1Institute of Pharmacology and Toxicology, University of Tübingen, 72074 Tübingen; 2Max-Planck-Institute for Experimental Endocrinology, 30625 Hannover, Germany; and 3Departments of Medicine and Pharmacology, University of California, and Veterans Affairs Medical Center, San Diego, California 92161
Submitted 4 August 2003 ; accepted in final form 18 March 2004
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ABSTRACT |
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mouse; collecting duct; knockout
PKC-, which is a member of the "conventional" PKC class, appears to be one of the predominant isoenzymes in the kidney (9, 11, 27, 29). Employing immunohistochemistry and confocal laser-scanning microscopy in the mouse kidney, PKC-
was localized to glomeruli, intercalated cells of the cortical collecting duct, as well as the medullary collecting duct (31a). Gene knockout can be a powerful approach to delineate respective gene function, and therefore, using homologous recombination, we recently generated a mouse that is deficient for PKC-
(22). Despite the fact that PKC-
is presumed to be the most ubiquitously expressed PKC isoenzyme, the PKC-
knockout mouse appears normal with regard to external characteristics, viability, and fertility. A preliminary closer look revealed that insulin signaling to insulin receptor substrate 1-dependent phosphoinositide 3-kinase, PKB, and PKC-
and downstream processes like glucose transport and activation of extracellular signal-regulated kinases (ERK) are enhanced in skeletal muscles and adipocytes from PKC-
knockout mice. Serum glucose or insulin levels, however, were not significantly different in fed PKC-
knockout mice compared with fed control PKC-
wild-type mice (22). Here, we used PKC-
knockout mice to gain insights into the role of PKC-
in kidney function.
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METHODS |
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Metabolic cage experiments in conscious mice. To assess basal renal function, mice had free access to tap water and a standard diet (2.5 g Na+/kg). Mice were placed in metabolic cages (Tecniplast, Hohenpeissenberg, Germany) to assess urinary excretion of fluid and sodium as well as urinary osmolality. After adaptation of the mice to metabolic cages over a period of 3 days, a 24-h urine collection was performed. To ensure quantitative urine collection, metabolic cages were siliconized, and urine was collected under water-saturated oil. The concentration of Na+ in urine was determined using a flame photometer (ELEX 6361, Eppendorf, Hamburg, Germany) and osmolality by freezing-point depression. The concentrations of albumin and vasopressin were determined using a commercial ELISA kit for mouse albumin (CellTrend, Luckenwalde, Germany) or a commercial RIA kit for vasopressin (IBL, Hamburg, Germany), respectively. After urine collection was completed, the mice were anesthetized with ether and blood was drawn from the retrobulbar plexus for determination of hematocrit and plasma osmolality.
Twenty-four-hour urine collections were performed after an adaptation period of 2 days under a standard sodium diet (2.5 g Na+/kg) and, subsequently, in response to a sodium-deficient diet (0 g Na+/kg) for 6 days.
Urinary flow rate and urinary osmolality were determined in conscious mice in response to 1) 36-h water deprivation with urine collection being performed over the last 14 h of water deprivation; 2) application of the vasopressin V2-receptor antagonist SR-121463 (1 mg/kg ip), which in the rat induces diuresis through internalization of aquaporin-2 from the luminal membrane and subsequent inhibition of water transport in medullary collecting duct (14); and 3) acute water loading (1 ml/16 g body wt) using a gastric tube. In the latter experiments, access to food had been withdrawn 4 h before to facilitate water application. To prevent contamination with the concentrated urine being present in the bladder before administration of SR-121463 or water loading, osmolality was measured in the second portion of spontaneously voided urine after drug application or water loading, respectively.
Blood pressure measurements and clearance experiments in anesthetized mice. As described before (3436, 39), mice were anesthetized for terminal clearance experiments (100 mg/kg Inactin ip and 100 mg/kg ketamine im). After every hour, 20% of this dose was readministered intraperitoneally to maintain anesthesia until euthanization. The femoral artery was cannulated for blood pressure measurement and blood sample withdrawal. The jugular vein was cannulated for a continuous maintenance infusion of 2.25 g/dl BSA in 0.9% NaCl at a rate of 0.35 ml/h. For assessment of glomerular filtration rate (GFR), [3H]inulin was added to deliver 20 µCi/h. After surgery, the mice were allowed to stabilize for 60 min. Then, a timed urine collection was performed for 60 min using a bladder catheter. Blood was withdrawn at the beginning and end of the collection period to determine [3H]inulin. Urine was analyzed for [3H]inulin. Before the mice were euthanized by intravenous application of saturated KCl at the end of the clearance experiment, the kidneys were excised and decapsulated and the wet weight was determined. After the kidneys were dried overnight at 50°C, the dry weight was determined.
Aquaporin-2 expression in inner medulla and thickness of inner medulla.
