The Kidney

1. Stress (hypoxaemia/dehydration) results in ↓urine volume and ↑urine osmolarity. Mediated by AVP and sympathetic nerves.

   Annotations:

   • 1. Infusing an AVP antagonist prevents the ↑urine osmolarity caused by dehydration. 2. Renal denervation reduces the ↓RBF caused by hypoxaemia.
2. Can't produce hypertonic urine because: 1) Immature LoH; 2) Preferential blood flow through cortex; and 3) Unresponsive to AVP and ANP.

1. Markers: Lim1, Odd1, Pax2, Pax8.
2. Forms from intermediate mesoderm.
3. Specification due to signals from: 1) Paraxial mesoderm and 2) Overlying ectoderm.

   Annotations:

   • 1. Separating paraxial from intermediate mesoderm prevents kidney specification - both necessary and sufficient. 2. BMP gradient from overlying ectoderm specifies intermediate mesoderm as kidney progenitor. Intermediate levels of BMP → kidney progenitor marker gene expression and inhibition of FoxC expression. 

1. Reciprocal RET/GDNF signalling between UB and MM responsible for growth of nephric duct.

   Annotations:

   • 1. Transfilter induction assays (separating MM and UB) show that kidney development does not occur without both present. 2. GDNF knockout prevents UB outgrowth. 3. RET -/- and WT chimaeras form normal kidneys but the RET -/- cells don't form tip cells, suggesting RET/GDNF signalling required for tip cell formation.
   1. RET/GDNF → Wnt11 expression by MM. Upregulates GDNF expression in positive feedback loop.

      Annotations:

      • 1. Wnt11 mutant kidneys are 36% smaller than WT kidneys, whilst RET + Wnt11 mutant kidneys are 52% smaller.
   2. RET/GDNF → Sprouty expression. Inhibits RET/GDNF signalling to prevent additional UB outgrowth.
   3. RET/GDNF → Etv4 and Etv5 expression. These promote branching morphogenesis by increasing the expression of genes involved in:
      1. Differential proliferation (Myb).
      2. Localised ECM remodelling (MMP14).
      3. Directed cell movements (Cxcr4).
      4. Oriented cell division.
      5. Differential adhesion.
      6. Cell shape changes.

1. MM undergoes MET, which involves: 1) Condensation; 2) Epithelialisation; 3) Early morphogenesis; and 4) Tubule maturation.
2. MET driven by Wnt9b expression by UB.

   Annotations:

   • 1. Transfilter induction assays showed that Wnt proteins able to induce MET in isolated MM. 2. Wnt9b expressed by UB. 3. MET doesn't occur in Wnt9b mutants.
   1. Wnt9b promotes Lim1, FGF8, Pax9, and Wnt4 expression.
      1. Wnt4 maintains and propagates inductive signal.

         Annotations:

         • 1. Wnt9b is insufficient to induce MET in Wnt4 mutant mice.

1. Lim1 promotes the expression of:
   1. Dll1 - promotes Notch signalling, leading to expression of proximal genes e.g. Wt1. Wt1 promotes the expression of:

      Annotations:

      • 1. In Notch mutants, proximal nephron regions don't differentiate.
      1. Nephrin - forms slit diaphragm.
      2. VEGF - guides endothelial cells to glomerulus.

1. Endothelial cells guided into cleft of S-shaped body by VEGF, form vascular tuft, then differentiate into mature glomeruli.

1. PC1 and PC2 localise to primary cilium and form mechanosensory Ca channel that opens in response to shear stress.
2. PC1 and PC2 regulate orientation of cell division - PKD associated with disoriented cell division → ↑tubule diameter.

1. Associated with ↓fetal urine production, due to urinary tract obstruction or ↓renal blood flow.
2. Can lead to umbilical cord compression.

Annotations:

• 1. Epidemiological studies.

