α-Intercalated cells defend the urinary system from bacterial infection

Abstract

α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

Figure 1. LCN2 is markedly upregulated in human and mouse UTI.

(A) uLCN2 expression in LE⁺Cx⁺ (100 ng/ml; IQR 30–300 ng/ml; n = 43) versus LE^–Cx^– patients (14.2 ng/ml; IQR 7.4–35.3 ng/ml; n = 514) in an Emergency Department cohort lacking other forms of kidney disease. (B) Relationship between uLCN2 and uCFU in all LE⁺ Emergency Department patients (LE⁺ and <10⁴ uCFU [limit of detection], 45 ng/ml uLCN2, IQR 20–150 ng/ml, n = 75; LE⁺ and 10⁴–10⁵ uCFU, 250 ng/ml, IQR 40–425 ng/ml, n = 21; LE⁺ and >10⁵ uCFU, 520.2 ng/ml, IQR 236.7–1,126 ng/ml, n = 45). (C) Suppression of uLCN2 by antibiotics (Pre, 600 ng/ml, IQR 533–700 ng/ml; Post, 83.0 ng/ml, IQR 21–129 ng/ml) in patients presenting to clinic with dysuria, frequency, urgency and LE⁺ (>30 wbc per high-powered field), hematuria, and mucus threads (n = 4). Values represent median and IQR.

Figure 2. LCN2 is necessary and sufficient to suppress UTI.

(A) Longitudinally sampled WT C57BL/6 mouse urine (n = 7 per time point) demonstrated faster clearance of Ent⁺ uCFUs than that of sibling-matched Lcn2^–/– mice (n = 6 per time point). *P < 0.05, Mann-Whitney test. (B) Longitudinal measurements of uLCN2 by immunoblot, in the same urine samples as in A, correlated with uCFUs. Lanes for 0–3 days and for 4–7 days were run on different gels, but with the same in-gel LCN2 standards. (C) In contrast, there was no significant difference in uCFUs in Lcn2^–/– versus WT mice inoculated with Ent-null UPEC. (D) Bladder CFUs in Lcn2^–/– mice (n = 9) exceeded those in WT mice (n = 5) and mirrored the difference in uCFUs at 1 day after infection with Ent⁺ UPEC. (E) LCN2 (5 μM; n = 3) suppressed the growth of UPEC in urine (pH 5.8). P < 0.0001.

Figure 3. Response of UPEC to LCN2 and DFO.

UPEC were grown in M9 medium with (A) LCN2 (5 μM; n = 5) or (B) DFO (50 μM; n = 5) for 30 minutes. Note the upregulation of Ent synthetic enzymes (entA and entF) and receptors (fepA and iroN), aerobactin synthetic enzymes (iucA and iucD) and receptors (iutA), and heme receptors (chuS), which indicates that LCN2 induced Fe starvation and widespread activation of compensatory pathways, similar to Fe restriction by DFO.

Figure 4. Temporal and spatial expression of LCN2 in the kidney.

(A–D and G) Cystitis model. (A and B) Lcn2-Luc2 (C57BL/6 background) reporter mice were inoculated with UPEC, and luciferase was quantified (n = 11). (C) Excised urogenital tracts (1 day after inoculation) verified that LCN2 luminescence (arrowheads) originated from the kidney medulla. (D) Lcn2 copy number in C57BL/6 kidney and bladder before and 1 day after inoculation. (E and F) C57BL/6 and C3H/HeN mice inoculated with bioluminescent CFT073 UPEC-lux (20 μl of 5 × 10⁸ CFU/ml; n = 4 each) were imaged on the dorsal and ventral sides for 3 days. UPEC-lux were detected 1–3 days after inoculation in C3H kidney (cystitis and pyelonephritis), but not in C57BL/6 kidney (cystitis). (G) Cytokine activation in C57BL/6 kidney (cystitis model; n = 10). *P < 0.05; ^#P < 0.001. Scale bar: 1 cm. Kd, kidney; g, gonad; Blad, bladder.

Figure 5. Temporal and spatial expression of LCN2 in bladder.

(A) GFP-labeled UPEC in C3H bladder wall. (B) Lcn2 expression by uroepithelium (1 day after inoculation). (C) Cytokine activation in C3H/HeN Lpsⁿ bladder (pyelonephritis model; n = 6). Note the consistent expression of Il1a and Il1b in bladder and kidney (see Figure 4G). Scale bars: 100 μm.