Semiquantitative Western blot analysis was adapted from the rat (14, 29, 30) to the mouse. PKC- +/+ and / mice (n = 5/group) were anesthetized (ketamine, 60 mg/kg ip and xylazine, 9 mg/kg ip). After opening of the abdominal cavity, the renal artery and vein of the left kidney were clamped and the kidney was rapidly excised. The inner medulla was dissected and immediately immersed in liquid nitrogen for later Western blot analysis. Subsequently, the left cardiac ventricle was exposed. The tip of the perfusion system was inserted into the left ventricle, and the arterial system was perfused for 1 min with 35 ml PBS to clear the kidney of blood and subsequently for 10 min with 1015 ml of a fixation solution (2% paraformaldehyde and 3% sucrose in phosphate buffer). Both solutions were at room temperature (RT). Then, the right kidney was removed for morphological examination.
For determination of aquaporin-2 expression in the inner medulla by Western blotting, total cellular proteins of the inner medulla of the left kidney were obtained by pulverizing the tissue and dissolving the powder in lysis buffer. Homogenization was followed by centrifugation (1,000 g, 10 min, 4°C) to remove nuclei and unbroken cells, and the supernatant was used for further analysis. Protein content was determined by the method of Bradford using a commercial protein assay and bovine serum albumin as a standard. Samples were diluted 1:3 with Roti-Load sample buffer and boiled for 10 min at 65°C. The following steps were performed at RT. Samples of protein (10 µg) were subjected to SDS-PAGE using 12% acrylamide gels in a Mini-PROTEAN II Electrophoresis Cell. For determination of molecular mass, a prestained protein ladder was used. After gel electrophoresis (60 mA/gel, 70 min), proteins were transferred to polyvinylidene difluoride membranes of 0.45-µm pore size. Membranes were blocked for 90 min with blocking buffer and rinsed twice with TBS containing 0.1% Tween 20 (TBST). Thereafter, the aquaporin-2 antibodies (0.5 µg/ml) were incubated overnight in TBST. The secondary alkaline phosphate-conjugated antibody was incubated at a concentration of 0.1 µg/ml for 2 h at RT in TBST. Blots were rinsed twice with TBST and washed three times for 15 min with TBST, then twice in assay buffer at RT. Development was done with CDP-Star ready-to-use solution. Immunoreactive bands were visualized by chemiluminescence system with CSC1.1 program software and then quantitated by TINA program (Raytest, Straubenhardt, Germany).
For determination of inner medulla thickness and morphology, the perfusion-fixated right kidney was cut into slices (3 mm in thickness) displaying cortex and outer and inner medulla and incubated for 3 h in the fixation at 4°C. After rinsing in PBS for 15 min, kidney slices were dehydrated in 30% sucrose in PBS overnight at 4°C. Thereafter, kidney slices were frozen in isopentane precooled by liquid nitrogen and stored at 80°C until further use. Cryosections of 10 µm were made at 20°C and transferred onto gelatin-coated glass slides. Cortex and inner medulla thicknesses were determined in the maximal cross-sectional area of frontal sections, and 10 sections/animal in all were evaluated (20).
Statistical methods.
Results are presented as means ± SE. The statistical significance of differences between PKC- / and PKC-
+/+ mice was assessed by an unpaired Student's t-test.
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RESULTS |
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Basal kidney function in clearance experiments in anesthetized mice.
Mean arterial blood pressure (see Fig. 3) and heart rate (375 ± 13 vs. 391 ± 12 beats/min; n = 8/group, NS) were not different between PKC- / and +/+ mice. PKC-
-deficient mice, however, exhibited a modestly lower GFR, which was associated with an enhanced absolute and fractional fluid excretion (see Fig. 3) and a modestly lower kidney wet weight (0.443 ± 0.017 vs. 0.497 ± 0.013 g for 2 kidneys; n = 8/group; P < 0.05) and kidney dry weight (see Fig. 3), such that GFR per gram kidney weight was preserved (3.1 ± 0.2 vs. 3.2 ± 0.2 ml·min1·g kidney dry wt1, NS).
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Response to a sodium-deficient diet.
As illustrated in Fig. 4, urinary sodium excretion was not different under a standard diet (day 0). Dietary sodium restriction for 6 days also did not reveal significant differences in urinary sodium excretion or in body weight change between groups. The greater urine flow rate and lower urinary osmolality, however, persisted in PKC- / mice over the 6 days of sodium restriction (data not shown).
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DISCUSSION |
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The greater urinary flow rate and lower urinary osmolality observed under basal conditions in PKC- / mice were associated with a greater urinary vasopressin-to-creatinine ratio as determined from 24-h urine collection in metabolic cages and a tendency for greater plasma osmolality. The urinary vasopressin-to-creatinine ratio has been used before as a surrogate parameter for mean daily plasma vasopressin concentration in mice (2) and was also used in the present study because precise measurements of plasma vasopressin in mice were not possible due to the relatively large volume of plasma required for commercial assays and that could not be obtained by decapitation of the mice. A tendency for greater plasma osmolality and a greater urinary vasopressin-to-creatinine ratio, however, are not compatible with a greater intake of water or a reduced central release of vasopressin as the primary cause of greater renal water excretion in PKC-
/ mice. In accordance, a significantly lower urinary osmolality persisted in PKC-
/ mice compared with PKC-
+/+ mice during 36-h water deprivation. This finding shows that the phenotype is independent of water intake and also indicates a lower urinary concentrating ability in PKC-
/ mice.