1. Potential mechanisms
   1. Altered Na handling by the kidney

      Annotations:

      • 1. Manning et al. - Low protein diet in rats associated with increased expression of NKCC and NaCl transporters in kidney.
   2. Increased apoptosis

      Annotations:

      • 1. Welham, Wade, and Woolf - Low protein diet associated with ↑metanephric apoptosis and ↓kidney progenitors.
   3. ↓Expression of RAS components

      Annotations:

      • 1. Vehaskari et al. - ↓expression of RAS components associated with ↓nephron number
   4. ↑Glucocorticoid exposure
   5. ↓RET/GDNF function

Annotations:

• 1. Brenner et al. - animals born with ↓nephron number develop hypertension. 2. Bhathena et al. - humans with unilateral lateral agenesis of the kidney develop hypertension. 3. Keller et al. - autopsies showed ↓nephron number associated with hypertension.

Annotations:

• 1. Gilbert et al. - animal model. 2. Barker and Osmond - epidemiological study. Showed inverse relationship between birth weight and hypertension.

Annotations:

• Low nephron number → ↓surface area for excretion → ↑sodium and water retention → ↑blood volume → ↑blood pressure → initial glomerular growth → eventual glomerular sclerosis → chronic kidney disease.

1. Pc can be altered by:
   1. Changing renal arteriolar resistance.
   2. Changing renal arterial pressure.
      1. ↑ABP would be expected to cause ↑RBF and GFR but response dampened down due to renal autoregulation.

Annotations:

• 1. Some autoregulation persists in hydronephrotic kidneys. 2. Some autoregulation persists in isolated afferent arterioles. 3. Some autoregulation persists in animal models of hydronephrotic kidneys.

1. ↑ABP
   1. ↑Transmural tension
      1. Activation of stretch-activated non-selective cation channels.

         Annotations:

         • Are these ENaC channels? FOR 1. Amiloride (ENaC blocker) in high but not low concentrations prevented AA constriction in response to KCl depolarisation. 2. Immunostaining showed all ENaC subunits expressed in AA. 3. Knockout of ENaC channel impaired myogenic autoregulation. AGAINST 1. Amiloride at 1ul and 3ul did not affect AA constriction, but may have been too low a dose to have an effect. 2. Northern blots found ENaC subunits only all present in 11/20 cases.
         1. Depolarisation
            1. VOCC activation
               1. AA SM contraction
               2. Ca binds to Ca-calmodulin, which ↑ the affinity of MLCK for myosin, increasing cross-bridge cycling and causing SM contraction.
2. 'On' kinetics faster than 'off' kinetics, allowing autoregulation in response to cyclical changes in ABP.
3. Thought to protect against hypertensive injury rather than maintain ECF volume.

1. Independent control of afferent and efferent arterioles possible due to VOCCs being involved in AA constriction but not EA constriction.

   Annotations:

   • 1. Depolarising renal arteriles with KCl (↑ EC Ca conc.) causes AA SM contraction but not EA SM contraction. 2. Carmines and Navar - Diltiazem (VOCC blocker) prevents AA SM contraction but not EA SM contraction in response to ATII.

1. Studied using in vitro blood-perfused juxtamedullary nephron preparation.
   1. Kidney isolated, hemisected, and juxtamedullary circulation exposed.
   2. Can see vessel diameter.
   3. Tubulovascular relationships preserved.
   4. Continuous oxygenation of tissue.
   5. Can control hormone concentrations in perfusate and superfusate.
   6. Can measure SFP (pressure required to stop flow at glomerulus).
2. ↑ABP
   1. ↑GFR
      1. ↑NaCl uptake by MD
         1. ↑Basolateral Cl efflux by MD

            Annotations:

            • 1. Ren et al. - Blocked Cl channels with NPPB and found that ↑NaCl conc. did not cause AA constriction.
            1. MD depolarisation

               Annotations:

               • 1. Ren et al. - Depolarisation using nystatin when NKCC transporter blocked still causes AA constriction, showing that depolarisation is involved in TGF.
               1. VOCC activation

                  Annotations:

                  • 1. Ren et al. - Treatment with Ca ionophore caused ↓SFP even when NaCl conc. was low, whilst treatment with Ca chelator prevented ↓SFP even when NaCl conc. was high.
                  1. Chemical signal released
                     1. AA SM contraction → ↓Pc
                     2. Intraglomerular mesangial cell contraction → ↓Kf
                     3. Nature of Chemical Signal
                        1. Angiotensin II
                           1. Now thought to be modulator, acting synergistically with adenosine.

