Cell-type specific gene silencing in the Thick Ascending Limb with CRISPR/Cas9 to study NKCC2 kinases

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas) is a highly effective gene-editing tool. However, few studies have used this method to target genes in the nephron. In the nephron, abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitive hypertension. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at the Thr96,101 by upstream kinases SPAK (Stk39), OSR1 (Oxsr1) and TNIK (Tnik). We showed that NKCC2 phosphorylation is 4-fold higher in TALs of Dahl SS rats and it is accompanied by a greater phosphorylated SPAK/OSR1 in the TAL, suggesting these kinases may be involved in salt sensitivity. However, there are no methods that allow studying the effect of silencing one or the three kinases simultaneously in rats.We hypothesized that using the NKCC2 promoter to drive High-Fidelity (HF) Cas9 expression in kidney TALs would allow us to chronically silence kinases that regulate NKCC2 and reduce its phosphorylation at the Thr96,101. First, we designed several gRNA sequences targeting SPAK or TNIK and tested their efficacy in Cas9 expressing cell lines. The most efficient guides for each kinase, showing over 50% decrease in target protein expression, were packaged into Adeno-associated Virus (AAV) serotype 9. To silence the kinases in TALs in vivo, we injected the renal medulla of the left kidney of rats with AAV-gRNA-SPAK or AAV-gRNA-TNIK together with AV-NKCC2 promoter-HFCas9. After 2 weeks, we isolated medullary TALs and cortical tubules and measured the kinase expression and NKCC2 phosphorylation. In cultured cells expressing Cas9, gRNAs designed for each kinase decreased the expression of SPAK by 78 ± 6%, or TNIK by 48 ± 22%, respectively, compared to the control (p< 0.5, n=3). In medullary tubule suspensions that contain mostly TALs, SPAK was silenced by 55 ± 2% with AAV-gRNA-SPAK (n=3, p< 0.01), and pThr-96,101-NKCC2 was decreased by 37 ± 8% compared to kidneys transduced with AV-control (p< 0.5, n=3). The expression of SPAK in cortical tubules was not changed (Control: 100 vs Cas9-SPAK in cortical tubules: 117±22 %, n=4) supporting specificity of NKCC2-promoter to drive Cas9 expression in medullary TALs. The expression of TNIK and OSR1 in medullary TALs was not modified in kidneys transduced with AAV-SPAK-gRNAs (Control: 100, TNIK: 101 ± 45%, OSR1 127 ± 43% of control, n=3, ns), supporting selectivity of the gRNA for the targeted SPAK. In medullary TALs transduced with gRNA-TNIK, TNIK expression was decreased by 50 ± 6% (n=4, p< 0.01), and pThr-96,101-NKCC2 was decreased by 38 ± 10% compared to kidneys transduced with AV-control (n=4, p< 0.05). The expression of the related kinases, SPAK and OSR1, was not modified (SPAK: 89 ± 18%, OSR1: 98 ± 19% of control, n=4, ns). The expression of TNIK in cortical tubules was not changed (Control: 100 vs Cas9-TNIK in cortical tubules: 78±32 %, n=4). We conclude that driving Cas9 expression in medullary TALs allows for TAL-specific Cas9 mediated gene silencing in vivo, to silence the kinases that regulate NKCC2 activity. NKCC2promoter-driven Cas9 was specific to medullary TALs because it did not alter expression of the targeted kinases in cortical tubules. Our data suggests that this method is effective and specific to study NKCC2 interacting-kinases in the kidney. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.