P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells.

Luminal nucleotide stimulation is known to reduce Na(+) transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca(2+) transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200 bp was not regulated by changes in [Ca(2+)]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.

The role of the BK channel in potassium homeostasis and flow-induced renal potassium excretion.

The kidney is the major regulator of potassium homeostasis. In addition to the ROMK channels, large conductance Ca(2+)-activated K(+) (BK) channels are expressed in the apical membrane of the aldosterone sensitive distal nephron where they could contribute to renal K(+) secretion. We studied flow-induced K(+) secretion in BK channel alpha-subunit knockout (BK(-/-)) mice by acute pharmacologic blockade of vasopressin V(2) receptors, which caused similar diuresis in wild-type and knockout mice. However, wild-type mice, unlike the BK(-/-), had a concomitant increase in urinary K(+) excretion and a significant correlation between urinary flow rate and K(+) excretion. Both genotypes excreted similar urinary amounts of K(+) irrespective of K(+) diet. This was associated, however, with higher plasma aldosterone and stronger expression of ROMK in the apical membrane of the aldosterone-sensitive portions of the distal nephron in the knockout than in the wild-type under control diet and even more so with the high-K(+) diet. High-K(+) intake significantly increased the renal expression of the BK channel in the wild-type mouse. Finally, despite the higher plasma K(+) and aldosterone levels, BK(-/-) mice restrict urinary K(+) excretion when placed on a low-K(+) diet to the same extent as the wild-type. These studies suggest a role of the BK channel alpha-subunit in flow-induced K(+) secretion and in K(+) homeostasis. Higher aldosterone and an upregulation of ROMK may compensate for the absence of functional BK channels.

Tubuloglomerular feedback responses of the downstream efferent resistance: unmasking a role for adenosine?

This Commentary aims to integrate or interrelate the available in vivo data with the in vitro study by Ren and co-workers, which comes to the somewhat surprising conclusion that tubuloglomerular feedback activation vasodilates the efferent arteriole by an adenosine-dependent mechanism.

Primary kidney growth and its consequences at the onset of diabetes mellitus.

Diabetes mellitus is a primary contributor to progressive kidney dysfunction leading to end-stage renal disease (ESRD). In the early phase of diabetes, prior to the onset of further complications, both kidney size and glomerular filtration rate (GFR) increase. Glomerular hyperfiltration is considered a risk factor for downstream complications and progression to ESRD. Abnormalities in vascular control have been purported to account for the glomerular hyperfiltration in early diabetes. In this review we discuss a tubulo-centric concept in which tubular growth and subsequent hyper-reabsorption contribute to the onset of glomerular hyperfiltration that demarks the early stage of diabetes. Kidney growth, in this concept, is no longer relegated to a compensatory response to hyperfiltration, but rather plays a primary and active role in its genesis and progression. As such, components of kidney growth, such as the polyamines, may provide a means of early detection of diabetic kidney dysfunction and more effective therapeutic intervention.

Transport regulation by the serum- and glucocorticoid-inducible kinase SGK1.

The serum- and glucocorticoid-inducible kinase SGK1 is an ubiquitously expressed kinase with the ability to regulate a variety of transport systems. Recent observations point to a role of SGK1 in the regulation of diverse physiological functions such as epithelial transport and cardiac and neuronal excitability. At least partially through its effect on transport, SGK1 contributes to a number of pathophysiological conditions including metabolic syndrome and fibrosing disease.

Kidney function in mice: thiobutabarbital versus alpha-chloralose anesthesia.