                              Annotations:

                              • FOR 1. Isolated rabbit AAs had greater constrictory response to adenosine and ATII than to either alone. 2. The constrictor actions of adenosine reduced by ATII antagonist. 3. TGF response abolished in ATII receptor KO mice.
                        2. Nitric oxide
                           1. Thought to attenuate TGF response by dilating AA.
                        3. Adenosine
                           1. Thought to cause AA constriction by acting on A1 receptors.

                              Annotations:

                              • FOR 1. A1 receptor antagonists prevent TGF in some studies. 2. HOM A1 receptor mutants have no TGF response, whilst HET A1 receptor mutants have a reduced TGF response. 3. Thomson et al. - Inhibiting conversion of AMP to adenosine prevents TGF. 4. RT-PCR and in situ hybridisation have shown that A1 receptor is expressed by AA. 5. Exogenous adenosine causes AA constriction. 6. Receptor-specific agonists have shown that vasoconstrictor effect of adenosine is mediated by A1 receptors. 7. Magnitude of TGF response rose with ↑adenosine concentration.
                              • AGAINST 1. Prolonged adenosine application causes vasconstriction to wane or revert. 2. Adenosine doesn't increase IC Ca conc. in isolated AA SM cells and mesangial cells. 3. A1 antagonist has no effect on calcium wave spreading from MD to AA and mesangial cells.
                           2. May also cause EA dilation by acting on AT2 receptors.
                        4. ATP
                           1. Thought to cause AA constriction by acting on P2X1 receptors.

                              Annotations:

                              • FOR 1. ↑NaCl delivery to MD causes ATP release via maxi-anion channel. 2. P2 receptor desensitisation results in ↓TGF response. 3. P2 antagonists prevent TGF. 4. P2X KO mice don't exhibit TGF. 5. ATP evokes much faster TGF response than adenosine. 6. ATP causes ↑ IC Ca conc. in isolated AA SM cells. 7. ATP and UTP cause mesangial cells to contract, suggesting role of P2Y receptor. 8. P2 antagonist or ATP scavenger prevents Ca wave from spreading from MD to AA and mesangial cells. 9. Immunostaining showed P2X receptors expressed in AA but not EA.
                        5. ATP and Adenosine
                           1. ATP released from MD cells and converted to adenosine by NTPDase and ecto-5'-nucleotidase.

                              Annotations:

                              • FOR 1. KO of NTPDase and ecto-5'-nucleotidase associated with ↓TGF response.
                     4. May act indirectly on EG mesangial cells, with signals being transmitted to IG mesangial cells and AA via gap junctions.

                        Annotations:

                        • FOR 1. Anti-mesangial cell antibodies block TGF. 2. Gap junction blocking agents prevent TGF.
         2. Basolateral Na/K pump sets up Na gradient.

            Annotations:

            • Lorenz et al. - Ouabain-sensitive Na/K pump blocked using ouabain prevented ↓SFP in response to ↑flow rate. 
         3. NKCC transporter drives NaCl uptake by MD.

            Annotations:

            • 1. Loop diuretics prevent ↓SFP in response to ↑flow rate. 2. Schnerrman and Briggs - TGF occurs in both NKCC2A and NKCC2B knockouts but at different thresholds due to different affinities for Cl. Shows that both isoforms involved in TGF.
         4. ROMK recycles K.

            Annotations:

            • 1. Schnerrmann and Briggs - ROMK knockout mice do not show ↓SFP in response to ↑flow rate.
         5. MD cells may also release a chemical signal in response to ↑flow rate independent of NaCl concentration.

            Annotations:

            • 1. Sipos et al. - Superfusing NaCl-free solution over MD caused AA constriction, which ↑ if NaCl added. 2. Sipos et al. - Mechanical displacement of primary cilium caused AA constriction.

