Orjan W. Peterson

Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat.

Until recently, conversion of arginine to agmatine by arginine decarboxylase (ADC) was considered important only in plants and bacteria. In the following, we demonstrate ADC activity in the membrane-enriched fraction of brain, liver, and kidney cortex and medulla by radiochemical assay. Diamine oxidase, an enzyme shown here to metabolize agmatine, was localized by immunohistochemistry in kidney glomeruli and other nonrenal cells. Production of labeled agmatine, citrulline, and ornithine from [3H]arginine was demonstrated and endogenous agmatine levels (10(-6)M) in plasma ultrafiltrate and kidney were measured by HPLC. Microperfusion of agmatine into renal interstitium and into the urinary space of surface glomeruli of Wistar-Frömter rats produced reversible increases in nephron filtration rate (SNGFR) and absolute proximal reabsorption (APR). Renal denervation did not alter SNGFR effects but prevented APR changes. Yohimbine (an alpha 2 antagonist) microperfusion into the urinary space produced opposite effects to that of agmatine. Microperfusion of urinary space with BU-224 (microM), a synthetic imidazoline2 (I2) agonist, duplicated agmatine effects on SNGFR but not APR whereas an I1 agonist had no effect. Agmatine effects on SNGFR and APR are not only dissociable but appear to be mediated by different mechanisms. The production and degradation of this biologically active substance derived from arginine constitutes a novel endogenous regulatory system in the kidney.

Temporal adjustment of the juxtaglomerular apparatus during sustained inhibition of proximal reabsorption

Tubuloglomerular feedback (TGF) stabilizes nephron function by causing changes in single-nephron GFR (SNGFR) to compensate for changes in late proximal flow (VLP). TGF responds within seconds and reacts over a narrow range of VLP that surrounds normal VLP. To accommodate sustained increases in VLP, TGF must reset around the new flow. We studied TGF resetting by inhibiting proximal reabsorption with benzolamide (BNZ; administered repeatedly over a 24-hour period) in Wistar-Froemter rats. BNZ acutely activates TGF, thereby reducing SNGFR. Micropuncture was performed 6-10 hours after the fourth BNZ dose, when diuresis had subsided. BNZ caused glomerular hyperfiltration, which was prevented with inhibitors of macula densa nitric oxide synthase (NOS). Because of hyperfiltration, BNZ increased VLP and distal flow, but did not affect the basal TGF stimulus (early distal salt concentration). BNZ slightly blunted normalized maximum TGF response and the basal state of TGF activation. BNZ sensitized SNGFR to reduction by S-methyl-thiocitrulline (SMTC) and caused the maximum TGF response to be strengthened by SMTC. Sensitization to type I NOS (NOS-I) blockers correlated with increased macula densa NOS-I immunoreactivity. Tubular transport measurements confirmed that BNZ affected TGF within the juxtaglomerular apparatus. During reduced proximal reabsorption, TGF resets to accommodate increased flow and SNGFR through a mechanism involving macula densa NOS.

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

An analysis of glomerular-tubular balance in the rat proximal tubule

An analysis of glomerulo-tubular balance in the rat proximal tubule. Flow dependence of absolute proximal reabsorption (APR) or glomerulo-tubular balance (GTB) has been observed with spontaneous alterations in flow and attributed to both intraluminal and extraluminal factors. Flow dependent alterations in APR were demonstrated when 1. nephron filtration rate (SNGFR) was decreased by tubulo-glomerular feedback mechanisms by increasing late proximal tubular microperfusion rates, and 2. when SNGFR was increased by addition of [Sar1, Ala8] angiotensin II to the adjacent peritubular capillary flow. Selective reduction in early proximal tubular flow rate by pump aspiration also resulted in flow dependent reductions in APR. However, selective additions of perfusion fluids of various native and artificial constituency to the early proximal tubule did not result in flow dependent increase in APR.Conclusions. 1. GTB with both increases and decreases in SNGFR can be demonstrated at the level of the single nephron, 2. selective reductions in luminal flow rate produces parallel reductions in APR; however, 3. increases in flow rate with either artificial or native fluids of different ionic concentrations did not result in increases in APR. This lack GTB may be due to lack of parallel changes in peritubular physical factors or that APR in the S2 segment is less sensitive to increase in flow rate.

Effects on kidney filtration rate by agmatine requires activation of ryanodine channels for nitric oxide generation.

Agmatine, decarboxylated arginine, is produced in the kidney and can increase nephron and kidney filtration rate via renal vasodilatation and increases in plasma flow. This increase in filtration rate after agmatine is prevented by administration of nitric oxide synthase (NOS) inhibitors. In endothelial cells, agmatine-stimulated nitrite production is accompanied by induction of cytosolic calcium. NOS activity requires calcium for activation; however, the source of this calcium remains unknown. Ryanodine receptor (RyR) calcium-activated calcium release channels are present in the kidney cortex, and we evaluated if RyR contributes to the agmatine response. Agmatine microperfused into Bowmans space reversibly increases nephron filtration rate (SNGFR) by approximately 30%. cADP-ribose (cADPR) regulates RyR channel activity. Concurrent infusion of agmatine with the cADPR blocker 8-bromo-cADPR (2 microM) prevents the increase in filtration rate. Furthermore, direct activation of the RyR channel with ryanodine at agonist concentrations (5 microM) increases SNGFR, and, like agmatine, this increase is prevented by administration of N(G)-monomethyl-l-arginine, a nonselective NOS blocker. We demonstrate that agmatine does not elicit ADPR cyclase activity in vascular smooth muscle membranes and does not directly affect RyR calcium channel responses using sea urchin egg homogenates. These results imply interplay between endothelial cell cADPR/RyR/Ca(2+)/NO and the cADPR/RyR/Ca(2+) pathways in vascular smooth muscle cells in arterioles in the regulation of kidney filtration rate. In conclusion, we show that agmatine-induced effects require activation of cADPR and RyR calcium release channels for NO generation, vasodilation, and increased filtration rate.

