Survey of tissues
Since urea transporters are expressed in numerous extra-renal tissues, a comprehensive pathological survey of numerous tissues from UT-A1/3−/− mice was performed to determine if any abnormalities were present. Observations were made from 33 tissues (see Methods for full list) by both gross (wet weight) and microscopic anatomy from three age-matched UT-A1/3−/− or wildtype mice. Except for the kidney and testis, no abnormalities were apparent. When normalized by brain weight, the kidneys were significantly smaller and the testis significantly larger in knockout animals compared to controls (). However, microscopic analysis could not detect any differences in cell number, cell type or morphology. Resected kidneys of UT-A1/3−/− mice also had greater blood congestion than kidneys from wild-type animals (representative images are shown in ), especially in the renal medulla ().
Blood congestion in kidneys of UT-A1/3−/− mice. Representative images of whole kidney and inner medulla from wildtype (A and C) or UT-A1/3−/− mice (B and D) show greater blood congestion in knockout mice.
Urinary NO excretion
Measurements of total urinary nitrate and nitrite (NOx) excretion rates under basal conditions on a 20% protein diet (24 hour urine collections) were performed to determine if the blood congestion observed in UT-A1/3−/− mice kidneys may be related to abnormal nitric oxide (NO) production by the kidney. In knockout mice, NOx excretion (127.0 ± 7.8 nmol/gBW/day) was more than double that in wildtype controls (52.5 ± 9.5 nmol/gBW/day) ().
Figure 2 Twenty-four hour total urinary nitrate and nitrite (NOx) excretion. Values are means ± SE, n = 6. Significant difference (p<0.01) between the groups is indicated by *. UT-A1/3−/− mice (solid bars) have a significantly greater (more ...)
Circulating vasopressin levels
Another factor that could alter renal blood flow dynamics is vasopressin. Consequently, we measured plasma vasopressin levels in UT-A1/3−/− and wildtype mice. Under basal conditions (free access to water) on a 20% protein diet, we observed no difference in plasma vasopressin levels between the groups ().
Plasma vasopressin levels of wildtype (clear bars) and UT-A1/3−/− mice (solid bars) under basal conditions. Values are means ± SE; n=3.
Total renal blood flow
Since greater blood congestion was observed in the kidneys of UT-A1/3−/− mice, we carried out measurements of total renal blood flow in anesthetized mice () using an ultrasonic flow probe. Total renal blood flow in the UT-A1/3−/− mice was not significantly different from that observed in age-matched wildtype control mice. Inhibition of NO production by infusion of L-NAME resulted in a significant decrease in total renal blood flow in both knockout mice and controls. However, the percentage decrease due to L-NAME was significantly greater in UT-A1/3−/− mice (39.2 ± 2.4% versus 27.1 ± 4.9%), suggesting a greater role for NO in the maintenance of blood flow.
Figure 4 Total renal blood flow (RBF) in wildtype (clear bars) and UT-A1/3−/− mice (solid bars). Values are means ± SE. Administration of NG-Nitro-L-arginine Methyl Ester Hydrochloride (L-NAME, 1μg/g bodyweight) statistically (ANOVA) (more ...)
Glomerular filtration rate (GFR)
We determined GFR in conscious UT-A1/3−/− and wildtype mice on two levels of protein intake, 4% and 40%, using FITC-inulin clearance (). Increasing the protein content of the diet more than doubled the FITC-inulin clearance in both UT-A1/3−/− mice and wildtype controls ( and ). However, no significant differences were observed in inulin clearance between UT-A1/3−/− and wildtype mice under either dietary condition, even when corrected for bodyweight ( and ).
Figure 5 Glomerular filtration rate (GFR) in conscious male mice. FITC-inulin clearance for wildtype (clear bars) and UT-A1/3−/− mice (solid bars) was determined by a microosmotic minipump method. Representative values are mean ± SE and (more ...)
Urinary concentrating ability, plasma electrolytes and urinary excretion rates
Urinary excretion values
A summary of the excretion of water, monovalent cations and urea in wildtype and UT-A1/3−/− mice on a low (4%) or high (40%) protein diet is shown in . Urine volume was greater in UT-A1/3−/− mice only on the high protein diet, resulting in a marked decrease in the urine/plasma (U/P) inulin ratio and a corresponding reduction in urinary osmolality. However, there were no differences in osmolar excretion in UT-A1/3−/− mice versus wildtype controls.
On a high protein diet, plasma urea concentration was significantly lower in UT-A1/3−/− mice than in wildtype controls (). Plasma concentrations of Na, K, and Cl were not different between UT-A1/3−/− mice and controls.
Fractional urea excretion (FEurea) was markedly elevated in UT-A1/3−/− versus wildtype controls on both the 4% and 40% protein diets. In fact, FEurea reached 102.4 ± 8.8% of the filtered urea in the UT-A1/3−/− mice on the 40% protein diet, a level that may be indicative of net active urea secretion along the renal tubule (see Discussion). Fractional excretion rates of sodium and potassium were not significantly different in UT-A1/3−/− mice versus wildtype controls.
