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Macrophage inflammatory protein 1α (MIP-1α)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain ofCryptococcus neoformans. The present study evaluated the interaction of MIP-1α with other innate immune system cytokines by comparing the immune responses that followed pulmonary infections with high- (C. neoformans145A) and low (C. neoformans52D)-virulence strains. In contrast to what was found forC. neoformans145A infection, lack of MIP-1α inC. neoformans52D infection did not cause the development of EP.C. neoformans52D induced tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1α knockout (KO) mice by day 7 postinfection. Both WT and MIP-1α KO mice subsequently cleared this infection. Thus, the robust expression of early inflammatory cytokines inC. neoformans52D-infected mice promoted the development of protective immunity even in the absence of MIP-1α. Alternatively,C. neoformans145A-infected WT and MIP-1α KO mice had diminished TNF-α, IFN-γ, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulentC. neoformans145A evaded early innate host defenses. HoweverC. neoformans145A-infected WT mice had an early induction of MIP-1α and subsequently did not develop EP. In contrast,C. neoformans145A-infected MIP-1α KO mice developed EP and had increasedC. neoformansdissemination into the brain by day 35. We conclude that, in the absence of other innate immune response effector molecules, MIP-1α is crucial to prevent the development of EP and to controlC. neoformansdissemination to the brain.

Leukocyte recruitment to the site of infection by the encapsulated yeast Cryptococcus neoformans is critical for clearance of the infection. We review data from our lab that chemokines, such as the CC chemokines MCP-1 and MIP-1alpha, are important mediators of leukocyte recruitment during C. neoformans infection. In addition, studies in CC chemokine receptor knockout mice have demonstrated that CCR2 and CCR5 are required not only for leukocyte recruitment but also for other aspects of immune response development and innate imunity to C. neoformans.

The Na/H exchanger isoform 3 (NHE3) is expressed in the proximal tubule and thick ascending limb and contributes to the reabsorption of fluid and electrolytes in these segments. The contribution of NHE3 to fluid reabsorption was assessed by micropuncture in homozygous ( Nhe3−/−) and heterozygous ( Nhe3+/−) knockout mice, and in their wild-type (WT, Nhe3+/+) littermates. Arterial pressure was lower in the Nhe3−/−mice (89 ± 6 mmHg) compared with Nhe3+/+(118 ± 4) and Nhe3+/−(108 ± 5). Collections from proximal and distal tubules demonstrated that proximal fluid reabsorption was blunted in both Nhe3+/−and Nhe3−/−mice (WT, 4.2 ± 0.3; Nhe3+/−, 3.4 ± 0.2; and Nhe3−/−, 2.6 ± 0.3 nl/min; P < 0.05). However, distal delivery of fluid was not different among the three groups of mice (WT, 3.3 ± 0.4 nl/min; Nhe3+/−, 3.3 ± 0.2 nl/min; and Nhe3−/−, 3.0 ± 0.4 nl/min; P < 0.05). In Nhe3−/−mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 ± 0.9 nl/min in the Nhe3+/+vs. 6.6 ± 0.8 nl/min in the Nhe3−/−, measured distally. Proximal-distal SNGFR differences in Nhe3−/−mice indicated that much of the decrease in SNGFR was due to activation of tubuloglomerular feedback (TGF), and measurements of stop-flow pressure confirmed that TGF is intact in Nhe3−/−animals. In contrast to Nhe3−/−mice, normalization of early distal flow rate in Nhe3+/−mice was not related to decreased SNGFR (9.9 ± 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment ( Nhe3+/+, 2.6 ± 0.2; Nhe3+/−, 3.6 ± 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating Na+and fluid reabsorption in the proximal tubule. In animals with NHE3 deficiency, normalization of fluid delivery to the distal tubule is achieved through alterations in filtration rate and/or downstream transport processes.