Interactive Control of Renal Function by α2-Adrenergic System and Nitric Oxide: Role of Angiotensin II

We studied the role of angiotensin II (AII) in the interactive control of renal function by the alpha 2-adrenergic system and nitric oxide (NO) in adult male Munich Wistar rats 5-7 days after ipsilateral renal denervation (DNX). Renal micropuncture was used under euvolemic conditions before (period 1) and during (period 2) systemic inhibition of NO synthase (NOS) with NG-monomethyl-L-arginine (L-NMMA) in three groups. Group 1 served as a DNX control. In group 2, the alpha 2-adrenergic agonist B-HT 933 (BHT) was infused systemically throughout the experiment. In group 3, the AII-receptor blocker, Iosartan (LOS), was infused before period 2 as well as throughout infusion of BHT. L-NMMA increased blood pressure (BP) to a similar degree in all three groups. In group 1, infusion of L-NMMA did not affect glomerular hemodynamics or tubular function. With BHT in group 2, L-NMMA reduced absolute proximal tubular reabsorption (APR) and by reducing nephron plasma flow (SNPF) and glomerular ultrafiltration coefficient (LpA) caused nephron filtration rate (SNGFR) to decrease, a response described in innervated kidneys. LOS in group 3 abrogated the BHT-facilitated reduction of LpA and SNGFR but not of SNPF and APR in response to L-NMMA. In group 1, urinary sodium excretion (UNaV) did not change and urinary flow rate (UV) increased slightly in period 2. L-NMMA combined with BHT, however, exerted a profound diuresis and natriuresis in group 2. These effects were further exaggerated with LOS. In a fourth group of DNX rats. LOS given alone before period 2 did not affect SNGFR, SNPF, LpA, APR, UV, or UNaV. We conclude that after subacute renal denervation alpha 2-adrenergic activation sensitizes (a) LpA to reduction by NOS inhibition through an AII-dependent mechanism, and (b) SNPF and proximal tubular reabsorption to reduction by L-NMMA regardless of the AII activity. Furthermore, our results suggest a potential role for the alpha 2-adrenergic system and AII in the diuretic and natriuretic effect of systemic NOS inhibition.

Glycine prevents toxic tubular cell injury.

Glycine prevents tubular injury as suggested by in vitro cell culture studies, studies in the isolated perfused kidney, and in vivo studies. We have previously demonstrated that intratubular administration of uranyl nitrate (UN) produces proximal tubular cell injury and decreases proximal tubular reabsorption (APR). The decrease in APR activates tubuloglomerular feedback and lowers nephron filtration rate (SNGFR). This study was designed to evaluate if glycine administration could prevent the decrease in SNGFR after UN administration and if maintenance of SNGFR was due to tubular cell cytoprotection or suppression of the tubuloglomerular feedback. Administration of 0.65 ng of UN into the early proximal tubule was associated with a decrease in distal SNGFR (SNGFRD) from 29 +/- 2 to 24 +/- 2 nL/min (p < .05) and late proximal SNGFR (SNGFRLP) from 37 +/- 2 to 26 +/- 2 nL/min, and APR from 14 +/- 1 to 10 +/- 1 nL/min. Systemic administration of glycine (20 g/dL, 1.4 mL/h) was associated with significant increases in SNGFRD and SNGFRLP, and APR (38 +/- 3, 44 +/- 3, and 15 +/- 2 nL/min). UN administration did not affect APR or SNGFR in glycine-treated rats. These findings demonstrate that glycine prevents UN-induced decreases in SNGFR through a cytoprotective effect on proximal tubular cells.

Mechanisms of Glomerular Immune Injury: Effects of Antioxidant Treatment

Significant glomerular vasoconstriction and production of reactive oxygen species has been known to occur with exposure to anti-glomerular basement membrane antibody (AGBM-Ab) in the rat model. Previously published studies have demonstrated that such effects can be reduced by therapy with phentolamine, an alpha-adrenergic antagonist. It was hypothesized that antioxidant pretreatment with water-soluble probucol would improve glomerular hemodynamics 60 to 90 min after the administration of AGBM-Ab. These relationships were examined with both in vivo renal micropuncture and in vitro studies in rats. Single-nephron GFR (SNGFR) decreased markedly in untreated rats after AGBM-Ab as a result of afferent and efferent arteriolar vasoconstriction with consequent reductions in nephron plasma flow (SNPF) and decreases in the glomerular ultrafiltration coefficient (LpA). Basal SNGFR was increased, and SNGFR was significantly higher after AGBM-Ab in probucol-treated versus untreated rats. This finding was due solely to higher values for SNPF and prevention of afferent arteriolar constriction. A reduction in LpA after AGBM-Ab was not prevented by probucol treatment. In vitro analyses of glomeruli revealed reduced myeloperoxidase activity in antioxidant-treated rats. Lipoxygenase activity and leukotriene products, however, were not changed by antioxidant therapy, yet vasoconstriction was prevented. H(2)O(2) generation before and after formyl-methionyl-leucyl-phenylalanine stimulation was significantly reduced before and after AGBM-Ab in glomeruli harvested from rats that were treated with the antioxidant. Antioxidant therapy in this model of AGBM-Ab injury did not prevent reductions in LpA, an index of glomerular membrane damage, but did prevent afferent arteriolar vasoconstriction. Reactive oxygen species generation was reduced by probucol. The specific mechanisms whereby antioxidant therapy ameliorates glomerular hemodynamic effects will be defined in additional studies and is likely to involve either enhanced vasodilator or diminished vasoconstrictor activity.