Mice that lack or over-express a gene of interest are important tools for unraveling gene function. The determination of single nephron function by micropuncture or precise determination of glomerular filtration rate (GFR) by inulin clearance method require experiments under anesthesia. A good anesthetic protocol should allow for reasonable and stable glomerular and tubular function. The aim of this study was to compare the commonly used thiobutabarbital (TBB) versus alpha-chloralose (CHL) anesthesia with regard to absolute levels and the stability of blood pressure, heart rate, and kidney function. Male CD1 mice were anesthetized with TBB (100 mg/kg body weight i.p.) or CHL (120 mg/kg body weight i.p.), plus ketamine (100 mg/kg body weight i.m.) given to every mouse for analgesia. After preparation for clearance experiments, two 30-min urine collections were performed at periods 1 and 2 (P1 and P2). It was observed that heart rate and mean arterial blood pressure did not differ between TBB ( n=9) vs. CHL ( n=9) and were stable through P1 and P2. In CHL, GFR as well as fractional excretion of fluid, Na(+) and K(+) were stable from P1 to P2 (P1: 190+/-15 microl/min, 1.6+/-0.2%, 0.7+/-0.1%, 35+/-5%; percent change in P2: 1+/-6, 26+/-10, 29+/-15, 6+/-10 respectively). In TBB, GFR was significantly greater vs. CHL in P1 and did not significantly change in P2 (246+/-8 microl/min, p<0.05; percent change: -6.5+/-4). Fractional excretion of fluid, Na(+) and K(+) were not significantly different vs. CHL in P1, but significantly increased in P2 (P1: 1.5+/-0.2%, 1.1+/-0.2%, 31+/-3%; percent change in P2: 122+/-23, 128+/-21 and 29+/-6 respectively; each p<0.05 vs. P1). In conclusion, mice under both anesthetic regimens present reasonable and stable blood pressure and reasonable kidney function, but kidney reabsorption is more stable under CHL than under TBB anesthesia, which may facilitate study of the response in kidney function to acute interventions.

Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice.

Mice deficient for adenosine A1 receptors (A1AR) lack tubuloglomerular feedback (TGF). In vivo micropuncture experiments were performed under anesthesia in A1AR-deficient and wild-type littermate mice to study the effects of chronic absence of A1AR on fluid and Na(+) reabsorption along the nephron, as well as the functional consequences at the single-nephron level of the lack TGF. Evidence is provided for an A1AR-mediated tonic inhibition of Na(+) reabsorption in a water-impermeable segment of the loop of Henle, possibly the thick ascending limb. In contrast, proximal tubular reabsorption of fluid, Na(+) and K(+) was unaffected by the chronic absence of A1AR. Experiments in which artificial tubular fluid was added to free-flowing late-proximal tubules demonstrated an essential role of A1AR/TGF in the stabilization of fluid and Na(+) delivery to the distal nephron. Further, the occurrence of spontaneous oscillations of hydrostatic pressure in proximal tubule ( P(PT)) at a frequency of about 32 mHz depended on intact A1AR/TGF. In comparison, the normal, stabilizing reduction in P(PT) following the initial rise in P(PT) during sustained small increases in proximal tubular flow rate does not require A1AR/TGF; TGF-independent mechanisms appear to compensate in this regard for a lack of TGF under physiological conditions and the lack of TGF is unmasked only when supraphysiological flow rates overwhelm TGF-independent compensation.

The salt paradox of the early diabetic kidney is independent of renal innervation.

Glomerular filtration rate (GFR) is inversely and thus paradoxically related to dietary NaCl intake in rats and patients with early type 1 diabetes mellitus (DM). Enhanced sensitivity of proximal reabsorption to NaCl diet inducing secondary adaptations in GFR through actions of tubuloglomerular feedback causes this salt paradox. We studied the role of renal nerves for the salt paradox in rats with streptozotocin (STZ)-induced DM since a regulatory influence of renal nerves on proximal reabsorption is well established. The left kidney (LK) was denervated before induction of STZ-DM. Subsequently, the normal diet was continued or a low NaCl diet was initiated and 1 week later animals were prepared for clearance experiments under anesthesia including ureter catheterization to measure GFR for each kidney. In diabetic rats, the right innervated as well as the left denervated kidney showed higher values for GFR and kidney weight in animals on a low versus a normal NaCl diet indicating that the salt paradox occurs independent of renal innervation. In addition, evidence is provided that the renal nerves of non-diabetic rats do not contribute to renal Na(+) retention during dietary NaCl restriction but modulate renal hemodynamics and kidney weight under these conditions.

Ornithine decarboxylase, kidney size, and the tubular hypothesis of glomerular hyperfiltration in experimental diabetes.

