletion causes a significant elevation of bicarbonate absorption among partially nephrectomized rats through the stimulation of the hydrogen-ATPase activity.

The transport mechanisms that mediate the ability of angiotensin II (ANG II) to increase bicarbonate reabsorption was analyzed in the late distal tubules (DT) of rats that were exposed to five-sixths nephrectomy (Nx). Western and immunocytochemical analysis in Nx rats indicated the presence of enhanced H+-adenosine triphosphatase expression in the renal cortical tissue. Nx rats also exhibited normal plasma bicarbonate concentrations and ANG II-dependent bicarbonate reabsorption was inhibited by losartan.

The renal cortical collecting ducts respond to low extracellular potassium levels and acidosis by activating the intercalated cells to extrude their H+-ATPase apparatus from their apical membranes. The H+-ATPase vesicles form studs along the luminal membrane. This phenomenon was investigated in the intercalated cells by quantitative electron microscopial studies. The apical membranes of the intercalated cells showed a greater degree of exposed H+-ATPase in reponse to potassium depletion and acidosis.

A study of the adaptive responses of unidirectional bicarbonate reabsorption components Jreab and Jsec during acid loading in the distal tubules of rats through in vivo microperfusion experiments shows that metabolic acidosis suppresses Jsec but activates bafilomycin A1-resistant Jreab. Jreab also undergoes reduction after 3 days of severe acidosis. Sch-28080 inhibitor affects the bafilomycin sensitive Jreab, while in overnight fasted rats amiloride and BAF suppress Jreab.

Measurements of distal tubule unidirectional bicarbonate secretion and reabsorption in rats reveal the unidirectional compounds of net bicarbonate reabsorption during metabolic alkalosis (MA). Transition of chloride depletion from the acute secretory phase to the chronic phase, whose increased levels of plasma and bicarbonate are maintained, does not require inhibition of distal tubule J(sec), but involves improved J(reab), implying sensitivity to bafilomycin A1, Sch-28080 and amiloride.

Microperfusion studies of the distal tubules of normally-fed rats analyze the in vivo effects of agonists that influence bicarbonate flux. The agonists which maintain bicarbonate flux act as potential regulators of systemic acid-base balance. The action of 1-desamino-8-D-arginine vasopressin on antidiuretic hormone improves bicarbonate reabsorption in normal rats through an elevation in adenosine monophosphate levels in interspersed rat cells.

A study examined the effects of maternal acid-base alterations on intercalated cell morphology and groups in collecting ducts and in newborn and two-week-old pups. A bigger percentage of B-type intercalated cells and a lower percentage of A-type intercalated cells was present in newborn and two-week-old pups from dams with metabolic alkalosis. The percentage of A-type intercalated cells was not increased, but the percentage of B-type intercalated cells was decreased by acid loading with NaCl. Metabolic alkalosis is connected with enhancement in the B-type intercalated cell percentage, indicating that early variations of intercalated cells respond to maternal acid-base alterations.

Research findings indicate that adenosine 1 receptors are essential for eliciting tubuloglomerular feedback (TGF) responses. Adenosine triphosphate (ATP) may activate P2 receptors in preglomerular vessels that are involved in the autoregulation of renal vascular resistance. Furthemore, nitric oxide from macula densa cells decreases the constrictor effect of adenosine.

Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo. Am J Physiol Renal Physiol 281: F189-F194, 2001.--To directly determine intratubular nitric oxide concentrations ([NO]) in vivo, we modified amperometric integrated electrodes (WPI P/N ISO-NOP007), which are highly sensitive to NO and not affected by ascorbic acid, nitrite, L-arginine, or dopamine. Although reactive lengths were as short as 5 [micro]m long, the electrode still responded rapidly. With the use of kidney surface fluid as the 'zero point,' the electrode tip was inserted into tubular segments along the track of a perforation made by a beveled glass pipette. The surface fluid zero point was usually stable as distal, late proximal, and early proximal tubule [NO] levels were measured sequentially in the same nephron. In eight normal rats, distal, late proximal, and early proximal [NO] concentrations were each ~110 nM. In contrast, in nine 5/6 nephrectomized rats 2 wk postsurgery, although [NO] also did not differ among distal, late proximal, and early proximal segments, levels were approximately fourfold higher than those in normal rats and were significantly reduced after [N.sup.G]-monomethyl-L-arginine administration. These are the first quantitative in vivo tubular fluid [NO] measurements and show a significant increase in tubular fluid [NO] after renal ablation. kidney tubules; in vivo nitric oxide measurements; remnant kidney