In these experiments, we used a strain of angiotensin converting enzyme (ACE) germline null mutant mice, generated by J. H. Krege and co-workers (J. H. Krege, S. W. M. John, L. L. Langenbach, J. B. Hodgin, J. R. Hagaman, E. S. Bachman, J. C. Jennette, D. A. O’Brien, and O. Smithies. Nature 375: 146–148, 1995), to examine the effect of chronic ACE deficiency on the magnitude of tubuloglomerular feedback (TGF) responses. The genotype was determined by PCR on DNA extracted from the tail and was verified after each experiment by assessment of the blood pressure response to an injection of ANG I. To assess TGF responsiveness, we determined the change in stop-flow pressure (PSF) caused by increasing NaCl concentration at the macula densa by using micropuncture techniques. When loop of Henle flow rate was increased from 0 to 40 nl/min, PSF fell from a mean of 42.3 ± 1.95 to 33.6 ± 2.09 mmHg ( n = 6, P = 0.005) in wild-type mice (+/+), fell from 40.6 ± 2.35 to 38.6 ± 1.93 mmHg in heterozygous (+/−) mice ( n = 7, P = 0.014), and did not change in homozygous ACE (−/−) mice [36.7 ± 2.02 mmHg vs. 36.4 ± 2.01 mmHg; n = 4, P = not significant (NS)]. During an infusion of ANG II at a dose that did not significantly elevate blood pressure (70 ng ⋅ kg−1 ⋅ min−1), TGF response magnitude (PSF 0 − PSF 40) increased from 6.5 ± 1.4 to 9.8 ± 1.19 mmHg in +/+ ( P = 0.006), from 1.14 ± 0.42 to 4.6 ± 1.3 mmHg in +/− ( P = 0.016), and from 0.42 ± 0.25 to 4.02 ± 1.06 in −/− mice ( P = 0.05). Absence of TGF responses in ACE null mutant mice and restoration of near-normal responses during an acute infusion of ANG II supports previous conclusions that ANG II is an essential component in the signal transmission pathway that links the macula densa with the glomerular vascular pole.

For further elucidation of the role of neuronal nitric oxide synthase (nNOS) in macula densa (MD) cells, experiments were performed in anesthetized nNOS knockout mice (nNOS -/-). At comparable levels of arterial BP, renal blood flow was not significantly different between nNOS +/+ and nNOS -/- (1.7 +/- 0.2 versus 1.4 +/- 0.1 ml/min), and autoregulation of renal blood flow was maintained to a pressure level of approximately 85 mmHg in both groups of mice (n = 6 in each group). The fall in proximal tubular stop-flow pressure in response to an increase in loop of Henle perfusion rate from 0 to 30 nl/min was comparable in nNOS +/+ and -/- mice (40.7 +/- 1.6 to 32 +/- 2 mmHg versus 40.6 +/- 1.6 to 31.6 +/- 2 mmHg; not significant; n = 13 versus 18 nephrons). Luminal application of the nonselective NOS inhibitor nitro-L-arginine (10(-3) and 10(-2) M) enhanced the perfusion-dependent fall in stop-flow pressure in nNOS +/+ (7 +/- 1 to 13 +/- 2 mmHg; P < 0.05) but not in nNOS -/- (7 +/- 1 to 8 +/- 1 mmHg; not significant) mice. nNOS -/- mice exhibited a lower nephron filtration rate, compared with nNOS +/+, during free-flow collections from early distal tubules (influence of MD intact, 7 +/- 0.7 versus 10.9 +/- 1 nl/min; P = 0.002) but not from late proximal tubule (influence of MD minimized, 10.1 +/- 1 versus 11.7 +/- 1 nl/min; not significant; n = 16 nephrons). Distal Cl concentration and fractional absorption of fluid or chloride up to the early distal tubule was not different between nNOS -/- and +/+ mice. The data indicate that nNOS in MD tonically attenuates the GFR-lowering influence of ambient luminal NaCl, which may serve to increase the fluid and electrolyte load to the distal tubule, consistent with a role of MD nNOS in tubuloglomerular feedback resetting.