In early diabetes, the kidney grows and the glomerular filtration rate (GFR) increases. This growth is linked to ornithine decarboxylase (ODC). The study of hyperfiltration has focused on microvascular abnormalities, but hyperfiltration may actually result from a prior increase in capacity for proximal reabsorption which reduces the signal for tubuloglomerular feedback (TGF). Experiments were performed in Wistar rats after 1 week of streptozotocin diabetes. Kidney weight, ODC activity, and GFR were correlated in diabetic and control rats given difluoromethylornithine (DFMO; Marion Merrell Dow, Cincinnati, Ohio, USA) to inhibit ODC. We assessed proximal reabsorption by micropuncture, using TGF as a tool for manipulating single-nephron GFR (SNGFR), then plotting proximal reabsorption versus SNGFR. ODC activity was elevated 15-fold in diabetic kidneys and normalized by DFMO, which also attenuated hyperfiltration and hypertrophy. Micropuncture data revealed an overall increase in proximal reabsorption in diabetic rats too great to be accounted for by glomerulotubular balance. DFMO prevented the overall increase in proximal reabsorption. These data confirm that ODC is required for the full effect of diabetes on kidney size and proximal reabsorption in early streptozotocin diabetes and are consistent with the hypothesis that diabetic hyperfiltration results from normal physiologic actions of TGF operating in a larger kidney, independent of any primary malfunction of the glomerular microvasculature.

Acute renal response to the non-peptide vasopressin V2-receptor antagonist SR 121463B in anesthetized rats.

Vasopressin V2-receptor antagonists are promising agents for the use in water-retaining diseases. Potential renal mechanisms of action include effects on water permeability in the collecting duct as well as on electrolyte transport in the thick ascending limb of Henle's loop (TALH). To elucidate sites of action upstream of the distal tubule, e.g., in TALH, micropuncture experiments were performed in anesthetized rats during application of the V2-receptor antagonist SR 121463B. As compared to vehicle-treated rats, SR 121463B (0.3 mg/kg i.v.) did not affect mean arterial blood pressure (means +/- SEM, n=10 rats per group: 108+/-4 mmHg vs. 107+/-4 mmHg), whole kidney GFR (1.1+/-0.1 ml/min vs. 1.1+/-0.1 ml/min), or whole kidney fractional reabsorption (FR) of potassium (66+/-5% vs. 68+/-4%). The drug, however, reduced whole kidney FR of fluid (92+/-1% vs. 99+/-1%), increased urinary flow rate (84+/-7 microl/min vs. 8+/-1 microl/min) and electrolyte-free-water clearance (72+/-8 microl/min vs. 2+/-1 microl/min), and reduced urinary osmolality (148+/-11 mosmol/kg vs. 1,200+/-185 mosmol/kg). This pronounced diuretic response was associated with a minor reduction in whole kidney FR of sodium (99.6+/-0.1% vs. 99.9+/-0.1%) and chloride (98.3+/-0.2% vs. 98.9+/-0.1%). As compared to vehicle application, SR 121463B did not significantly alter single nephron GFR (39+/-2 nl/min vs. 39+/-1 nl/min, n=22 and 23 nephrons, respectively) or the FR up to the early distal tubule of fluid (76+/-2% vs. 76+/-1%), sodium (92+/-1% vs. 93+/-1%), potassium (91+/-1% vs. 90+/-1%) or chloride (90+/-1% vs. 91+/-1%). Together these data indicate a predominant aquaretic effect of SR 121463B which is located downstream of the early distal tubule. This response is compatible with blockade of vasopressin V2-receptors in the collecting duct and, as directly demonstrated by immunohistochemistry, subsequent retrieval of aquaporin-2 from apical plasma membrane, which inhibits water permeability and transport.

Adenosine formed by 5'-nucleotidase mediates tubuloglomerular feedback.

Nephron function is stabilized by tubuloglomerular feedback (TGF). TGF operates within the juxtaglomerular apparatus, sensing changes in tubular flow and eliciting compensatory changes in single nephron GFR (SNGFR). The mediator(s) of TGF remains unconfirmed. One theory is that ATP consumed in active transport by the macula densa leads to formation of adenosine, which causes glomerular vasoconstriction. We performed micropuncture in rats to test this hypothesis. Adenosine activity was manipulated by microperfusing nephrons with adenosine A1 receptor blocker, A1-agonist, or 5'-nucleotidase inhibitor. Effects on TGF were characterized by changes in TGF efficiency (the compensation for small perturbations in tubular flow) and by changes in the maximum range over which TGF can cause SNGFR to change. These data were further applied to generate TGF profiles [SNGFR versus late proximal flow (V(LP))]. TGF efficiency was significantly reduced by blocking A1-receptors. TGF efficiency, TGF range, and the slope of the TGF profile (DeltaSNGFR/DeltaV(LP)) were all significantly reduced by blocking 5'-nucleotidase. When adenosine activity was clamped by combining 5'-nucleotidase inhibitor with A1-agonist to determine whether TGF requires adenosine to be present or to fluctuate, the TGF slope was reduced by 83%, indicating that adenosine activity must fluctuate for normal TGF to occur and that adenosine is a mediator of TGF.