Early streptozotocin-diabetes mellitus downregulates rat kidney [AT.sub.2] receptors. Am J Physiol Renal Physiol 280: F254-F265, 2001.--The interaction of ANG II with intrarenal [AT.sub.2] receptors has been implicated in the progression of diabetic nephropathy, but the role of intrarenal [AT.sub.2] receptors is unknown. The present studies determined the effect of early diabetes on components of the glomerular renin-angiotensin system and on expression of kidney [AT.sub.2] receptors. Three groups of rats were studied after 2 wk: 1) control (C), 2) streptozotocin (STZ)-induced diabetic (D), and 3) STZ-induced diabetic with insulin implant (D+I), to maintain normoglycemia. By competitive RT-PCR, early diabetes had no significant effect on glomerular mRNA expression for renin, angiotensinogen, or angiotensin-converting enzyme (ACE). In isolated glomeruli, nonglycosylated (41-kDa) [AT.sub.1] receptor protein expression ([AT.sub.1A] and [AT.sub.1B]) was increased in D rats, with no change in glycosylated (53-kDa)[AT.sub.1] receptor protein or in [AT.sub.1] receptor mRNA. By contrast, STZ diabetes caused a significant decrease in glomerular [AT.sub.2] receptor protein expression (47.0 [+ or -] 6.5% of C; P [is less than] 0.001; n = 6), with partial reversal in D+I rats. In normal rat kidney, [AT.sub.2] receptor immunostaining was localized to glomerular endothelial cells and tubular epithelial cells in the cortex, interstitial, and tubular cells in the outer medulla, and inner medullary collecting duct cells. STZ diabetes caused a significant decrease in [AT.sub.2] receptor immunostaining in all kidney regions, an effect partially reversed in D+I rats. In summary, early diabetes has no effect on glomerular mRNA expression for renin, angiotensinogen, or ACE. [AT.sub.2] receptors are present in glomeruli and are downregulated in early diabetes, as are ell kidney [AT.sub.2] receptors. Our data suggest that alterations in the balance of kidney [AT.sub.1] and [AT.sub.2] receptor expression may contribute to ANG II-mediated glomerular injury in progressive diabetic nephropathy. renin-angiotensin system; glomerulus; immunohistochemistry; hyperglycemia; angiotensin type 1 receptor

Localization of protein inhibitor of neuronal nitric oxide synthase in rat kidney. Am J Physiol Renal Physiol 278: F702-F707, 2000.--We have recently demonstrated that in rats with 5/6 nephrectomy (5/6 Nx), renal cortical and inner medullary neuronal NOS (nNOS) expression is downregulated, associated with decreased urinary excretion of nitric oxide (NO) products. Recently, a novel 89-amino acid protein [protein inhibitor of nNOS (PIN)] was isolated from rat brain and shown to inhibit nNOS activity. The present studies localized PIN in the rat kidney and determined the effect of 5/6 Nx on PIN expression. By RT-PCR, PIN mRNA was detected in the kidney cortex and inner medulla. Immunohistochemistry revealed staining for PIN in glomerular and vasa rectae endothelial cells. PIN was also localized to the apical membranes of inner medullary collecting duct (IMCD) cells. Two weeks after 5/6 Nx, inner medullary PIN expression was significantly upregulated (sham, 0.18 [+ or -] 0.07 vs. 5/6 Nx, 0.58 [+ or -] 0.13 arbitrary units; n = 6, P [is less than] 0.02), as determined by Western blotting. In summary, our data show that PIN, a specific inhibitor of nNOS activity, is expressed in the IMCD, a site of high nNOS expression in the kidney. PIN expression is upregulated in the inner medulla of 5/6 Nx rats. Inhibition of nNOS activity by PIN may have important implications for the regulation of NO synthesis in the IMCD of normal and remnant kidneys. inner medulla; chronic renal failure

Levine, David Z., Michelle Iacovitti, Brian Luck, Maxwell T. Hincke, Kevin D. Burns, and James N. Fryer. Surviving rat distal tubule bicarbonate reabsorption: effects of chronic [AT.sub.1] blockade. Am. J. Physiol. Renal Physiol. 278: F476-F483, 2000.--To determine the in vivo effects of chronic ANG II type 1 ([AT.sub.1])-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive [H.sup.+]-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of [H.sup.+]-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that [H.sup.+]-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], we perfused three inhibitors simultaneously [5-(N,N,-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] of surviving nephrons is largely mediated by [AT.sub.1] receptor-dependent synthesis and insertion of apical [H.sup.+]-ATPase pumps in A-type intercalated cells. kidney failure; receptors; losartan; proton-transporting adenosine 5'-triphosphate synthase; concanamycin A

Distal tubules (DT) from sham or five-sixths nephrectomized (Nx) rats were perfused in vivo to evaluate the hypothesis that, after Nx, endogenous angiotensin II (ANG II) modulates DT in vivo bicarbonate reabsorption (JtCO2 ) via H+-adenosinetriphosphatase (H+-ATPase) and Na+/H+ exchange. In Nx rats JtCO2 was increased (65 ± 7 vs. - 24 ± 21 pmol·min-1·mm-1, P < 0.01). Both luminal and intravenous AT1-receptor blockade by losartan reduced Nx DT JtCO2 (41 ± 6 and 34 ± 4 pmol·min-1·mm-1, respectively, P < 0.05), whereas neither 10-9 M nor 10-11 M ANG II luminal perfusion increased JtCO2, suggesting preexisting high endogenous ANG II levels. The Na+/H+ antiporter inhibitors 5-(iV-ethyl-iV-isopropyl)- amiloride and 5-(A/yV-dimethyl)-amiloride were without effect. Luminal perfusion of 5 nM concanamycin A, a V-type H+-ATPase inhibitor, reduced Nx DT JtCO2 (45 ± 8 pmol · min-1 · mm-1, P < 0.05). In Nx A-type intercalated cells, we demonstrated cellular hypertrophy, elaboration of apical microplicae, and enhanced expression/apical polarization of H+-ATPase. Thus ANG II is an important determinant in sustaining brisk DT JtCO2 following Nx and is associated with enhanced expression ancf A-type intercalated cell apical polarization of H+-ATPase. [ABSTRACT FROM AUTHOR]

Examines the mechanism behind angiotensin II-dependent bicarbonate reabsorption in surviving rat distal tubules. Cellular distribution and expression of transport proteins in distal tubules; Determinants in sustaining brisk distal tubules in vivo bicarbonate reabsorption.