1. Isosmotic reabsorption
2. Basolateral Na/K pump sets up Na gradient.

   Annotations:

   • 1. Dinitrophenol (microtubule uncoupler) prevents water reabsorption in PT, showing active transport is involved. 2. Ouabain (Na/K pump inhibitor) prevents water reabsorption in PT, showing Na/K pump involved.
   1. Na diffuses into PT cells via Na-glucose, Na-aa, and Na/H transporters.
      1. Water follows solute across leaky tight junctions.

         Annotations:

         • 1. Inulin concentration increases along length of PT, despite no reabsorption or secretion, showing water reabsorption must have taken place. 2. Split oil drops move closer together along PT, indicating water reabsorption. 3. Changing NaCl concentration of filtrate shows that water reabsorption is dependent on NaCl conc.
3. Cl reabsorbed paracellularly and transcellularly via apical Cl/organic anion and basolateral Cl-K transporters.
   1. Organic anions recycled via protonation and reabsorption - tertiary active transport.

1. Diluting Kidney
   1. NaCl reabsorbed via NKCC transporter in TAL of LoH.

      Annotations:

      • 1. Gregor et al. - Found that NKCC involved in NaCl reabsorption by TAL of LoH.
      1. K recycled via K-Cl symporters and K channels.
      2. Water reabsorbed in TD of LoH, making filtrate more hypertonic and allowing more NaCl to be reabsorbed by NKCC. Countercurrent multiplication.
   2. Most urea excreted in urine.
2. Concentrating Kidney
   1. Countercurrent multiplication as in diluting kidney.
   2. Urea concentration high as it enters IMCD due to water reabsorption.
      1. IMCD highly permeable to urea, urea reabsorbed.

         Annotations:

         • 1. Kokko and Rector - Demonstrated differences in urea permeability between different segments of nephron: IMCD highly permeable to urea in presence of ADH. 2. Morgan and Berliner - Demonstrated changes in permeability of collecting duct to water and urea in response to ADH. 3. Lassiter et al. - Showed urea reabsorbed by collecting duct.
         1. Hypertonic medullary interstitium drives water reabsorption from TD, which ↑NaCl conc. in TA.

            Annotations:

            • 1. Morgan and Berliner showed that TD more permeable to water than TA or TAL.  2. Lassiter et al. - Micropuncture studies showed that water reabsorption occurred in TD.
            1. NaCl reabsorption in TA makes interstitium even more hypertonic, driving water reabsorption from IMCD, resulting in concentrated urine.

               Annotations:

               • 1. Fenton et al. - Genetic deletion of urea transporters results in large volumes of hypotonic urine, showing that urea involved in producing concentrated urine. However, deletion of urea transporters did not affect NaCl concentration in inner medulla, suggesting that urea does not ↑NaCl reabsorption from TA of LoH. 2. Micropuncture studies have shown that TA is permeable to solute but not water. 3. Lassiter et al. - Micropuncture showed that water reabsorption occurs in TA.
3. Vasa Recta
   1. Maintain concentration gradients in interstitium.
   2. NaCl and urea secreted on descent, reabsorbed on ascent.
   3. Water reabsorbed on descent, secreted on ascent.
4. Osmotic gradient from renal cortex to medulla - hypotonic interstitium in cortex, hypertonic interstitium in medulla.

   Annotations:

   • 1. Wirz, Hargitay, and Kuhn - Demonstrated increasing osmotic gradient from cortex to medulla using freezing point studies. 2. Gottschalk and Mylle - Osmolarity same in different sections of nephron at same level. 

1. ↑Water permeability of all sections of nephron and ↑urea permeability of IMCD and TD, leading to production of concentrated urine.
2. ADH secretion promoted by ↓blood volume and ↓ABP
3. ADH secretion inhibited by: ↑ABP, ↑blood volume, drinking, and emotional factors.
4. Secreted from posterior pituitary gland.

   Annotations:

   • 1. Shafer and Magnus - Extracts from PPG capable of inhibiting diuresis. 2. Shafer - Damage to PPG associated with diuresis. 3. Frank - Damage to PPG associated with diabetes insipidus.