Sodium reabsorption in thick ascending limb of Henle's loop: effect of potassium channel blockade in vivo.

1. Based on previous in vitro studies, inhibition of K(+) recycling in thick ascending limb (TAL) is expected to lower Na(+) reabsorption through (i) reducing the luminal availability of K(+) to reload the Na(+)-2Cl(-)-K(+) cotransporter and (ii) diminishing the lumen positive transepithelial potential difference which drives paracellular cation transport. 2. This issue was investigated in anaesthetized rats employing microperfusion of Henle's loop downstream from late proximal tubular site with K(+)-free artificial tubular fluid in nephrons with superficial glomeruli. 3. The unselective K(+) channel blocker Cs(+) (5 - 40 mM) dose-dependently increased early distal tubular delivery of fluid and Na(+) with a maximum increase of approximately 20 and 185%, respectively, indicating predominant effects on water-impermeable TAL. 4. The modest inhibition of Na(+) reabsorption in response to the 15 mM of Cs(+) but not the enhanced inhibition by 20 mM Cs(+) was prevented by luminal K(+) supplementation. Furthermore, pretreatment with 20 mM Cs(+) did not attenuate the inhibitory effect of furosemide (100 microM) on Na(+)-2Cl(-)-K(+) cotransport. 5. Neither inhibitors of large (charybdotoxin 1 microM) nor low (glibenclamide 250 microM; U37883A 100 microM) conductance K(+) channels altered loop of Henle fluid or Na(+) reabsorption. 6. The intermediate conductance K(+) channel blockers verapamil and quinine (100 microM) modestly increased early distal tubular Na(+) but not fluid delivery, indicating a role for this K(+) channel in Na(+) reabsorption in TAL. As observed for equieffective concentrations of Cs(+) (15 mM), Na(+) reabsorption was preserved by K(+) supplementation. 7. The results indicate that modest inhibition of K(+) channels lowers the luminal availability of K(+) and thus transcellular Na(+) reabsorption in TAL. More complete inhibition lowers paracellular Na(+) transport probably by reducing or even abolishing the lumen positive transepithelial potential difference. Under the latter conditions, transcellular Na(+) transport may be restored by paracellular K(+) backleak.

Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice.

To examine the role of aquaporin-1 (AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we compared osmolalities in micropuncture samples of late proximal tubular fluid and plasma in wild-type (+/+) and AQP1-knockout (-/-) mice. Compared with matched wild-type mice, the -/- animals produce a relatively hypotonic urine (607 +/- 42 vs. 1,856 +/- 101 mosmol/kgH(2)O) and have a higher plasma osmolality under micropuncture conditions (346 +/- 11 vs. 318 +/- 5 mosmol/kgH(2)O; P < 0.05). Measurements of tubular fluid osmolality were done in three groups of mice, +/+, -/-, and hydrated -/- mice in which plasma osmolality was reduced to 323 +/- 1 mosmol/kgH(2)O. Late proximal tubular fluid osmolalities were 309 +/- 5 (+/+, n = 21), 309 +/- 4 (-/-, n = 24), and 284 +/- 3 mosmol/kgH(2)O (hydrated -/-, n = 19). Tubular fluid chloride concentration averaged 152 +/- 1 (+/+), 154 +/- 1 (-/-), and 140 +/- 1 mM (hydrated -/-). Transtubular osmotic gradients in untreated and hydrated AQP1 -/- mice were 39 +/- 4 (n = 25) and 39 +/- 3 mosmol/kgH(2)O (n = 19), values significantly higher than in +/+ mice (12 +/- 2 mosmol/kgH(2)O; n = 24; both P < 0.001). AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules, resulting from the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1.