The effect of the adenosine type 1 receptor agonist N 6-cyclohexyladenosine (CHA) on glomerular vascular reactivity was studied in male angiotensin II type 1A (AT1A) receptor knockout mice (9). Vascular reactivity was assessed as the response of stop-flow pressure (PSF) to infusion of CHA into loops of Henle using micropuncture techniques. In AT1A +/+ mice at ambient arterial blood pressure (96.7 ± 2.8 mmHg), the presence of CHA (10−5 M) in the perfusate increased PSF responses from 6.8 ± 0.6 to 14.3 ± 0.9 mmHg when the loop of Henle of the index nephron was perfused and from 0.7 ± 0.3 to 12.3 ± 1.0 mmHg when the loop of an adjacent nephron was perfused. At reduced arterial blood pressure (82.8 ± 1.3 mmHg), index nephron perfusion with CHA increased PSF responses from 4.5 ± 0.3 to 9.4 ± 0.4 mmHg. In AT1A −/− mice with a mean arterial blood pressure of 80 ± 1.9 mmHg, CHA increased PSF responses only from 0.1 ± 0.3 to 3.6 ± 0.54 mmHg during index nephron perfusion and from 0.25 ± 0.2 to 2.7 ± 0.55 mmHg during adjacent nephron perfusion, significantly less than in wild-type animals ( P < 0.001). Responses to CHA were intermediate in AT1A +/− mice. Thus AT1A receptor knockout mice show a markedly reduced constrictor response to CHA both in the presence and absence of simultaneous activation of the tubuloglomerular feedback system. These data support the notion of a functional interaction between adenosine and angiotensin II in the regulation of afferent arteriolar tone.

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 ± 40 vs. 1081 ± 68 milliosmolar). Transepithelial osmotic water permeability (P f ) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [−/−] mice was 0.033 ± 0.005 cm/s (SE, n = 6 mice, 37°C), much lower than that of 0.15 ± 0.03 cm/s ( n = 8) in tubules from wild-type [+/+] mice ( P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 ± 0.12 (−/−, n = 5) and 0.64 ± 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 ± 0.05 (−/−, n = 8 mice [53 tubules]) and 1.95 ± 0.09 (+/+, n = 7 mice [40 tubules]) ( P < 0.001), corresponding to 26 ± 3% [−/−] and 48 ± 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 ± 0.3 [−/−] and 4.4 ± 0.5 [+/+], corresponding to 62 ± 4% [−/−] and 76 ± 3% [+/+] absorption ( P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.

Experiments were performed in a recently generated strain of mice with an angiotensin II AT1A-receptor null mutation (M. Ito, M. I. Oliverio, P. J. Mannon, C. F. Best, N. Maeda, O. Smithies, and T. M. coffman. Proc. Natl. Acad. Sci. USA 92: 3521-3525, 1995) to examine the effects of chronic AT1A receptor deficiency on tubuloglomerular feeback (TGF) responses. All animals were genotyped by polymerase chain reaction using primers designed to amplify sequences from the deleted AT1A gene and from the neomycin resistance gene. Normal mice (AT1A +/+) and mice heterozygous (AT1A +/-) and homozygous (AT1A -/-) for the gene disruption were anesthetized, and stop-flow pressures (PSF) were determined during changes in loop perfusion rate with previously established micropuncture methods. In five AT1A +/+ mice (26 tubules) mean PSF at zero loop flow was 37.2 +/- 1.5 mmHg, falling to 28.2 +/- 1.9 mmHg at a flow of 45 nl/min (P < 0.0001). Flow rate causing the half-maximum response (V1/2) was 8.7 +/- 0.4 nl/min. In four AT1A +/- animals (19 tubules) mean PSF at zero flow was 39.9 +/- 2.4 mmHg, falling to 34.8 +/- 2.7 mmHg at 45 nl/min (mean V1/2 8.6 +/- 1.04 nl/min). In five AT1A -/- mice (24 tubules) PSF was not significantly affected by loop flow with PSF averaging 33.9 +/- 1.7 mmHg at zero flow and 33.2 +/- 1.6 mmHg at 45 nl/min (not significant). Mean arterial blood pressures in the anesthetized and laparotomized mice were 91.8 +/- 2.2, 97.1 +/- 3, and 80.7 +/- 3.2 mmHg in the AT1A +/+, AT1A +/-, and AT1A -/- animals, respectively. Blood pressure responses to exogenous angiotensin II were greatly blunted in the AT1A -/- mice. We conclude that AT1A receptor-mediated effects of angiotensin II are in essential component of TGF responsiveness under chronic conditions. Our studies show the feasibility of using complex micropuncture methods in mice, an approach that widens the potential of genetically altered mouse strains as experimental models.