Figure 6. UPEC bound to A-ICs.

(A) In the C3H/HeN model of pyelonephritis, GFP-labeled UPEC (green) specifically bound A-ICs (marked by apical v-ATPase; red; n = 4). Original magnification, ×40. (B) High-resolution image of UPEC-GFP bound to A-ICs. Original magnification, ×100. (C) UPEC induced Lcn2 (arrowheads) in C3H medullary cells/ICs in kidney (paraffin in situ hybridization). (D) Treatment of an IC line with killed UPEC (24 hours; n = 4) induced Lcn2 message. NF-κB inhibitor (5 μM) reversed the LCN2 signal. (E) Coculture of Lcn2-Luc2 kidney medullary cells with living UPECs (3 hours) induced LCN2-Luc2 reporters. Gentamicin reversed the LCN2 signal (n = 3). Note the luminescent wells (blue, baseline; green/red, activated). Scale bars: 50 μm (A and C); 10 μm (B).

Figure 7. A-ICs regulate uLCN2 and urinary pH.

(A) ICs in sibling-matched Tcfcp2l1^fl/fl and (B) IC-knockout Tcfcp2l1^fl/^fl;Ksp-Cre mice were visualized by immunostaining with Troma1-Krt8 (red), v-H⁺ATP6v1b1 (blue), and Tcfcp2l1 (green). (C and D) Deletion of IC Tcfcp2l1 suppressed (C) uLCN2 (n = 6) and (D) acidification in response to LPS (n = 15) compared with Tcfcp2l1^fl/fl littermates. (E) UPEC also induced urinary acidification in C57BL/6 mice (n = 6). (F) IC-knockout Tcfcp2l1^fl/^fl;Ksp-Cre mice were significantly less able to clear bladder and urinary bacteria 1 day after TU inoculation than Tcfcp2l1^fl/fl mice (n = 8). *P < 0.05. Scale bars: 10 μm.

Figure 8. Acidification inhibits bacterial growth.

UPEC growth was significantly suppressed by acidification in both (A) M9 media (n = 4; P < 0.0001) and (B) acidified urine (n = 4; P < 0.0001, pH 5.0 vs. pH 5.5, pH 5.5 vs. pH 6.0, and pH 6.0 vs. pH 6.5).

Figure 9. TLR4 is required to induce LCN2 in UTI.

(A) Gram^– infections, but not Gram⁺ infections, induced high levels of uLCN2 in the subset of the Emergency Department patients with documented speciation data, CFU counts, and no other renal disease (Gram⁺, 200.0 ng/ml, IQR 30.0–425.0 ng/ml, n = 9; Gram^–, 400.0 ng/ml, IQR 135.0–775.0 ng/ml, n = 77). Both Gram⁺ and Gram^– patients had >100,000 CFU/ml urine. (B) uCFUs were significantly lower, and (C) kidney Lcn2 and (D) kidney Il1 levels were significantly higher, in C3H Lpsⁿ (n = 9) versus Lps^d (n = 15) mice after inoculation (pyelonephritis model). (E) Lpsⁿ kidneys transplanted into Lps^d hosts demonstrated a 3-fold greater response to LPS (as assessed by kidney Lcn2 levels) than the reciprocal transplantation (n = 3 each). (F) In situ hybridization showing Lcn2 signal in medullary cells and ICs in LPS-challenged mice (paraffin in situ hybridization). Lcn2 RNA signal (dark purple stain) is denoted by arrowheads. (G) UPEC-GFP (green) bound TLR4⁺ (blue) cells in the CDs of C3H mice 1 day after inoculation (original magnification, ×100). *P < 0.05; **P < 0.001. Scale bars: 10 μm.

Figure 10. LCN2 induction.

(A) CD (boxed region) and connecting segments contain A-ICs. (B) Systemic sepsis, cystitis, and pyelonephritis (UTI) activate A-ICs. Kidney TLR4 expression is critical for the secretion of uLCN2 and cytokines and for the suppression of UTI. LCN2 is secreted into the urine (apically) and is potentially also secreted into the circulation (basolaterally). Note that LCN2 and H⁺ secretion are both activated in UTI.