Postglomerular vasoconstriction induced by dopamine D(3) receptor activation in anesthetized rats.

In the present study we investigated the renal hemodynamic effects of dopamine D(3) receptor activation by R(+)-7-hydroxy-dipropylaminotetraline (7-OH-DPAT) in thiopental-anesthetized Sprague-Dawley rats. In clearance experiments infusion of 7-OH-DPAT (0.01-1.0 microg. kg(-1). min(-1)) dose-dependently elevated glomerular filtration rate (GFR) without affecting mean arterial blood pressure (MAP). In renal blood flow experiments 7-OH-DPAT infusion (1.0 microg. kg(-1). min(-1)) increased GFR by 16 +/- 2%, associated with an unexpected fall in renal blood flow by 20 +/- 3% and a significant elevation of renal vascular resistance by 18 +/- 3%. The renal hemodynamic changes were not influenced by pretreatment with the D(2)-receptor antagonist S(-)-sulpiride but were completely abolished during D(3) receptor inhibition by 5,6-dimethoxy-2-(di-n-propylamino)indane (U-99194A). In micropuncture experiments 7-OH-DPAT (1.0 microg. kg(-1). min(-1)) significantly elevated stop-flow pressure measured in the early proximal tubules and reduced hydrostatic pressure at the first branching point of the efferent arteriole without altering MAP. We conclude from these data that pharmacological activation of dopamine D(3) receptors affects renal hemodynamics in anesthetized rats by preferential postglomerular vasoconstriction.

Role of Na(+)/H(+) exchanger NHE3 in nephron function: micropuncture studies with S3226, an inhibitor of NHE3.

Na(+)/H(+) exchanger NHE3 is expressed in the luminal membrane of proximal tubule and thin and thick ascending limb of Henle's loop. To further define its role, the novel NHE3 inhibitor S3226 was employed in micropuncture experiments in nephrons with superficial glomeruli of anesthetized rats. Microperfusion of proximal convoluted tubule with S3226 revealed a dose-dependent inhibition of reabsorption (IC(50) of 4-5 microM) with a maximum inhibition of 30% for fluid and Na(+). During microperfusion of Henle's loop (last superficial proximal to first superficial distal tubular loop), no effect of S3226 (10 or 30 microM) on the reabsorption of fluid or Na(+) was observed. Finally, S3226 (30 microM) left the tubuloglomerular feedback response unaltered as determined by the fall in proximal tubular stop-flow pressure in response to increasing loop of Henle perfusion rate. These studies indicate that NHE3 significantly contributes to fluid and Na(+) reabsorption in proximal convoluted tubule. NHE3 appears not to significantly contribute to fluid or Na(+) reabsorption in the loop of Henle (including the S3 segment of proximal tubule) or macula densa control of nephron filtration.

Glomerular hyperfiltration in experimental diabetes mellitus: potential role of tubular reabsorption.

An increase in Na+/glucose cotransport upstream to the macula densa might contribute to the increase in single nephron GFR (SNGFR) in early diabetes mellitus by lowering the signal of the tubuloglomerular feedback, i.e., the luminal Na+, Cl-, and K+ concentration sensed by the macula densa. To examine this issue, micropuncture experiments were performed in nephrons with superficial glomeruli of streptozotocin-induced diabetes mellitus in rats. First, in nondiabetic control rats, ambient early distal tubular concentrations of Na+, Cl-, and K+ were about 21, 20, and 1.2 mM, respectively, suggesting collection sites relatively close to the macula densa. Second, glomerular hyperfiltration in diabetic rats was associated with a reduction in ambient early distal tubular concentrations of Na+, Cl-, and K+ by 20 to 28%, reflecting an increase in fractional reabsorption of these ions up to the early distal tubule. Third, in diabetic rats, early proximal tubular application of phlorizin, an inhibitor of Na+/glucose cotransport, elicited (1) a greater reduction in absolute and fractional reabsorption of Na+, Cl-, and K+ up to the early distal tubule, and (2) a greater increase in early distal tubular concentration of these ions, which was associated with a more pronounced reduction in SNGFR. These findings support the concept that stimulation of tubular Na+/glucose cotransport by reducing the tubuloglomerular feedback signal at the macula densa may contribute to glomerular hyperfiltration in diabetic rats. Glomerular hyperfiltration in diabetic rats serves to compensate for the rise in fractional tubular reabsorption to partly restore the electrolyte load to the distal nephron.