Urinary concentrating ability of UT-A1/3−/− mice
mice have a urinary concentrating defect that is dependent on the level of urea excretion [4
]. Since the main determinant of urea excretion rate is protein intake, in the present study, the effects of three different dietary protein intakes on urinary concentrating ability were determined. Initial observations were made without restriction of water intake (). On a low protein intake (4% protein diet), there were no significant differences in fluid consumption, urine flow or urine osmolality between wildtype and UT-A1/3−/−
mice. However, on a normal protein intake (20% protein diet), UT-A1/3−/−
mice exhibited significantly greater fluid consumption and urine flow than wildtype mice, resulting in a decreased urine osmolality. This decrease in urinary concentrating ability was even greater on a high protein intake (40% protein diet). Furthermore, after an 18hr water restriction (2ml of water per day per 20gBW), UT-A1/3−/−
mice on either a 20% or 40% protein diet (but not a 4% protein diet) were unable to reduce their urine flow and could not raise their maximal urinary osmolality above that observed under basal conditions (data not shown). During this 18hr water restriction, the body weight of UT-A1/3−/−
mice on a 20% or 40% protein diet decreased by 18.2 ± 0.4% and 24.6% ± 0.3%, respectively. In contrast, UT-A1/3−/−
mice on a 4% protein diet were able to maintain fluid balance without a marked loss of body weight (3.0 ± 0.2%).
Figure 6 Water conservation and urinary concentrating ability of UT-A1/3−/− mice. For all graphs, values are mean ± SE and a significant difference (ANOVA) between wild-type mice (clear bars) and UT-A1/3−/− mice (solid bars) (more ...)
Expression of sodium transporters and aquaporins
The greater urine flow in UT-A1/3−/−
mice can be explained by a urea-dependent osmotic diuresis [4
]. We hypothesized that changes in the expression of other channels and transporters may, in part, compensate for the observed polyuria. Therefore, we examined the abundance (semiquantitative immunoblotting) of the major Na transporters and aquaporins in kidneys from wildtype and UT-A1/3−/−
mice either under normal conditions, where animals had free-access to water, or after water restriction.
Under basal conditions without water restriction, apart from a significant reduction in the expression of the type 2 Na-dependent phosphate transporter (NaPi-2), no other major differences in Na transporter abundances were observed between the groups (). However, after 36 hr of water restriction, the abundances of both the thiazide-sensitive Na-Cl cotransporter (NCC) and all three subunits of ENaC were significantly greater in knockout animals compared to controls (). In contrast, the expression of NaPi-2 was further reduced in knockout animals to a level that was virtually undetectable by immunoblotting.
Figure 7 Immunoblots assessing sodium transporter and sodium channel abundances in whole kidney homogenates from wildtype and UT-A1/3−/− mice. Mice received either free access to water (control) or were water restricted for 36hrs. Each lane was (more ...)
Under basal conditions without water restriction (), UT-A1/3−/− mice had greater aquaporin 3 expression levels than wildtype controls. After water restriction, the abundances of both aquaporin 2 and aquaporin 3 increased in both UT-A1/3−/− and wildtype mice, however, the increase in abundances of aquaporins 2 and 3 were greater in knockout mice (). Thus, the concentrating defect in UT-A1/3−/− mice cannot be attributed to a defect in long-term regulation of aquaporin 2 or 3.
Figure 8 Immunoblots assessing aquaporin water channel abundances in whole kidney homogenates from wildtype and UT-A1/3−/− mice. Mice received either free access to water (control) or were water restricted for 36hrs. Each lane was loaded with a (more ...)
Medullary solute gradients
Changes in urea transport in the collecting duct and changes in NO production in the kidney could both have potential effects on corticomedullary solute gradients. Consequently, we carried out analysis of tissue solute composition in dissected cortex, outer medulla, and two levels of inner medulla in UT-A1/3−/− and wildtype mice on 4% and 40% protein diets. In knockout mice on a high protein intake, there was a marked depletion of both osmolality and urea in the inner medulla compared to controls (). However, there were no differences between UT-A1/3−/− mice and wildtype control mice in Na concentrations measured in cortex, outer medulla, or inner medulla (). In addition, tissue K concentration was not different between UT-A1/3−/− and wildtype mice (not shown). Furthermore, the Na concentration profiles were virtually identical when mice on a 4% diet are compared to mice on a 40% protein diet. Thus, sodium accumulation in both the outer and inner zones of the kidney medulla appears to occur largely independently of tissue urea concentrations.
Figure 10 Comparison of kidney solute composition of wild-type and UT-A1/3−/− mice. For all graphs, values are mean ± SE and a significant difference (ANOVA) between wild-type mice (clear bars) and UT-A1/3−/− mice (solid (more ...)