Clearance experiments established that the modestly greater urine flow rate in PKC- / mice was in fact the consequence of reduced fluid reabsorption in the kidney. The question remained of which segment of the tubular and collecting duct system contributed to this phenotype. Notably, the dilution of urine was not impaired in PKC-
/ mice as indicated by experiments with acute water loading and application of a vasopressin V2-receptor antagonist. Because intact function of the thick ascending limb is required for not only urine concentration but also hypotonic dilution, these findings argue against a significant primary defect in PKC-
/ mice in this nephron segment. Furthermore, the ability of the kidney to retain sodium in response to dietary sodium restriction was unaffected in PKC-
/ mice, arguing against a significant defect in the major sodium-retaining segments of the nephron, including the proximal tubule, thick ascending limb, distal tubule, and cortical collecting duct. This leaves the medullary collecting duct as the most likely candidate for the defective nephron segment in PKC-
/ mice, and this notion matches perfectly the renal localization of the isoenzyme in PKC +/+ mice: PKC-
is highly expressed in the medullary collecting duct, whereas it is not detectable in the proximal tubule, thick ascending limb, distal tubule, or principal cells of the cortical collecting duct (31a).
The observed modestly lower GFR may be part of a water-conserving/compensating mechanism in PKC- / mice. Alternatively, Bankir and colleagues (6) have proposed before that vasopressin-mediated urinary concentrating mechanisms are related to GFR, with high concentrating activity being related to high GFR and vice versa. Thus the lower GFR observed in PKC-
/ mice may be related to an impaired urinary concentrating ability independent of body fluid loss. The modestly lower kidney wet and dry weight, on the other hand, may be the consequence of the lower GFR, which requires less absolute salt reabsorption and is in accordance with the preserved GFR-to-kidney weight ratio. Furthermore, PKC-
/ mice did not present significant differences in the ratio of the thickness of the inner medulla vs. the cortex compared with PKC-
+/+ mice or alterations in the morphology of the inner medulla, which could have explained differences in urinary concentration.
The mechanism of how PKC- can potentially contribute to urinary concentration in the medullary collecting duct, i.e., what target molecules are phosphorylated and what activates PKC-
, remains to be determined. Using semiquantitative immunoblotting, we observed no significant differences in the expression of aquaporin-2 in the inner medulla between PKC-
/ vs. PKC-
+/+ mice. Interestingly, there are reports on rabbit and rat collecting duct that PKC in this nephron segment can mediate an inhibitory influence on water channels and transport (1, 13, 32), i.e., an influence that is opposite of the one proposed here for PKC-
in mouse medullary collecting duct. On the other hand, a study by Kato et al. (19) in rat terminal inner medullary collecting duct showed that angiotensin II increases vasopressin-stimulated facilitated urea permeability via a PKC-mediated signaling pathway, and the authors proposed that this may play a physiological role in the urinary concentrating mechanism by augmenting the maximal response to vasopressin. It remains to be determined whether vasopressin-stimulated facilitated urea permeability is affected in PKC-
/ mice.
Notably, neither PKC-, -
I, -
II, -
, nor -
was detected in principal cells of the cortical collecting duct in mice (31a), and therefore the identification and functional characterization of the PKC isoenzyme expressed at this site will be of significant interest. Furthermore, it is of interest that besides PKC-
, PKC-
I and -
II, which also belong to the group of conventional PKC isoenzymes, are similarly expressed in the medullary collecting duct of the mouse (31a). The fact that PKC-
knockout mice show a phenotype that presumably is localized to the medullary collecting duct indicates that classic PKC isoenzymes cannot fully compensate for each other in vivo even though they are expressed within the same cell.
Besides the medullary collecting duct, PKC- in mouse kidney is expressed in intercalated cells of the cortical collecting duct and in glomeruli. In the present studies, we did not follow up on the potential role of PKC-
in intercalated cells. Because PKC-
expression is enhanced in mesangial cells by high glucose and in whole rat kidney in response to streptozotocin-induced diabetes mellitus and because there is evidence for a link between glomerular PKC and albuminuria (7, 17, 30), we determined urinary albumin excretion under basal conditions but could not detect differences between PKC-
knockout and wild-type mice. Whether glomerular PKC-
plays a role in albuminuria under pathophysiological conditions remains to be determined.
In summary, evidence is provided that PKC-, which is expressed in the medullary collecting duct, is involved in urinary concentration in mice. Considering that PKC-
I and -
II are coexpressed with PKC-
in mouse medullary collecting duct, the observed phenotype of PKC-
knockout mice in urinary concentration indicates that conventional PKC isoenzymes cannot fully compensate for each other.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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