Eukaliuric diuresis and natriuresis in response to the KATP channel blocker U37883A: micropuncture studies on the tubular site of action.

1. Systemic application of U37883A, a blocker of ATP sensitive potassium (KATP) channels, elicits diuresis and natriuresis without significantly altering urinary potassium excretion. 2. To elucidate tubular sites of action upstream to the distal nephron, micropuncture experiments were performed in nephrons with superficial glomeruli of anaesthetized Munich-Wistar-Frömter rats during systemic application of U37883A (1, 5 or 15 mg kg-1 i.v.). 3. The observed eukaliuric diuresis and natriuresis in response to U37883A at 15 mg kg-1 was accompanied by an increase in early distal tubular flow rate (VED) from 10 - 18 nl min(-1) reflecting a reduction in fractional reabsorption of fluid up to this site (FR-fluid) of 13%. The latter proposed an effect on water-permeable segments such as the proximal tubule which could fully account for the observed reduction in fractional reabsorption of Na+ up to the early distal tubule (FR-Na+) of 8% and the increase in early distal tubular Na+ concentration ([Na+]ED) from 35 - 51 mM whereas [K+]ED was left unaltered. 4. In comparison, furosemide (3 mg kg-1 i.v.), which acts in the water-impermeable thick ascending limb, elicited diuresis, natriuresis and kaliuresis which were associated with a fall in FR-Na+ of 10% with no change in FR-fluid, and a rise in [Na+]ED from 42 - 117 mM and [K+]ED from 1.2 - 5.7 mM with no change in VED. 5. Direct late proximal tubular fluid collections confirmed a significant inhibition of fluid reabsorption in proximal convoluted tubule in response to systemic application of U37883A. 6. These findings suggest that the diuretic and natriuretic effect upstream to the distal tubule in response to systemic application of U37883A involves actions on water-permeable segments such as the proximal convoluted tubule.

Immunolocalization of protein kinase C isoenzymes alpha, beta1 and betaII in rat kidney.

Protein kinase C (PKC) significantly contributes to the control of renal function, but little is known about the renal function or localization of PKC isoenzymes. Therefore, the localization of PKC isoenzymes alpha, betaI, and betaII was studied in rat kidney. Immunoblot analysis identified immunoreactive bands corresponding to PKC a, betaI, and betaII in total cell extracts of both renal cortex and medulla. Immunohistochemistry using confocal laser scanning microscopy revealed immunostaining for PKC alpha within the glomerulus including podocytes and mesangial cells. PKC betaI was detected in mesangial cells, whereas anti-PKC betaII labeled neither podocytes nor mesangial cells. PKC betaII, however, was detected in cells within the mesangial area, which expressed MHC II, a marker for antigen-presenting cells. None of the three isoforms was detected in glomerular endothelial cells. A prominent immunostaining with anti-PKC alpha and betaI was localized to the brush border of S2 and S3 segments of proximal tubule, whereas S 1 segments were not stained. Along the loop of Henle, both PKC a and PKC betaI were found in the luminal membrane of cortical and medullary thick ascending limb. In addition, anti-PKC betaI labeled the luminal membrane of thin limbs. In the cortical collecting duct (CCD), immunofluorescence for PKC alpha was observed at the apical membrane of both peanut agglutinin (PNA)-negative cells and part of PNA-positive cells, whereas in the medullary collecting duct (MCD), PKC a was detected at the basolateral membrane. In comparison, PKC betaI was localized at the luminal membrane of PNA-positive cells only in CCD and at the luminal membrane of MCD. Unlike PKC a or betaI, there was (1) no detectable immunostaining with anti-PKC betaII in the proximal tubule, the loop of Henle, or the CCD and (2) a distinct staining for PKC betaII of interstitial cells in cortex and medulla (including MHC II-positive dendritic cells). Furthermore, PKC betaII was detected in the luminal membrane of MCD. In summary, a distinct and differential expression pattern for PKC alpha, betaI, and betaII was shown in rat kidney, which may contribute to a better understanding of the specific role of these isoenzymes in the control of renal function.